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} | pes2o/s2orc | Chemical profile and larvicidal activity of essential oil obtained from the leaves of Plectranthus amboinicus (Lour.) Spreng
This study aimed to determine the chemical constituents and larvicidal activity of the essential oil of Plectranthus amboinicus (Lour.) Spreng against larvae of the Aedes aegypti mosquito. The essential oil (EO) was extracted by hydrodistillation at 100 ° C for 3 hours. The chemical composition was obtained by Gas Chromatography coupled to Mass Spectrometry (GC / MS). To assess larvicidal activity, Aedes aegypti larvae were subjected to EO solutions in concentrations of 10-100 mg L-1, where larval mortality was assessed and the LC50 was determined using the Probit method. The main chemical constituent found in the EO was carvacrol, which is considered to be very promising for pharmaceutical synthesis. The EO showed larvicidal activity with an LC50 of 28.52 mg L-1. According to the results found, it was possible to evaluate that the analyzed EO is composed of substances that have an efficient larvicidal effect against Aedes aegypti, thus encouraging its potential for application.
Introduction
Plants are considered one of the main natural resources for medicinal use, due to their biological potential, whether due to the action of deadly diseases or diseases that affect living beings, therefore, according to the World Health Organization, almost 80% of the population in developing countries development uses them directly or indirectly for their basic health needs, either because of cultural tradition or because there are no other options, due to the high cost of traditional medicines for this population (Bermúdez,Oliveira-Miranda & Velázquez, 2005;Alitonou et al., 2012;de Souza et al., 2020 ).
Multidisciplinary efforts have also led to an increase in the number of researches to obtain greater knowledge about a medicinal plant (de Souza et al., 2020). The study of substances extracted from plants has been shown to be indispensable over time, whether due to the great biological diversity in Brazil or the potential of this extraction. Thus, essential oils represent a viable alternative in several studies involving substances of plant origin (Gomes et al., 2019;Minott & Brown, 2007).
Specific analyzes are necessary around the validation process, which includes chemical composition, proof of pharmacological, molluscicidal, microbial activity and possible toxicity in humans parameters that ensure the quality of raw materials of plant origin (Gomes et al., 2019).
The toxic potential of essential oils (EOs) and their compounds can vary significantly according to intrinsic and extrinsic factors (Kim et al., 2016). The chemical composition of EOs contributes significantly to the determination of the pharmacological potential attributed to plant species. In the case of EOs, it is recommended to regularly use the gas chromatography system coupled to the mass spectrum (GC / MS), in order to identify substances derived from secondary metabolism. EO extracted from medicinal plants have been widely used successfully in research aimed at epidemiological control (Mirzahosseini et al., 2017).
According to the Ministry of Health (2019), it was declared that in 2019, until the 12th Epidemiological Week (12/30/2018 to 03/23/2019), there were 273,193 probable cases of dengue in the country, which had a growth of approximately 382% compared to the same period in 2018 (71.525 thousand).
In the last decades, it is noted that the diseases transmitted by the Aedes aegypti mosquito, specifically dengue, have grown exponentially worldwide. There are approximately 390 million dengue infections per year, including 96 million with clinical manifestations (Brahtt et al., 2013).
Among the methods used to control the larvae, the use of temephos organophosphate insecticide is the main measure adopted by the National Dengue Prevention Program in Brazil and by the World Health Organization (Carvalho et al., 2004;Crivelenti et al ., 2011;Prophiro et al., 2011).
In view of the importance of EOs and their wide application, the present study aims to determine the chemical constituents and the larvicidal potential of the essential oil of Plectranthus amboinicus (Lour.) Spreng against the larvae of Aedes aegypti, aiming at a safe, ecologically viable and efficient alternative fighting and controlling the population of Aedes aegypti in the country.
Phytochemical screening
A hydroalcoholic extract of the plant material obtained was prepared and it was subjected to chemical tests based on the methodology presented by Matos (2009). The tests performed to identify alkaloids, steroids, phenolics, flavonoids, glycosides, cardiac glycosides, saponins and tannins are described below:
Steroids (Salkowsk test)
About 100 mg of dry extract was dissolved in 2 ml of chloroform. Sulfuric acid was carefully added to form a lower layer. A reddish-brown color at the interface indicated the presence of a steroid ring.
Alkaloids (Mayer's test)
1.36 mg of mercury chloride were dissolved in 60 ml and 5 mg of potassium iodide dissolved in 10 ml of distilled water, respectively. These two solvents were mixed and diluted to 100 ml using distilled water. To 1 ml of the aqueous acidic solution of the samples, a few drops of the reagent previously prepared were added. The formation ofwhite or pale precipitation showed the presence of alkaloids.
Flavonoids
In a test tube containing 0.5 mL of alcoholic extract from the samples, 5 to 10 drops of diluted HCl were added and a small amount of Zn or Mg was added to the solution, which was then boiled for a few minutes. The appearance of a reddish Research, Society and Development, v. 10, n. 4, e15410413846, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.13846 4 pink or dark brown color indicated the presence of flavonoids.
Glycosides
One small amount of alcoholic extract from samples was dissolved in 1 ml of water and then aqueous sodium hydroxide was added. The formation of a yellow color indicated the presence of glycids.
Cardiac glycosides
About 100mg of extract was dissolved in 1ml of glacial acetic acid containing a drop of ferric chloride solution and 1ml of concentrated sulfuric acid was added. A brown ring obtained at the interface indicated the presence of an oxy sugar characteristic of cardenolides.
Saponins
A drop of sodium bicarbonate was added to a test tube containing about 50 ml of an aqueous extract from the sample.
The mixture was shaken vigorously and held for 3 minutes. A honey comb like foam was formed and showed the presence of saponins.
Phenols
For 1 mL of alcoholic sample solution, 2 mL of distilled water was added followed by a few drops of 10% aqueous ferric chloride solution. The formation of a blue or green color indicated the presence of phenols.
Tannins
In a test tube containing about 5 ml of an aqueous extract, a few drops of 1% lead acetate solution were added. The formation of a yellow or red precipitate indicated the presence of tannins.
Obtaining essential oils
For the extraction of essential oils, the hydrodistillation technique was used with a Clevenger glass extractor coupled to a round bottom flask wrapped in an electric blanket as a heat generating source. 200g of P. amboinicus leaves were used, adding distilled water (1:10). Hydrodistillation was carried out at 100 ° C for 3 hours, collecting the extracted essential oil.
Each essential oil was dried by percolation with anhydrous sodium sulfate (Na2SO4) and stored in a refrigerator until further analysis.
Chemical constituents
The EO constituents were identified by gas chromatography coupled to mass spectrometry (GC-MS).
Egg collection
The eggs were collected at the Federal University of Maranhão, Campus Bacanga in São Luís / MA, using traps called ovitrampas. These consist of brown buckets (500 mL), made of polyethylene, with 1 mL of brewer's yeast and 300 mL of running water and two Eucatex straws are inserted for the mosquito's oviposition. The traps were inspected weekly for the replacement of straws and collection of eggs and sent to the Laboratory of Research and Application of Essential Oils (LOEPAV / UFMA) of the Technological Pavilion of the Federal University of Maranhão -UFMA.
Initially, the eggs of Aedes aegypti were placed to hatch at room temperature in a circular glass aquarium containing mineral water. The identification of the species followed the methodology proposed by Forattini (1962). The obtained larvae were fed with cat food according to the methodology of Silva et al., (1995) until they reached the third and fourth stages, the age at which the experiments were carried out.
Larvicidal activity
The tests for larvicidal activity were performed according to the adapted methodology proposed by Silva (2006). After 24h the counting of alive and dead was performed, being considered dead, the larvae that did not react to the touch after 24 hours of the beginning of the experiment. To quantify the efficiency of the EOs, the statistical test of Probit (Finney, 1952) was applied.
Phytochemical screening
Phytochemical analysis enabled the determination of secondary metabolites present in the analyzed plant materials, shown in Table 1. Research, Society andDevelopment, v. 10, n. 4, e15410413846, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.13846 6 conditions.According to Menezes Filho and Castro (2019), temperature, water availability, ultraviolet radiation, addition of nutrients, environmental pollution and attack of pathogens are factors that can influence the amount of secondary metabolites present in plant extracts.According to Alfaia (2016), the natural function of many secondary metabolites has been investigated with greater tenacity, being recognized that these are essential for the existence of plants and in several biotechnological applications.
Chemical constituents
According to Table 2, the substances identified in GC / MS, it is possible to highlight that Carvacrol is the major component (69.93%), followed by Cyclohexanone (11.07%) and Caryophyllene (5.64%).
These results are in agreement with the study by (Monzote et al., 2020), who also found a high percentage in the aerial parts of P. amboinicus with a 71% content of carvacrol. The chemical composition of the essential oil of P. amboinicus However, in a study by Santos et al., (2015), results inferior to this study were quantified in relation to the chemical composition of the leaves of P. amboinoicus, with carvacrol being 37.7%, caryophyllene and cyclohexanone were not quantified. The same was presented by Huang et al., (2019), with a content of carvracol of 61.53%, and the chemical components caryophyllene and cyclohexanone were also not found in this study. These results confirm the presence of a satisfactory percentage of the chemical components observed in the EO of this study. Table 3 shows the LC50 and LC90 referring to the action of the EO against the larvae of Aedes aegypti calculated through the log of the intersection of the curves. Regarding the larval activity of P. amboinicus EO, it is possible to observe that through Table 3, the plant presented LC50 of 28.52 mg L -1 and LC90 of 49.55 mg L -1 against the larvae of the mosquito Aedes aegypti.
Larvicidal activity in vitro front Aedes aegypti
According to the criteria of Dias and Moraes (2014), essential oils with lethal concentrations (LC50) below 50 mg L -1 are classified as highly active, to be considered active they must have LC50 below 100 mg L -1 and inactive when they have higher LC50 at 100 mg L -1 , confirming the larvicidal potential of the EO assessed in this study.
These results are in agreement with the findings in the literature, which confirm the potential of P. amboinicus EO. Paramasivam et al., (2020) when assessing the larvicidal activity of extracts of P. amboinicus, collected in India, observed maximum activity with minimum concentrations against the larvae of the mosquito of the 4th instar of Aedes aegypti, with LC50 ranging between 53, 36 and 13.64 µg mL -1 and LC90 of 92.51 and 86.09 µg mL -1 .
In a recent study by Huang et al., (2019), they obtained a higher value than this study, with an LC50 of 42.90 mg L -1 , in the evaluation of the larvicidal action of the peppermint EO, against the larvae of the mosquito Aedes aegypti. The same was reported by dos Santos et al., (2020) who obtained an LC50 of 41.7 mg L -1 for the larvicidal activity of P. amboinicus EO, however, they are classified as highly active, as in this study.
In a study by Jayaraman, Senthilkumar and Venkatesalu (2015), they observed that after 12h, the larvicidal potential of the methanol extract of P. amboinicus against Aedes aegypti larvae was 322.67 ppm (LC50) and 601.93 ppm (LC90), showing a lower action when purchased at this time. study.
The use of P. amboinoicus in traditional medicine, in other words, its biological action is related to the presence of some chemical components, such as carvracol, thymol, phenols and aromatic acids, being very common its application as suddenly natural, as it has a high content of EO on its leaves ( Huang et al., 2019).
Conclusion
These findings encourage the application of the EO of P. amboinicus in this study as larvicide, as it presents in greater quantity one of the chemical substances (carvracol), which may be associated with its satisfactory biological action, thus allowing the development of an insecticide without effects collateral. | v3-fos-license |
2019-04-02T13:09:33.878Z | 2016-01-01T00:00:00.000 | 89873500 | {
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} | pes2o/s2orc | A . ENDOTHELIO-AND CARDIOPROTECTIVE EFFECTS OF VITAMIN В 6 AND FOLIC ACID IN MODELLING METHIONINE-INDUCED HYPERHOMOCYSTEINEMIA
The endotelioand cardioprotective effects of vitamin B6 (2 mg/kg) and folic acid (0.2 mg/kg) upon modeling of methionine-induced hyperhomocysteinemia via methionine intragastric administration at a dose of 3 g/kg were studied. It was shown that the combined use of vitamin B6 and folic acid allows on the background of a significant reduction in homocysteine concentrations normalizing the endothelial dysfunction coefficient and the parameters of maximum left ventricular pressure in response to intravenous administration of adrenaline. The research was partially supported by the grant of the President of the Russian Federation NoMD-4711.2015.7.
Introduction.
By now, a number of factors of different nature has been determined [1], contributing to the development and progression of cardiovascular disease such as dyslipidemia, hypertension, overweight, smoking, physical inactivity, and diabetes.In addition, a group of so-called "new" risks can be distinguished, which primarily includes an increase in homocysteine levels in the blood.More than 80 clinical and epidemiological studies have confirmed that hyperhomocysteinemia is a significant, independent risk factors for early development and rapid progression of atherosclerosis, and thrombosis of the coronary, cerebral and peripheral arteries, and may be a predictor of death [2].The obtained reliable evidences have served as the basis for creating homocysteic theory of pathogenesis of atherosclerosis development [3].The first report on the role of homocysteine as a possible risk factor for cardio-vascular diseases goes back to 1964.S.H.Mudd, T.Gerritsen, H.A.Waismann et al. demonstrated that high concentration of homocysteine in the blood and, consequently, in urine, resulting in homocystinuria is a consequence of deficiency of the cystathionine-beta-synthase enzyme.
Subject to the above, the objective of the study was to investigate the endotelio-and cardioprotective effects of vitamin B 6 and folic acid upon modeling of methionine-induced hyperhomocysteinemia.
Materials and methods.Experiments were conducted on white male Wistar rats weighing 200-250 g.Hyperhomocysteinemia was simulated by intragastrical administration of methionine (JSC "Synthesis") at a dose of 3 g/kg/day for 7 days.Solution for intragastric administration of methionine was prepared ex tempore with polysorbate TWEEN-80 and 1% starch solution.Data obtained by intragastric administration of an equivalent amount of polysorbate of 10% TWEEN-80 solution were used as control.Folic acid (Valenta Pharmaceuticals, OJSC) was administered intragastrically at a dose of 0.2 mg/kg/day for 7 days.Vitamin B 6 (Veropharm) RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY was administered intraperitoneally at a dose of 2 mg/kg/day for 7 days.
Animals were divided into groups (n=10): Idaily, once a day, for 7 days, intragastric administration of 10% Tween-80 solution at a dose of 1 ml/kg (control, n=10 animals); II -daily, once a day, intragastric administration of methionine at a dose of 3 g/kg for 7 days (n=10 animals); IIIadministration of folic acid on the background of methionine (0.2 mg/kg, intraperitoneally) and vitamin B 6 (2 mg/kg, i.p.) once a day for 7 days.
On day 8 of the experiment, a catheter was inserted under anesthetisia (chloral hydrate 300 mg/kg) into the left carotid artery to record blood pressure (BP); bolus administration of pharmacological agents was into the femoral vein.Hemodynamic parameters: systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were measured continuously with the use of a sensor and the computer program "Biopac".In addition to blood pressure measurements a series of functional tests was carried out in the following sequence: 1. Test for endothelium-dependent vascular relaxation (intravenous solution of acetylcholine (ACh) at a dose of 40 mg/kg at the rate of 0.1 ml per 100 g). 2. Test for endotheliumindependent vascular relaxation (intravenous solution of sodium nitroprusside (NP) at a dose of 30 mg/kg at the rate of 0.1 ml per 100 g) [4,5,6,7,8].
The degree of endothelial dysfunction in experimental animal, as well as the degree of its correction with the studied medications was assessed by the estimated coefficient of endothelial dysfunction (EDC).The coefficient was calculated by the formula: EDC = BPS NP / BPS AC, where BPS NP is an area of the triangle above the blood pressure recovery curve, where points of the smaller leg are the point of maximum blood pressure drop and the point of BP level egress to the plateau during the functional test with the administration of sodium nitroprusside, BPS AC -is an area of the triangle above the blood pressure recovery curve during the test with acetylcholine, where a smaller leg shall be the difference between the end point of bradycardiac cardiac component and a BP recovery point [6,9,10,11,12].
The development of hyperhomocysteinemia and its correction with the studied medications were assessed by the content of homocysteine in the blood serum of experimental animals.Homocysteine concentration was measured by immunoturbidimetric method with the use of a set by Pliva-Lachema Diagnostika s.r.o.
To assess the myocardial functionality in animals under controlled respiration, the cavity of the left ventricle was catheterized and stress tests were performed in the following sequence: 1. Test for adrenoreactivity (a one-time intravenous administration of epinephrine hydrochloride solution 1 .10 -5 mol/L at the rate of 0.1 ml per 100 g) [9,10,11].During this test, the maximum increase in LVP in response to adrenaline administration was assessed.
2. Resistance load (ascending aorta compression for 30 seconds) [1,3].After this test, index of myocardial reserve exhaustion was calculated (expressed as a percentage) equal to the ratio of increase in the LVP on the 5th second of compression to the increase in the LVP on the 25th second of compression.
The significance of changes in absolute parameters was determined by the difference method of variation statistics with finding the average values of the shifts (M), the arithmetic mean (±m) and the probability of possible error (p) by using the Student tables.Differences were evaluated as significant at p<0.05.Statistical calculations were performed with Microsoft Excel 7.0.
Results
According to the study design, a hyperhomocysteinemia-induced endothelial dysfunction was simulated by daily intragastric administration of methionine at a dose of 3 g/kg for 7 days.
Intragastric administration of the stated dose of methionine resulted in a significant increase in the endothelial dysfunction coefficient up to 3.3 ± 0.3, while the EDC in the control group was 0.9 ± 0.2.Systolic and diastolic blood pressure values remained within physiological limits in all series of the experiments (Table 1).
Simultaneous administration of methionine, vitamin B 6 and folic acid led to a significant reduction in EDC up to 1.7 ± 0.1 (Table 1).2. Intragastric administration of methionine resulted in significant increase in the concentration of homocysteine, while co-administration of vitamin B 6 and folic acid allowed significantly reducing this coefficient and making it closer to the values of the control group (Table 2).During test for adrenoreactivity, the group of animals, which received intragastrically vitamin B6 and folic acid on the background of methionine, showed a decrease in the absolute values of left ventricular pressure, which indicates the prevention of hyperhomocystein-induced increase of adrenoreactivity (Table 3).
During test for resistance load, neither vitamin B6 nor folic acid prevented the drop of contractility in the period from 5 to 25 second of aortic compression.For example, index of myocardial reserve exhaustion on the 25th second of the test was 85.4±3.1% in the control group.The same in the methionine-treated animals was 69.8±3.4%.Results in the group of animals treated with vitamin B6 and folic acid were equal to 72.4±4.1% (Table 3).The literature provides evidences that the use of vitamin B6 and folic acid can effectively reduce homocysteine levels [2].In our study, the use of therapeutic doses of vitamin B6 and folic acid allowed normalizing the ratio of endotheliumdependent and endothelium-independent vasodilation with a significant decrease in the endothelial dysfunction coefficient.During stress testing, intragastric administration of vitamin B6 and folic acid on the background of methionine allowed reducing the adrenoreactivity in the experiment with an open heart, however, has not resulted in the prevention of myocardial reserve exhaustion.Positive dynamics of functional performance was accompanied by a significant decrease in the concentration of homocysteine.
These facts confirm the important role of vitamin B6 and folic acid in the implementation of the protective effect in case of hyperhomocysteinemy, however, raise the question of the possibility of their combined application with conventional drugs used to treat cardiovascular disease (ATE inhibitors, AT-I receptor blockers, etc.).
Conclusions. 1. Combined application of vitamin B6 at a dose of 2 mg/kg and folic acid at a dose of 0.2 mg/kg has endothelioprotective effect shown in the model of methionine-induced hyperhomocysteinemia.
2. Combined application of vitamin B6 at a dose of 2 mg/kg and folic acid at a dose of 0.2 mg/kg on the background of simulated methionine-induced hyperhomocysteinemia reduces the maximum pressure in the cavity of the left ventricle during test for adrenoreactivity and has no effect on the myocardial reserve exhaustion during the test for resistance load.
Y., Korokin M.V., Pokrovsky M.V., Povetkin S.V., Lazareva G.A., Stepchenko A.A., Bystrova N.A. Endothelio-and cardioprotective effects of vitamin B6 and folic acid in modelling methionine-induced hyperhomocysteinemia. Research result: pharmacology and clinical pharmacology.2016.18 RESEARCH RESULT: PHARMACOLOGY AND CLINICAL PHARMACOLOGY The results of the study of homocysteine concentration in the blood serum of experimental animals are shown in Table
Discussion.
Possible role of homocysteine (HC) in the development of cardiovascular diseases (CVD) started to be investigated after K. McCully demonstrated in 1969 a predisposition to atherothrombotic events in patients with severe hyperhomocysteinemia (HHC) (HC> 100 µmol/l).The mechanism of development of atherosclerotic vascular lesions with hyperhomocysteinemia remains unclear.Experimental findings suggest that the products of homocysteine autoxidation proceeding with the formation of reactive oxygen species induce the formation of atherosclerotic plaques by damaging endothelium, destroying the integrity of the vascular wall and stimulating the proliferation of medial smooth muscle cells [2, 3].Homocysteine also impairs the normal NO production by endothelial cells, reduces the bioavailability of NO by decreasing its synthesis.Increased lipid peroxidation with homocysteine involvement leads both to a reduction in NO production by the NO-synthase enzyme, and to NO direct degradation. | v3-fos-license |
2018-04-03T04:29:28.077Z | 2015-04-29T00:00:00.000 | 633093 | {
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} | pes2o/s2orc | Transition Metal Complexes of Phyllobilins – A New Realm of Bioinorganic Chemistry
Natural cyclic tetrapyrroles feature an outstanding capacity for binding transition metal ions, furnishing Nature with the important metallo-porphyrinoid ‘Pigments of Life’, such as heme, chlorophyll (Chl) and vitamin B12. In contrast, linear tetrapyrroles are not generally ascribed a biologically relevant ability for metal-binding. Indeed, when heme or Chl are degraded to natural linear tetrapyrroles, their central Feor Mg-ions are set free. Some linear tetrapyrroles are, however, effective multi-dentate ligands and their transition metal complexes have remarkable chemical properties. The focus of this short review is centred on such complexes of the linear tetrapyrroles derived from natural Chl-breakdown, called phyllobilins. These natural bilin-type compounds are massively produced in Nature and in highly visible processes. Colourless non-fluorescing Chl-catabolites (NCCs) and the related dioxobilin-type NCCs, which typically accumulate in leaves as ‘final’ products of Chl-breakdown, show low affinity for transition metal-ions. However, NCCs are oxidized in leaves to give less saturated coloured phyllobilins, such as yellow or pink Chl-catabolites (YCCs or PiCCs). YCCs and PiCCs are ligands for various biologically relevant transition metal-ions, such as Zn(II)-, Ni(II)and Cu(II)-ions. Complexation of Zn(II)and Cd(II)-ions by the effectively tridentate PiCC produces blue metal-complexes that exhibit an intense red fluorescence, thus providing a tool for the sensitive detection of these metal ions. Outlined here are fundamental aspects of structure and metal coordination of phyllobilins, including a comparison with the corresponding properties of bilins. This knowledge may be valuable in the quest of finding possible biological roles of the phyllobilins. Thanks to their capacity for metal-ion coordination, phyllobilins could, e.g., be involved in heavy-metal transport and detoxification, and some of their metal-complexes could act as sensitizers for singlet oxygen or as plant toxins against pathogens. Introduction Cyclic tetrapyrroles feature an outstanding capacity for binding (transition) metal ions, furnishing Nature with the important metalloporphyrinoid ‘Pigments of Life’, such as heme, chlorophyll and vitamin B12. 2-4 Linear tetrapyrroles, in contrast, are not typically ascribed a biologically relevant ability for metal-binding. Most known natural linear tetrapyrrroles are products of the degradation of heme 10-12 or of chlorophyll, such as bilirubin (BR) 6 or, e.g., ‘non-fluorescent’ chlorophyll catabolites (NCCs), respectively. 9, 11, 14 When these linear tetrapyrrroles are produced via their respective catabolic pathways, the central Feand Mg-ions of heme or of chlorophyll are liberated from their porphyrinoid encasement, to be recuperated for the purpose of alternative metabolic uses. 12 The ‘bile-pigments’ (or natural heme-derived bilins) were already studied in ancient times, when they were puzzling, originally, as enigmatic secretions in animals and humans. 6 Nowadays, bile-pigments are ascribed intricate physiological roles (see, e.g. 6, ). In more recent times, attention has also been drawn to heme-derived bilins in plants and in other photosynthetic organisms, where they play important roles, e.g., in absorbing and sensing sunlight. 7, 18, 19 Phyllobilins, on the other hand, the natural degradation products of chlorophyll (Chl) in higher plants, are a related type of linear tetrapyrroles, 9, 11 which, surprisingly, has come into our focus only rather recently. 20 Chl-degradation is a natural phenomenon, commonly associated with the seasonal appearance of the fall colours. 21 Each year, it provides the biosphere the astounding estimated amount of about 10 tons of phyllobilins. Chl-derived bilin-type compounds are, thus, economically and ecologically relevant, and no less fascinating than other natural bilins. This ‘Perspective’ deals with phyllobilins, primarily, and compares them with bilins. It focuses on the role of both types of such linear tetrapyrroles as ligands for transition metal-ions, and on the structure and chemical properties of the resulting metalcomplexes. Phyllobilins – a new type of natural bilins. Phyllobilins, the tetrapyrrolic products of the natural degradation of chlorophyll (Chl), are generated in plants at a massive scale. Chl-breakdown was remarkably enigmatic until about 25 years ago, since up to then no trace of Chldegradation products had been found. In 1991 a non-fluorescent Chl-catabolite (NCC) was described as a 1-formyl-19-oxobilintype linear tetrapyrrole. This finding opened the door to the identification of the phyllobilins, and to the structure-guided discovery of the ‘PaO/phyllobilin’ pathway of Chl-breakdown in higher plants, 13, 23, 27-29 which is relevant, both in senescence, and in fruit ripening. More recently, the PaO/ Page 1 of 11 Dalton Transactions D al to n Tr an sa ct io ns A cc ep te d M an us cr ip t PERSPECTIVE Dalton Transactions 2 | Dalton Transactions., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 2015 phyllobilin pathway was recognized to ‘branch out’ and to furnish a second type of bilin-type Chl-catabolites. 35 The most widely occurring (colourless) representatives of the latter have been classified as ‘dioxobilin-type’ non-fluorescent Chlcatabolites (DNCCs), which share a common 1,19-dioxobilintype structure with the now ‘classical’ heme-derived bilins. DNCCs are formal deformylation products of NCCs, but do actually arise from oxidative enzymatic de-formylation of fluorescent Chl-catabolite (FCC) precursors, catalyzed by a new type of cytochromes P450. 36 The first formed FCC, or ‘primary’ FCC (pFCC), in turn, results from enzymatic reduction of the red Chl-catabolite (RCC). Most FCCs are only fleetingly existent, blue fluorescent 40, 41 intermediates of Chl-breakdown that are generally ‘programmed’ 42 for direct and rapid conversion (via acid catalysis) into the corresponding NCCs (see Figure 1). 43, 44 Figure 1. Typical Chl-catabolites (phyllobilins) of the PaO/phyllobilin pathway of Chl-breakdown. 1-Formyl-19-oxobilins (or type-I phyllobilins) are produced first, by oxygenolytic cleavage of the chlorin macrocycle. In a later step, the formyl group is removed, giving access to 1,19-dioxobilins (or type-II phyllobilins). In typical de-greening leaves, colourless ‘nonfluorescent’ Chl-catabolites (NCCs) and/or dioxobilin-type NCCs (DNCCs) accumulate temporarily as representative type-I or type-II phyllobilins, respectively. Recently, yellow Chl-catabolites (YCCs) and pink chlorophyll catabolites (PiCCs) were shown to constitute a new class of natural coloured phyllobilins, 46 which appear to be formed in leaves from NCCs via an enzymatic oxidation at later stages of senescence. The representative YCC 2 was also prepared from the NCC 1 by oxidation with dicyanodichlorobenzoquinone (DDQ). YCC 2 was converted into the corresponding PiCC 3 via an efficient two-step procedure involving spontaneous air oxidation in the presence of Zn-ions (Figure 2). Figure 2. Coloured Chl-catabolites (yellow and pink coloured YCCs and PiCCs) result from ‘biological’ and from ‘chemical’ oxidation of the colourless ‘non-fluorescent’ Chl-catabolites (NCCs): NCC 1 is thus oxidized to the yellow YCC 2. The latter is further oxidized to the PiCC 3 via the blue Zn-complex Zn-3. At this stage, more than a dozen NCCs 9, 32, 48 as well as a range of FCCs 9 and YCCs 9 with different structures have all been identified as 1-forml-19-oxo-bilins, now classified as ‘type-I phyllobilins’. Likewise, several DNCCs 32, 34-36, 48, 49 and related ‘type-II phyllobilins’ 9, 49 have meanwhile been discovered, expanding the repertoire of the known natural phyllobilins, as well as our knowledge on these tetrapyrrolic natural products. Coloured phyllobilins of the type of YCCs and PiCCs have -conjugated systems extending over two or three pyrrole-type rings, respectively. Remarkably, the main chromophores in YCCs and in bilirubin are virtually identical, 50 and close structural parallels also exist between PiCCs and some natural phycobilins, such as phycoviolobilin, (see Figures 3 and 4). 19, 51 Bilins – natural linear tetrapyrroles from heme-breakdown Oxidative cleavage of heme by heme oxygenase (HO) at its (‘northern’) -meso position gives ()-biliverdin (BV), CO and an Fe-ion, 10 providing a highly regio-selective entry to the bile pigments. Subsequent reduction of BV by NADPH, catalyzed by BV-reductase (BVR), generates bilirubin (BR) in animals and in humans. For the purpose of making BR more water soluble and available for excretion, it is then conjugated with Page 2 of 11 Dalton Transactions D al to n Tr an sa ct io ns A cc ep te d M an us cr ip t Dalton Transactions PERSPECTIVE This journal is © The Royal Society of Chemistry 2015 Dalton Transactions , 2015, 00, 1-3 | 3 Figure 3. Structural parallels between phyllobilins and heme-derived bilins, as exemplified by the common main chromophores of YCCs (such as 2) vs. bilirubin, and of PiCCs (such as 3) vs. phycoviolobilin. glucuronic acid in the liver. Phycobilins (e.g., phycocyanobilin, phyco-violobilin and phyco-urobilin, see, e.g. 8, 18, 19, ) form another widespread group of linear tetrapyrroles derived from BV and produced in photosynthetic organisms (plants, bacteria and algae) by enzyme-catalyzed reduction of BV by radical BVRs (rBVRs). 53 Alternative natural pathways of heme-degradation that furnish regio-isomers of ()-biliverdin (BV) have been discovered. 12 Furthermore, the oxygenase MhuD, a ‘noncanonical’ new type of oxygen-dependent heme-degrading enzyme was recently found in mycobacteria. 55 In contrast to ‘classical’ heme-oxygenase (HO), MhuD converted heme into a 1-formyl-oxobilin (mycobilin) regio-selectively, without generating CO (Figure 4). Interestingly, these heme-catabolites carry functionalities at the cleavage site that remind of some chlorophyll catabolites, now classified as type-I
Introduction
Cyclic tetrapyrroles feature outstanding capacity for binding (transition) metal ions, furnishing Nature with the important metallo-porphyrinoid 'Pigments of Life', 1 such as heme, chlorophyll and vitamin B 12 . 2-46][7][8][9] Most known natural linear tetrapyrrroles are products of the degradation of heme 5,[10][11][12] or of chlorophyll, 13 such as bilirubin (BR) 6 or, e.g., 'non-fluorescent' chlorophyll catabolites (NCCs), respectively. 9,11,14When these linear tetrapyrrroles are produced via their respective catabolic pathways, the central Fe-and Mg-ions of heme or of chlorophyll are liberated from their porphyrinoid encasement, to be recuperated for the purpose of alternative metabolic uses. 10,12e 'bile-pigments' (or natural heme-derived bilins) were already studied in ancient times, when they were puzzling, originally, as enigmatic secretions in animals and humans. 5,6owadays, bile-pigments are ascribed intricate physiological roles (see, e.g.ref. 6 and 15-17).In more recent times, attention has also been drawn to heme-derived bilins in plants and in other photosynthetic organisms, where they play important roles, e.g., in absorbing and sensing sunlight. 7,18,19Phyllobilins, on the other hand, the natural degradation products of chlorophyll (Chl) in higher plants, are a related type of linear tetrapyrroles, 9,11 which, surprisingly, has come into our focus only rather recently. 14,20Chl-degradation is a natural phenomenon, commonly associated with the seasonal appearance of the fall colours. 21Each year, it provides the biosphere the astounding estimated amount of about 10 9 tons of phyllobilins. 22Chl-derived bilin-type compounds are, thus, economically and ecologically relevant, and no less fascinating than other natural bilins. 23his 'Perspective' deals with phyllobilins, primarily, and compares them with bilins.[26] Phyllobilinsa new type of natural bilins Phyllobilins, the tetrapyrrolic products of the natural degradation of chlorophyll (Chl), are generated in plants at a massive scale. 9Chl-breakdown was remarkably enigmatic until about 25 years ago, since -up to then -no trace of Chl-degradation products had been found.In 1991 a non-fluorescent Chl-catabolite (NCC) was described as a 1-formyl-19-oxobilintype linear tetrapyrrole. 202][33] More recently, the PaO/phyllobilin pathway was recognized to 'branch out' and to furnish a second type of bilin-type Chl-catabolites. 34,35The most widely occurring (colourless) representatives of the latter have been classified as 'dioxobilin-type' non-fluorescent Chl-catabolites (DNCCs), which share a common 1,19-dioxobilin-type structure with the now 'classical' heme-derived bilins. 5DNCCs are formal deformylation products of NCCs, but do actually arise from oxidative enzymatic de-formylation of fluorescent Chlcatabolite (FCC) precursors, catalyzed by a new type of cytochromes P450. 23,368][39] Most FCCs are only fleetingly existent, blue fluorescent 40,41 intermediates of Chl-breakdown that are generally 'programmed' 42 for direct and rapid conversion (via acid catalysis) into the corresponding NCCs (see Fig. 1). 9,43,44cently, yellow Chl-catabolites (YCCs) and pink chlorophyll catabolites (PiCCs) were shown to constitute a new class of natural coloured phyllobilins, 45,46 which appear to be formed in leaves from NCCs via an enzymatic oxidation at later stages of senescence. 47The representative YCC 2 was also prepared from the NCC 1 by oxidation with dicyano-dichlorobenzoquinone (DDQ). 45YCC 2 was converted into the corresponding PiCC 3 via an efficient two-step procedure involving spontaneous air oxidation in the presence of Zn-ions (Fig. 2). 26t this stage, more than a dozen NCCs 9,32,48 as well as a range of FCCs 9 and YCCs 9 with different structures have all been identified as 1-forml-19-oxo-bilins, now classified as 'type-I phyllobilins'. 9Likewise, several DNCCs 32,[34][35][36]48,49 and related 'type-II phyllobilins' 9,49 have meanwhile been discovered, expanding the repertoire of the known natural phyllobilins, as well as our knowledge on these tetrapyrrolic natural products. Coloued phyllobilins of the type of YCCs and PiCCs have π-conjugated systems extending over two or three pyrroletype rings, respectively.51 Bilinsnatural linear tetrapyrroles from heme-breakdown Oxidative cleavage of heme by heme oxygenase (HO) at its ('northern') α-meso position gives (α)-biliverdin (BV), CO and an Fe-ion, 10 providing a highly regio-selective entry to the bile pigments.5 Subsequent reduction of BV by NADPH, catalyzed by BV-reductase (BVR), generates bilirubin (BR) in animals and in humans.52 For the purpose of making BR more water soluble and available for excretion, it is then conjugated with glucuronic acid in the liver.6 Phycobilins (e.g., phyco-cyanobi- 7,53 Alternative natural pathways of heme-degradation that furnish regio-isomers of (α)-biliverdin (BV) have been discovered.12 Furthermore, the oxygenase MhuD, a 'non-canonical' new type of oxygen-dependent heme-degrading enzyme was recently found in mycobacteria.54,55 In contrast to 'classical' heme-oxygenase (HO), MhuD converted heme into a 1-formyl-Fig.1 Typical Chl-catabolites ( phyllobilins) of the PaO/phyllobilin pathway of Chl-breakdown. 9 1-Formyl-19-oxobilins (or type-I phyllobilins) are produced first, by oxygenolytic cleavage of the chlorin macrocycle.In a later step, the formyl group is removed, giving access to 1,19dioxobilins (or type-II phyllobilins).In typical de-greening leaves, colourless 'non-fluorescent' Chl-catabolites (NCCs) and/or dioxobilin-type NCCs (DNCCs) accumulate temporarily as representative type-I or type-II phyllobilins, respectively.Fig. 2 Coloured Chl-catabolites (yellow and pink coloured YCCs and PiCCs) result from 'biological' and from 'chemical' oxidation of the colourless 'non-fluorescent' Chl-catabolites (NCCs): NCC 1 is thus oxidized to the yellow YCC 2. The latter is further oxidized to the PiCC 3 via the blue Zn-complex Zn-3.Fig. 3 Structural parallels between phyllobilins and heme-derived bilins, as exemplified by the common main chromophores of YCCs (such as 2) vs. bilirubin, and of PiCCs (such as 3) vs. phycoviolobilin.oxobilin (mycobilin) regio-selectively, without generating CO (Fig. 4).Interestingly, these heme-catabolites carry functionalities at the cleavage site that remind of some chlorophyll catabolites, now classified as type-I phyllobilins (see above).
Man-made linear tetrapyrroles
'Coupled oxidation' of heme (or of its dimethyl ester) with ascorbate and oxygen was studied as a model reaction for the oxidation of heme to BV by HO. 24 It showed insignificant regio-selectivity and the bilin-type products were obtained as a mixture of all four regio-isomers. 56The iron complex of (the symmetric) octaethylporphyrin underwent coupled oxidation to give a bilin-type tetrapyrrole with good yield. 57The regioselectivity of ring-opening of heme by HO to (α)-BV is thus explained by directing effects of the protein environment. 55][60] However, when the photo-oxygenation reaction of TPP was done in H 2 O or MeOH, biliviolin analogues were isolated as products of the further addition of water (or methanol) at one of bilitriene's meso-positions (see Fig. 15, below). 59,61n the context of the search for synthetic roads to (then still elusive) Chl-catabolites, photo-oxidation of Chl-derivatives was studied as a method for the preparation of formyl-oxo-bilintype tetrapyrroles. 22,62More recently, photo-oxygenation reactions with Zn-or Cd-complexes of methyl pheophorbide a 37 or of methyl pyropheophorbide a or b 63,64 were found useful and they allowed the partial synthesis of Chl-catabolites found in plants 37,44 or in a green alga. 65Indeed, this method provided 1-formyl-19-oxo-bilins with some regioselectivity, depending upon the coordinated metal ion, with preferential ring opening in the 'North' with Cd-complexes, and in the 'West' with Zn-complexes (see Fig. 6). 65
Transition metal complexes of phyllobilins
Metal complexes of the colourless NCCs are unknown and, indeed, NCCs are not expected to bind transition metal ions, 26 as deduced for other tetrapyrroles with isolated pyrrole units. 3owever, NCCs readily oxidize, and more unsaturated phyllobilins are obtained by oxidation of NCCs with DDQ. 45 By this approach, e.g.YCC 2 and PiCC 3 were prepared from the NCC 1, which feature two or three conjugated pyrrolic rings.YCC 2 and PiCC 3 are natural chlorophyll catabolites that are also found, e.g., in senescent leaves of the Katsura tree (Cercidiphyllum japonicum). 46The UV-Vis spectrum (in MeOH) of the pink coloured PiCC 3 has strong bands at 313 nm and 523 nm, and solutions of 3 only show a very weak luminescence near 615 nm. 26Unexpectedly, the solution structure of PiCC 3 was revealed by NMR analysis with double bonds C10vC11 and C15vC16 with E-configuration and Z-configuration, respect- ively. 46X-ray analysis of the crystal structure of the potassium salt of 3 (K-3) confirmed the NMR-derived structure and revealed bond-lengths consistent with a pattern of single and double bond alternation, as depicted by the formula used (see Fig. 7). 26In this first crystal structure of a phyllobilin from a higher plant, K-3 was revealed to be present as a H-bonded and K-bridged pair of enantiomers, which showed nearly parallel planes of the π-system extending over rings B to D. The three conjugated rings (B, C and D) form a planar structure.The fourth pyrrole (ring A) is stabilized in its 'out-of-plane' conformation by an H-bond between the carboxylic acid group and the NH group (of ring A), reminding of the structuring H-bonds observed in the crystal of bilirubin. 6,66n contrast to NCCs, the coloured phyllobila-c,d-diene 3 proved to be an excellent multi-dentate ligand for transient metal complexes. 26,67Deep blue metal complexes M-3 (M = Zn, Cd, Ni, Cu, Pd) of PiCC 3 could be prepared in excellent yields by treatment of 3 with corresponding transition metal salts (Fig. 8).Detailed structure analysis of these metal complexes by NMR, suggested a monomeric nature in solution and tridentate coordination of the metal-ion by the ligand nitrogen atoms.Observation of a ring A NH-signal in the 1 H-NMR spectrum of Zn-PiCC (Zn-3) was consistent with this.Polar solvent molecules or the OH group at C3 2 of ring A are likely fourth ligands (L) at the coordinated metal-ion.In order to achieve a tridentate coordination by PiCC, the metal complexes required a Z-configuration of the C10vC11 double bond, not directly compatible with the known, original structure of PiCC (see discussion below).Clearly, phyllobiladienes, such as 3, represent a new type of natural oligopyrrole that binds transition metal ions very well. 26ormation of transition metal complexes (M-3) from PiCC 3 was accompanied by colour changes from pink-red to blue, 26 revealing a notable bathochromic shift of the absorption maximum by roughly 100 nm (Fig. 9).Among the blue complexes M-3 prepared (with M = Zn, Cd, Ni, Cu and Pd), binding of Pd(II)-ions to 3 induced the largest bathochromic shift of the absorption maximum (to 645 nm). 67Such significant long wavelength shifts can be directly attributed to metal binding in combination with E to Z isomerization of the C10vC11 double bond.9][70] The capacity of PiCC 3 to bind different transition metal ions (such as Zn(II)-, Cd(II)-, Ni(II)-, Cu(II)-, and Pd(II)-ions) reminds of related properties of natural tripyrrolic alkaloids 71 and of artificial tripyrrones. 25he kinetics of formation of several metal-complexes M-3 of PiCC 3 was analysed qualitatively.At room temperature and in methanol as solvent over-all rates of roughly 600, 200, 10, 400, 1 M −1 s −1 were determined for Zn(II)-, Cd(II)-, Ni(II)-, Cu(II)-and Pd(II)-incorporation from the corresponding metal acetates. 26,67Due to the different configuration of the C10vC11 double bond in the ligand 3 and in complexes M-3 an E to Z isomerization of the C10vC11 double bond during the complex-formation was inferred. 26In the course of the fast formation of Zn-3, Cd-3 and Cu-3 the deduced double bond isomerization appears to be too fast, to allow the observation of separate intermediate states during complex-formation.However, a first fast interaction of 3 with Ni (indicated by partial spectral changes) in the formation of Ni-3 is followed by a slow product-forming step, which, presumably, is ratelimited by the isomerization.This result indicated weak bidentate coordination of the conjugated C-D-moiety to the metal-ion, as first step in the formation of M-3, followed by the double bond isomerization and tridentate coordination of the metal-ion, to afford the stable metal complexes.
PiCC 3 is barely luminescent (weak emission near 615 nm), as are YCC 2 and most linear tetrapyrroles, which de-excite by rapid isomerization processes. 5Coordination of Zn-ions by PiCC 3 (gave the blue metal complex Zn-3 and) lighted up an intense red luminescence (see Fig. 10): binding of Zn-ions and, likewise, of Cd-ions transformed the weakly luminescent PiCC 3 into the bright red fluorescent complexes Zn-3 and Cd-3.emission around 650 nm was almost two orders of magnitude more intense than that of 3. Appearance of such a strong luminescence by complex formation with closed-shell metal-ions can provide interesting insights and analytical applications with metal complexes M-3: as a consequence of the high affinity of 3 for transition metal-ions and high rates of binding to Zn(II)or to Cd(II)-ions, analysis of the fluorescence of solutions of 3 allowed for the quantitative detection of Zn-and Cd-ions down to nM concentrations (via the luminescence of Zn-3 or of Cd-3).A nearly linear correlation between the fluorescence intensity and the concentration of Zn(II)or of Cd(II)-ions was observed at concentrations down to <10 nM, which was consistent with a 1 : 1 stoichiometry in the complexes.PiCC 3, therefore, could serve as a reporter for Zn(II)-and Cd(II)-ions (and vice versa: Zn(II)-and Cd(II)-ions could be reporters for 3), even at very low concentrations of the analytes. 26Thus, the fluorescence of their Zn-or Cd-complexes could be used to detect and track such PiCCs (in vivo or ex vivo) in plants.
Cu(II)-ions displaced the Zn(II)-and Cd(II)-ions from the complexes Zn-3 and Cd-3, which indicated the stronger binding capacity of 3 to Cu(II).Ni(II)-and Cu(II)-complexes Ni-3 and Cu-3 showed negligible emission, as expected.Photoexcited Pd-3 displayed fluorescence, with a maximum at 668 nm with low intensity (Fig. 10).
The main chromophore of the yellow catabolite YCC 2 (or its methyl ester 2-Me) is the same as the one characteristic of bilirubin, 45 and may, thus, also have similar capacities to bind metal-ions (see below).Indeed, our ( preliminary) data suggest YCC 2 to have a significant affinity, e.g., to Zn(II)-ions. 72YCC 2 is only weakly luminescent.However, addition of Zn(II)-acetate to a deoxygenated solution of 2 in DMSO resulted in the formation of the Zn-complex Zn-2, as indicated by a red shift of the UV/Vis-absorption maximum from 431 to 498 nm and by the appearance of a bright green fluorescence, with an emission maximum at 540 nm. 72In the luminescent Zn-2 complex, the ligand is assumed to coordinate the Zn(II)-ion in a bidentate form.In the presence of air and of an excess of Zn(OAc) 2 , solutions of Zn-2 in methanol or in DMF undergo clean oxi- dation to the blue complex Zn-3. 26The presence of Zn(II)-ions appears to accelerate oxidation of 2, as similarly observed with BR. 73,74 As the Zn(II)-ion of Zn-3 was easily removed by addition of phosphate (which precipitated Zn-phosphate), YCC 2 could be oxidized efficiently to PiCC 3 (via Zn-2 and Zn-3). 26ikewise, addition of an excess of Zn(OAc) 2 to a deoxygenated (Ar-purged) solution of the methyl ester of YCC (2-Me) in DMSO led to the formation of a stable Zn(II)-complex of 2-Me, as indicated by a red shift of the absorption maximum from 430 to 484 nm, and by green luminescence with emission maximum at 538 nm (see Fig. 11).NMR-spectroscopic analysis in DMSO-d 6 provided evidence for the structure of the 1 : 2complex Zn(2-Me) 2 , in which the Zn(II)-ions were encased in a pseudo-tetrahedral coordination mode by two molecules of 2-Me that each acted as bidentate ligands.Upon binding of a Zn (II)-ion to 2-Me the signals of N23H and N24H disappeared in the 1 H-NMR spectra, indicating coordination to N23 and N24.Furthermore, an apparent long range NOE-correlation between the non-coordinated ring B pyrrole-NH to the vinyl group in ring D (which is not observed in the NMR spectra of 2-Me) is rationalized by an inter-ligand coupling between two coordinated molecules of 2-Me (see Fig. 12). 72An ESI-MS analysis of isolated Zn(2-Me) 2 supported the suggested 1 : 2 stoichiometry.
Transition metal complexes of bilins
Transition metal complexes of biliverdin Biliverdin (BV) has been thoroughly investigated as ligand for transition metal ions, as reviewed recently. 24,25The structure of BV (as its dimethyl ester) was analyzed in the crystal, where it was found in a Z,Z,Z-configurated bis-lactam form with a weakly nonplanar helical conformation.Two neighbouring BVdimethyl ester molecules were stitched together in the crystal in dimers by two lactam H-bonds. 75A non-natural bilindione, obtained from oxidation of tetra-mesophenyl-porphyrin (TPP), had similar structural characteristics. 76n early study reported a solution of meso-biliverdin (mBV) to change colour from blue to green upon addition of a solution of Zn(OAc) 2 in MeOH under N 2 , due to formation of the Zn(II)-complex of mBV. 77Analysis of the crystal structures of the Zn-complex of an 'octaethyl-formylbiliverdinate' ( prepared from photo-oxidation of Zn-octaethylporphyrin) revealed the presence of a monomeric penta-coordinate Zn-complex as mono-hydrate (with four N and one axial H 2 O coordinating to Zn, see Fig. 13), as well as a dimer involving alternative bonding to two tetra-coordinate Zn-ions by two pairs of N-atoms from each ligand. 78 Zn-1,19-dideoxy-1,2,3,7,8,12,13,17,18,19-decamethylbiladiene-a,c featured a similar dimeric structure. 79In water or DMSO and in the absence of O 2 , binding of BV to Zn-, Cd-and Cu-ions was observed in a 1 : 1 stoichiometry.In aqueous solution, further oxidation reactions of various BV-metal complexes were observed. 24,74,80ansition metal complexes of bilirubin Bilirubin (BR) tends to be a more capricious ligand than BV, due to the ease of oxidation at flexible meso-position linking the two dipyrromethene groups.Crystalline BR displayed C4vC5 and C15vC16 bonds in a Z configuration, and a ridge tile structure of the whole tetrapyrrolic molecule, in which the two dipyrromethene groups were linked by the CH 2 group. 6,24,66,81Various transition metal-ions, including Zn(II), Cu(II), Ni(II), Co(II), Fe(II), Fe(III), were tested for binding to BR or meso-bilirubin (mBR), as delineated in recent reviews. 24,25hen Zn(II)-, Cd(II)-or Co(II)-salts were added to a solution of BR in DMF or DMSO, fast colour changes to red were observed. 82A bathochromic shift of the band in the visible region (by approximately 80-100 nm) was observed, similar to the one seen when YCC 2 bound Zn(II)-ions.A range of structures have been discussed for metal complexes of BR, while essential structural data were hardly obtained. 25,82Coordination of BR with Zn(II)-, Cd(II)-and Cu(II)-ions was also studied in deoxygenated H 2 O, when formation of metal complexes was inefficient.Indeed, under O 2 , Zn-or Cd-complexes of the BR-oxidation product BV were obtained. 74
Zn-complexes of stercobilin and urobilins
Our knowledge is still scarce on metal complexes of partially reduced natural bilins, such as stercobilin (SB) and urobilin (UB). 24,25Titration of SB with Cu-ions gave UV/Vis-absorbance shifts that were interpreted by the formation of (a) Cu-SB complex(es).As with metal complexes of other bile pigments, treatment of Cu-complexes (e.g. of UB) with acid led to decomplexation. 83
Metal complexes of non-natural linear tetrapyrrole model compounds
Over a time of several decades, transition metal binding to a variety of non-natural bilins has been studied widely, as reviewed recently. 24,25Octaethylbilindione and the above-mentioned octaethylformyl-biliverdinate represented two easily accessible synthetic linear tetrapyrroles that were used as excellent models for BV, and binding, e.g., Zn-, Co-, Ni-and Cu-ions as effectively tetra-coordinate ligands. 24,25,57,85,86Depending on the metal-ion, monomeric or dimeric metal complexes were observed in the crystals.Thus, in contrast, e.g., to the complexes with Co-and Cu-ions, which were four-coordinate and monomeric, the five-coordinate Mn(III)-ion gave a dimeric complex with octaethylbilindione in the crystal, where the lactam-O of one monomer-unit acted as the bridge to bind with the Mn(III)-centre of the other moiety (see Fig. 14). 24,87n interesting, violet formyl-bilinone was isolated as main product of the photooxidation of meso-tetraphenylporphyrin (TPP) in H 2 O or MeOH, in which an OH or a MeO substituent was attached at one meso-position.This formyl-bilinone displayed a chromophore similar to the one of biliviolin, and of PiCC 3, as well.Formation of a blue solution was observed, when the OH-derivative was treated with Zn(II).On the basis of NMR data, the structure of the blue compound was proposed as the one of a biliviolin-type Zn complex. 59A crystal structure showed binding of three of the N-atoms and of the hydroxylgroup of the biliviolin-type ligand, confirming the proposed tridentate mode of N-coordination of the Zn(II)-ion in a dimeric arrangement (Fig. 15). 84g. 14 Octaethyl-biliverdinate model furnishes 4-coordinate Co-and Cu-complexes, and 5-coordinate Mn(III)-complexes (either pyridine coordinated mono-nuclear or O-bridged di-nuclear complexes). 24g. 15 Violobilin-type products are obtained from photo-oxygenation of tetra-meso-phenylporphyrin in the presence of water, which furnish dimeric, O-bridged Zn(II)-complexes, in which three N-atoms of the violobilin-type ligand coordinate the metal-ion (as deduced from a crystal structure analysis). 84etal-binding capacities of phyllobilins relate to those of hemederived bilins As a rule, the highly unsaturated cyclic tetrapyrroles ( porphyrins) act as tetra-coordinate ligands for transition metal ions.Indeed, the unsaturated linear derivative BV (a bilatriene obtained via heme-oxygenase) behaves in an analogous fashion. 24,25In contrast, typical ligands of the biliviolin-type provide only 3 N-atoms of the extended conjugated ligand chromophore for tridentate coordination to transition metal ions, keeping their isolated pyrrole unit de-coordinated. 59,84nterestingly, corresponding studies with the structurally related natural plant bilin phycoviolobilin appear to be unknown, so that the capacity for coordination of metal ions by the latter still remains to be established.
Among the phyllobilins, only PiCC 3 (a phyllobila-b,c-diene) has been studied extensively, so far, with respect to its capacity to bind transition metal ions. 26PiCC 3 is an effective tridentate ligand for biologically important transition metal ions.Free PiCC exhibits a remarkable 'stretched' structure (with E,Zconfiguration at the C10vC11 and C15vC16 double bonds) that needs to isomerize to the Z,Z-form, in order be able to complex and wrap around a metal ion in a tri-coordinate fashion. 26Probably, the observed E-configuration in PiCC is due to steric effects associated with the substituted, 'extra' ring E of phyllobilins, which is a characteristic of these Chl-derived bilin-type linear tetrapyrroles that is attached to a pyrrole ring and the γ-meso-position (see, e.g.Fig. 7).The presence of ring E of the phyllobilins appears to be of lesser consequence in other respects, although it imposes a further geometric restriction and inhibits any Z/E-isomerisation around the C9-C10 bond, which also features partial double bond character.Similar to some synthetic biliviolin-type tetrapyrroles, the bilab,c-diene PiCC 3 features a saturated, conformationally flexible 5-meso-position, which helps to avoid steric clashes between the 1-and 19-positions in the pseudo-cyclic structures of the 'wrapped-up' metal complexes.
The less unsaturated YCC 2 (a phyllobilene-c) exhibits a bidentate coordination pattern, as seen in its Zn-complex.So far, only Zn(II)-complexes (Zn-2 2 and Zn-(2-Me) 2 ) have been studied, 72 in which the coordination requirements of the Zn(II)-ion are satisfied by binding two (bidentate) YCC-units, i.e. with YCC : Zn(II) in 2 : 1 ratio.The conjugated system spanning rings C and D of YCC 2 occurs in a lactam form, which is indicated to undergo tautomerization to its lactim form in the neutral Zn-complexes Zn-2 2 and Zn-(2-Me) 2 .The suggested coordination of a lactim form in Zn-2 2 reminds of the proposed structure of the 'formyl-biliverdine' Zn-complex 78 and of dipyrromethenes, which are strong chelators for metal ions.
Thus, the coordination properties of PiCCs and of YCCs relate to those of the heme-derived violobilins and BR (see Fig. 16).In this respect, it still remains to study the behaviour of the corresponding, partially unsaturated type-II phyllobilins (1,19-dioxobilin-type Chl-catabolites), which would have main chromophore structures corresponding to those of PiCCs and YCCs (1-formyl-19-oxobilins or type-I phyllobilins).Clearly, the two types of linear tetrapyrroles may have a roughly similar behaviour as ligands in complexes with transition metal ions, comparable to the behaviour of heme-derived dioxo-bilins and formyl-oxobilins. 24,25nterestingly, 'non-fluorescent' Chl-catabolites (NCCs) and dioxobilin-type NCCs (DNCCs), the two most abundant classes of the natural phyllobilins, are not expected to bind metal ions strongly, as they feature only un-conjugated pyrrolic rings.Likewise, natural bilane-type tetrapyrroles (which occur in the course of the biosynthesis of the porphyrinoids) are not known to bind transition metal ions.In contrast, 'fluorescent' Chl-catabolites (FCCs), and the more unsaturated red Chl-catabolites (RCCs), exhibit structures that suggest a capacity for effective metal ion coordination.
Fig. 16 The coordination properties of phyllobilins and of corresponding bilins exhibit basic similarities.These are revealed here by a qualitative comparison of two representative types each, of PiCCs and YCCs (left) and of violobilins (VBs) and bilirubin (BR) (right).PiCCs and VBs provide a tridentate N-coordination pattern, YCCs and BR a bidentate N-coordination pattern (which is present twice in BR).Transition metal complexes of PiCCs require Z-configuration for the unsaturated bonds at the γ-meso-position (C10).This suggests a steric clash between the substituent R 2 at the extra ring E and the propionic acid group (R 3 ) at ring C. In metal complexes of heme-derived bilins a similar steric problem at the γ-meso-position would not exist.
Outlook
In the course of the last 25 years, the highly abundant catabolites of Chl, named 'phyllobilins', were discovered and explored as a new type of natural linear tetrapyrroles. 9Most of the original chemical work in this area centred on structure elucidation of the growing class of the phyllobilins.It revealed the biological importance of mainly colourless chlorophyll-catabolites that are hardly able to coordinate transition metal ions.However, in addition, partially unsaturated, coloured bilin-type chlorophyll catabolites were discovered in the course of this work.As reviewed here, these may display a capacity to complex metal ions comparable to, or even superior to that of well-investigated heme-catabolites, such as biliverdine (BV) or bilirubin (BR).
Among the phyllobilins examined with respect to binding of transition metal ions, the pink-coloured phyllobiladienes, called PiCCs, have been most thoroughly studied.Compared to BV or to BR, which may bind a transition metal ion in a tetra-dentate or (twice) bidentate fashion, PiCCs are effective tridentate ligands.When coordinating transition metal-ions that prefer to be tetra-coordinate, the PiCC ligand thus leaves one coordination site unoccupied.This 'vacancy' may be used for coordination by an external 'fourth' ligand.This feature offers an opportunity for attaching PiCC metal complexes to correspondingly dispositioned bio(macro)molecules, e.g., to proteins or to nucleobases, providing PiCC metal complexes with potentially interesting biological functions and applications.At the same time, coordination of closed-shell metal ions to the barely luminescent PiCCs, such as Zn(II)or Cd(II)ions, induce such phyllobilin metal complexes to exhibit bright fluorescence.Binding of Zn(II)or Cd(II)-ions to PiCC may occur in plants, where the strong luminescence could be used as diagnostic optical effects to detect these complexes in vivo.
Aside of the studies with PiCCs, the capacity of phyllobilins as ligands for transition metal ions has barely been investigated.As indicated here briefly, yellow chlorophyll catabolites (such as YCC 2 and its methyl ester 2-Me) are able to coordinate Zn(II)-ions and give green luminescent complexes.However, the presence of metal-ions and metal-chelation may enhance decomposition, or oxidation processes of phyllobilins, as seen with the oxygen sensitive YCC.
There are isolated reports on the natural occurrence of transition metal complexes of heme-derived bilins, and on presumed biological roles of their transition metal complexes. 24,68hus, a Zn-complex of BV was identified as pigment in the eggshells of birds. 24A Cu(II)-complex of BR appears to cut DNA in the presence of molecular oxygen, 88 a feature shared by the Cu-complex of tripyrrolic alkaloids, named prodigiosins. 71hyllobilins may be expected nowadays to have biological roles, as well, which are, however, still entirely elusive.Phyllobilins are linear tetrapyrroles that do represent an interesting new group of multi-dentate ligands for biologically important transition metal ions.In analogy to bilins, transition metal complexes of coloured phyllobilins have properties that may be physiologically relevant and beneficial e.g. in plants, as sensitizer for singlet oxygen, 89,90 act as additional toxins against pathogens, 71 or play a part in heavy metal transport and detoxification. 91Clearly, in that respect, only the 'top of the iceberg' has been uncovered by our studies, so far, and phyllobilins and their transition metal complexes are expected to remain the topic of further interesting discoveries.
Fig. 7
Fig. 7 The pink-coloured phyllobilin PiCC 3 exhibits an E-configurated C10vC11 double bond, giving it a 'stretched' structure in solution and in the crystal.Chemical formula (left), crystal structure (centre) and model structure with highlighted H-bonds (right) are depicted.
Fig. 10
Fig. 10 Solutions of metal complexes M-3 (from left to right, with M = Zn(II), Cd(II), Ni(II), Cu(II) and Pd(II)) of the PiCC 3 in MeOH, as observed under day light (top) or under UV-light (at 366 nm, bottom).
Fig. 11
Fig. 11 Absorption (full line, left scale), fluorescence (red broken line, right scale) and fluorescence excitation spectra (blue broken line, right scale) of solutions in DMSO of YCC methyl ester 2-Me before (top) and after addition of Zn(OAc) 2 (bottom).Complex formation with Zn(II)-ions is indicated by the shift of the maxima of the absorption and of the intense emission (note different scales for luminescence, right).72
Fig. 13
Fig.13Formula of a model Zn(II)-1-formylbiliverdinate, in which the Zn(II)-ion is pentacoordinate due to ligation of a water molecule (structure derived from X-ray crystal analysis).
Senior Lecturer at the Institute of Organic Chemistry in University of Innsbruck: Dr Li is interested in the chemistry of linear tetrapyrroles and porphyrins. | v3-fos-license |
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} | pes2o/s2orc | Characterization of Pancreatic Cancer Tissue Using Multiphoton Excitation Fluorescence and Polarization-Sensitive Harmonic Generation Microscopy
Thin tissue sections of normal and tumorous pancreatic tissues stained with hematoxylin and eosin were investigated using multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) microscopies. The cytoplasm, connective tissue, collagen and extracellular structures are visualized with MPF due to the eosin stain, whereas collagen is imaged with endogenous SHG contrast that does not require staining. Cellular structures, including membranous interfaces and nuclear components, are seen with THG due to the aggregation of hematoxylin dye. Changes in the collagen ultrastructure in pancreatic cancer were investigated by a polarization-sensitive SHG microscopy technique, polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of the SHG signal as a function of the linear polarization orientation of the incident laser radiation. From the PIPO SHG data, the second-order non-linear optical susceptibility ratio, χ(2)zzz'/χ(2)zxx', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C6 symmetry, an additional second-order non-linear optical susceptibility ratio, χ(2)xyz'/χ(2)zxx', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ(2)zzz'/χ(2)zxx' values were found between tumor and normal pancreatic tissues in periductal, lobular, and parenchymal regions, whereas statistically-significant differences in the full width at half maximum (FWHM) of χ(2)xyz'/χ(2)zxx' occurrence histograms were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Additionally, the PIPO SHG data were used to determine the degree of linear polarization (DOLP) of the SHG signal, which indicates the relative linear depolarization of the signal. Statistically-significant differences in DOLP values were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Hence, the differences observed in the χ(2)zzz'/χ(2)zxx' values, the FWHM of χ(2)xyz'/χ(2)zxx' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.
INTRODUCTION
Pancreatic cancer is currently the third-leading cause of death from cancer in the USA (1). It is rarely detected at an early stage, since symptoms are often not present until the cancer has spread to other organs, thereby making it one of the deadliest cancers. A diagnosis of cancer is confirmed by visualizing a stained thin section of tissue obtained at biopsy or surgical resection using bright-field microscopy. The initiation of cancer has been attributed to the progressive accumulation of somatic mutations in epithelial cells (2). In addition, recent studies also implicate the tumor microenvironment, including the extracellular matrix (ECM), blood vasculature and inflammatory cells and fibroblasts, in cancer promotion (3)(4)(5)(6)(7). During cancer initiation and progression, mechanisms responsible for ensuring normal organ development and function are deregulated, leading to ECM disorganization (8), so that a technique that identifies and quantifies structural details in the ECM as well as permits the distinction of cells present in the ECM would improve cancer diagnosis.
In this paper, SHG, MPF, THG, and polarization-sensitive SHG microscopy are used to visualize normal and tumorous pancreatic tissue. The SHG intensities and polarization parameters related to the structure of collagen are extracted including the χ (2) zzz '/χ (2) zxx ' value associated with the distribution of fibers, as well as the χ (2) xyz '/χ (2) zxx ' value related to the chiral structure of collagen. Additionally, the degree of linear polarization (DOLP) of SHG associated with the presence of disorder of collagen fibers is determined. The THG intensities of cells present in normal and tumorous pancreatic tissues are also measured. The study investigates the possibility of whether pancreatic cancer diagnosis by identifying cancer cells using THG imaging or polarization-resolved SHG imaging of the extracellular matrix is viable. For this, the SHG and THG intensities as well as the SHG polarization parameters in tumor tissue were compared with those in three distinct regions in normal pancreatic tissue all commonly found in a single tissue section. The architecture of normal pancreatic tissue consists of ducts, fibrous septa that delineate lobes and lobules, and parenchyma composed of acinar cells and scattered endocrine cells; where each tissue type has a distinct biological function. Due to the varying architecture of these units, it is important to compare any differences in the SHG and THG intensities, and the SHG polarization parameters amongst the normal tissue to understand their variability across normal tissue elements before comparing them to those measured from tumor taken from the same area of the pancreas. In this study, all tumors were adenocarcinomas that originate from ducts or ductules but infiltrate to involve all components of the pancreas. Other tumors that originate in pancreas may have different properties and a future study of in situ tumor is likely needed to determine whether the tumor tissue retains a structural remnant of its originating tissue.
Histology Sample Preparation
Samples of normal human pancreas from five patients and pancreatic ductal adenocarcinoma tissue samples from ten patients were obtained with informed consent and institutional approval (University Health Network Toronto, Canada). The tissues were handled as per standard clinical histology protocols. Thin sections (5 µm) were cut from formalin-fixed paraffinembedded tissues, mounted on glass slides and stained with H&E for histopathologic analysis. All slides were scanned at ×20 magnification using a whole-slide scanner (ScanScope XT: Leica Biosystems, Germany).
In each slide, 110 × 110 µm regions of interest, as identified by a pathologist (S.L.A), were scanned. Identification was performed by assessing the tissue architecture and cytology of the tumor cells in the high resolution bright-field microscopy images. A total of 47 tumor and 51 normal regions were imaged to determine quantitative differences between tumor and normal tissue. Twotailed t-tests of statistical significance were performed.
Non-linear Optical Microscope Setup
A custom-built Yb:KGW laser operating at 1028 nm, 14.3 MHz repetition rate and ∼450 fs pulse duration (49) was used for MPF, SHG and THG imaging. This was coupled to a custom-built non-linear optical microscope, described previously (50,51). Briefly, galvanometric scanning mirrors (VM1000, Cambridge Technology, USA) were used to raster scan the beam through a 0.75 numerical aperture (NA) air objective lens (Plan-Apochromat 20×, Carl Zeiss AG, Germany) that does not alter polarization at up to 10 frames per second. MPF, SHG and THG signals were collected in transmission mode through a custom-built 0.85 NA collection objective lens (Omex Technologies, USA). A pulse energy of ∼0.05 nJ was used for MPF imaging and a pulse energy of ∼1 nJ was used for SHG and THG imaging. For the MPF channel, the 525-630 nm band was collected (FF01-578/105 filter, Semrock Inc., USA), which included mostly eosin fluorescence. SHG was filtered with an interference filter centered at 514.5 nm with 10 nm bandwidth (F10-514.5, CVI Laser Optics, USA). For both MPF and SHG imaging, the intense excitation light requires additional filtering using a Schott color glass filter (BG39, CVI Laser Optics, USA). THG signals were selected by a single bandpass interference filter of 340 nm with 10 nm bandwidth (F10-340, CVI Laser Optics, USA). The signals were measured using single-photon-counting detectors (H7421-40, Hamamatsu Photonics K.K., Japan).
For polarization measurements of SHG signals, the polarization-in, polarization-out (PIPO) technique was used, as previously described (52). Briefly, the microscope was modified by addition of a polarization-state generator (PSG), consisting of a linear polarizer (IR 1100 BC4, Laser Components, Germany) followed by a half-wave plate (532GR-42, Comar Optics Ltd., United Kingdom) placed immediately before the excitation objective lens for rotation of the incident laser polarization. To measure the polarization of the SHG signal, a polarization-state analyzer (PSA) was used, consisting of a linear polarizer (10LP-VIS-B, Newport Corporation, USA) located after the collection objective lens. A typical PIPO SHG measurement consisted of recording an SHG image at 9 emission polarization angles for each of 9 half-wave plate angles. Every 9 images an additional image was obtained at reference polarizer and analyzer angles as a control. A pulse energy of ∼0.5 nJ was used for PIPO SHG imaging. To measure the variation in SHG intensity between normal and tumor tissues, the PSG was modified by adding a quarter-wave plate (WPQ05M-1064, Thorlabs, Inc., USA) just before the excitation objective lens in order to obtain circularly polarized light.
Second-Order Non-linear Optical Susceptibility Ratios and the Degree of Linear Polarization From Polarization-Sensitive SHG Measurements
The second-order non-linear optical susceptibility tensor components ratio of collagen fibers was determined from PIPO SHG measurements as described previously (52,53). Briefly, a laboratory Cartesian coordinate system (XYZ) was defined with respect to the principal propagation direction of the laser (Y), where XZ is defined as the image plane. The average orientation of collagen fibers in a voxel was defined by modified spherical angles, where δ is the average in-plane fiber orientation measured from the Z-axis, and α is the out-of-plane tilt angle of the fiber.
Assuming C 6 symmetry, the SHG intensity can be described as a function of the laser electric field polarization orientation (θ ) and the orientation of the analyzer (φ) (54): The primed coordinate denotes the molecular susceptibility projected onto the image plane. Equation (1) shows that 3 second-order non-linear optical susceptibility tensor component ratios of an arbitrarily oriented fiber could be deduced from PIPO SHG measurements. It was assumed that χ (2) xxz '/χ (2) zxx ' = 1 in order to reduce the free-parameter space for more accurate fitting, and is valid as long as collagen behaves as rod-like structures with a dominant χ (2) zzz (55). The molecular secondorder non-linear optical susceptibility tensor component ratios (χ (2) zzz /χ (2) zxx and χ (2) xyz /χ (2) zxx ) of an arbitrarily oriented fiber at an angle α from the imaging plane is related to the measured susceptibility ratios (R or χ (2) zzz '/χ (2) zxx ' and C or χ (2) xyz '/χ (2) zxx ', respectively) as: The equations show that at low α angles, R is representative of its molecular counterpart, while the measured C parameter would be near 0, conversely larger measured C values, closer to their molecular counterparts are only expected from fibers at higher α. Alternatively, when non-chiral cylindrical (C 6v ) symmetry is assumed, χ (2) xyz '/χ (2) zxx ' = 0 in Equation (1) (52,53). In addition to extracting the R and C values, a third parameter known as the degree of linear polarization (DOLP) was extracted from:
Non-linear Optical Imaging of Normal and Tumor Pancreatic Tissues
H&E stained histopathology sections of normal and tumorous human pancreatic tissue were imaged with MPF, SHG and THG microscopy (Figure 1). In general, the MPF signal from H&E stained tissues visualizes the entire extracellular matrix due to the eosin staining (Figures 1B,G,L,Q). The SHG signal is generated intrinsically from collagen ( Figures 1C,H,M,R), while the THG signal is predominantly generated by hemalum complexes from chromatin-bound hematoxylin (19). Hence, THG visualizes nuclear material, including nucleoli and the nuclear envelope. Structural cross-correlation image analysis between the 3 imaging modalities was performed using an overlap and threshold algorithm (57,58). In normal tissue, the SHG and THG signals were largely uncorrelated (Figures 1E,J,O), but more correlation was observed in tumor ( Figure 1T), likely due to the increased content of chromatin in the nuclei of collagenous tumor regions. Further rigorous analysis is needed in order to quantify and determine the statistical significance of this observation. Analysis of the correlation between MPF and the other signals was not useful, since the MPF from eosin is not selective for collagen or nuclei (Figures 1D,I,N,S).
SHG Intensity From Normal and Cancerous Pancreatic Tissues
Representative images of the SHG intensity of collagen in the normal periductal, lobular, and parenchymal tissues and in tumor are shown in Figures 2A-D. The values are independent of the fiber orientation angle, δ, since circularly polarized laser light was used. Furthermore, in order to perform valid intensity comparisons, identical laser powers and pixel dwell times were used. In general, the SHG intensity of periductal collagen (3,250 ± 1,750 photon counts: Figure 2A) was the highest, followed by lobular (2,250 ± 1,500: Figure 2B) and parenchymal (900 ± 700: Figure 2C) tissue. The higher SHG intensity of periductal and lobular collagen is attributed to the thick and densely arranged collagen around ducts and lobes as compared to the parenchymal region where the collagen fibers are thinner and sparsely arranged (59), although collagen disorder in the focal volume may also play a role. The SHG intensity of collagen in tumor (1,700 ± 800: Figure 2D) was less than in periductal and lobular regions and typically higher than parenchymal collagen however, a statistically-significant difference was not observed.
Although variations of the highest SHG intensities are easily visualized in Figure 2, in each tissue type, the SHG intensity is highly variable. For example, the row of images in Figure 2B shows intense SHG intensity from the thick lobular collagenous region while parenchymal collagen, next to the lobular collagen, has significantly lower intensities, complicating diagnosis by using SHG intensity. Hence, measurements of the ultrastructure of collagen in normal and cancerous human pancreas tissue were performed via polarization-sensitive SHG measurements.
Polarization-Resolved SHG Measurements
Analyzed With C 6v Symmetry Figure 3 shows typical results of PIPO SHG imaging of periductal, lobular, parenchymal, and tumor tissues. These were analyzed using C 6v symmetry, revealing for each pixel the structural parameter, R, which depends on several factors including: the ultrastructure of individual collagen fibers, the collagen disorder within the laser beam focal volume and the tilt angle, α, between the image plane and the collagen fibers Equation (2). Previously, R has been investigated in collagen of different animal species, including in human normal and tumor tissues to quantify the levels of collagen disorder (32,39,42). The R value ranges from ∼1.1 observed from well-ordered collagen in tibia (53) to a theoretical maximum of 3 for the most disordered structures (with the assumption that χ (2) xxz '/χ (2) zxx ' = 1). The maps of the fitted R values for typical normal and tumor pancreas tissue samples are seen in Figure 3 column 3, while the adjacent graphs in column 4 indicate the corresponding R-values occurrence histograms from which the mean R values as well as the width of the distribution can be calculated, as summarized in Table 1. The mean R values in normal periductal, lobular, and parenchymal pancreas were significantly smaller than in tumor (p < 0.05). The histogram widths were generally larger for normal tissue than tumor tissue, where those of periductal and lobular regions were significantly larger than those of tumor tissue (p < 0.02), indicating that the tumor tissue has a narrower collagen orientation distribution.
Polarization-Resolved SHG Measurements Analyzed With C 6 Symmetry
The polarization-sensitive SHG measurements were also analyzed using the more general C 6 symmetry, in order to evaluate whether the resultant additional chiral fitting component, C, would be informative in the characterization of pancreatic collagen. Fitting using Equation (1) also reveals the R parameter from the C 6 fit, which corresponds well with the R parameter from the C 6v fit (see Table 1). The fitted C values seen in Figure 3 polarity, meaning that the two colors indicate if the average tilt of fibers in the focal volume is above or below the imaging plane. These images show that collagen fibers are clustered into small positive and negative polarity regions similarly in normal and tumor samples.
The occurrence histograms of C values using C 6 symmetry are shown in Figure 3 column 6. The width of the occurrence histograms is related to the distribution of collagen tilt angles. The histograms were fit with a Gaussian function and the average C values and the occurrence widths were obtained. The average values for normal tissues were not significantly different than that for tumor tissue, which is unsurprising as this parameter depends on the angle at which the tissue was sectioned, which was arbitrary from sample to sample. However, the widths of the C value occurrence histograms for normal parenchymal pancreas was significantly smaller than that of tumor tissue (p < 0.05), while those for normal periductal and parenchymal tissues were significantly different from normal lobular pancreas tissue when the number of regions is taken into account (p < 0.02 and p < 0.001, respectively, see Table 1), likely related to structural variations in the collagen.
Disordered collagen fibers and the presence of small collagen fiber segments within the focal volume may produce depolarized SHG signals, which can be characterized by calculating the DOLP for each pixel of the PIPO SHG images. The influence of birefringence and scattering due to SHG propagation through birefringent tissue regions can be considered as negligible, since the tissue sections are sufficiently thin (5 µm) that there is minimal cumulative retardation of the light. An additional assumption that the scattering of the polarized SHG light is negligible is also reasonable, considering that the tissue samples are all the same thickness (60). The DOLP for normal periductal and parenchymal tissues were significantly different than for tumor (p < 0.002 and p < 0.01, respectively). The values for periductal tissues were higher than for tumor, while the values for parenchymal tissues were lower than for tumor. Lower average DOLP values in tumor tissues have previously been found in classical papillary thyroid
THG Signals in Nuclei of Normal and Cancerous Pancreatic Tissues
The H&E stained sections were also imaged with THG using linearly polarized laser light. The analysis is valid since the THG intensity images from H&E stain were found to be independent of the orientation of laser polarization (19). Figure 4 shows typical H&E-stained bright-field (Figures 4A1,B1,C1,D1) and THG (Figures 4A3,B3,C3,D3) images and the corresponding SHG images (Figures 4A2,B2,C2,D2) for periductal (A), lobular (B), parenchymal (C) and cancerous (D) tissue regions. The nuclear envelope and nucleoli can be seen in the THG images (Figures 4A3,B3,C3,D3), due to THG enhancement by hematoxylin aggregation (19). Hence, the regions around nuclei in the different tissue types were analyzed using SHG and THG contrast. SHG from collagen lying in between nuclei is seen in Figures 4A2,B2,C2,D2 and appears strongest for periductal tissue (A), indicating higher collagen density in the matrix around nuclei. Parenchymal tissue showed lower SHG intensity, indicating reduced collagen density, while tissue surrounding nuclei in lobular and tumor tissues had the lowest collagen density. The nuclei within normal periductal, lobular and parenchymal pancreas tissues appear round and small (Figures 4A3,B3,C3), while those within tumor cells appear elongated and larger ( Figure 4D3). We approximate nuclei as ellipses, and measured the length of the long axis (L) and the short axis (S) of the nuclei manually using imaging software (ImageJ 1.52a, NIH), and ellipticity (e) was calculated as: e = L 2 +S 2 S 2 . The average ellipticity was statistically-significantly larger for nuclei in the tumor regions (2.6 ± 0.4) compared the periductal (1.7 ± 0.1), lobular (1.6 ± 0.1), and parenchymal (1.6 ± 0.2) regions of normal tissue (p < 0.01, p < 0.002, and p < 0.01, respectively, based on the number of samples). There is not a significant difference in these parameters between nuclei imaged in the periductal, lobular, and parenchymal regions of normal tissue. The nuclear shape and size are easily observed with THG, with essentially no background signal from the extracellular matrix and thus, THG imaging could be used for automated analysis of nuclear size and shape for early cancer detection. The THG intensity was also compared between the different types of tissue, but there was no statistically-significant difference between normal (2,300 ± 1,000) periductal, lobular or parenchymal tissues compared with tumor (3,000 ± 1,500).
DISCUSSION
Quantitative polarization-resolved SHG values can be used to distinguish between normal and tumor pancreatic tissues. In particular, normal periductal, lobular and parenchymal tissues are significantly different from tumor tissue, as indicated by their R values. Additionally, periductal and lobular tissues are significantly different from tumor with respect to the FWHM of R occurrence, while parenchymal tissue is significantly different from tumor considering the FWHM of C occurrence. Moreover, parenchymal and periductal tissues have significantly different DOLP values from tumor tissue. A limitation of the present study was the relatively small number of patient samples available. However, considering instead the number of regions-of-interest measured, the DOLP values would statistically distinguish lobular tissue from tumor, while the FWHM of the C occurrence values would also segregate periductal tissue from tumor. Overall, these results indicate the potential of using polarization SHG parameters for automated cancer diagnosis, for example, by adding PIPO SHG based contrast to histopathology slide scanners, where the SHG parameters for each image pixel in the entire section is determined; regions of concern would then be flagged for pathologist review, based on standardized SHG parameters.
The observed changes in the R values for collagenous tissues have been previously attributed to variations in the amino acid content of the triple helices, the arrangement of the triple helices into fibrils and fibers, and the distribution of fibrils and fibers within the laser focal volume (39). We have previously reported similar variations in R between normal and tumor tissues in human lung (39), breast (32), and thyroid (42), indicative of increased structural disorder with malignancy. The FWHM of the C occurrence parameter is based on the intrinsic chirality of the collagen, but only collagen fibers pointing out of the image plane can have a significant C value, so that the observed variations in this parameter likely originate from different angular distributions of the collagen fibers (54). Several significant variations in the DOLP values were observed across the normal pancreatic tissue types and distinguishes parenchymal from lobular and periductal tissues. Parenchymal tissues had the lowest DOLP values, even lower than in tumor, indicating the most depolarized SHG. Since the tissue samples are thin, scattering of the laser or SHG light or birefringence that would induce elliptical polarization are unlikely to be the dominant effects. Notably, if birefringence was the cause of the DOLP variation, then periductal or lobular tissue would be expected to show a larger effect, since they both have significantly higher collagen content.
Parenchymal tissue is also significantly different than the other tissues when the number of regions measured is considered, rather than the number of patients. In particular, the FWHM of R occurrence statistically distinguishes parenchymal from lobular and periductal tissues, while the FWHM of C occurrence distinguishes parenchymal from lobular tissues. Interestingly, the R parameter does not distinguish between the different normal tissues, indicating that these have similar fiber ultrastructure. On the other hand, the FWHM of R occurrence distinguishes between the parenchymal, lobular and periductal tissues, showing some disparity in the collagen. Additionally, the FWHM of C occurrence indicates that the ultrastructure of the parenchymal collagen is closer to that of periductal tissue, and that these have differences in collagen fiber angle, α, or chirality as compared to lobular pancreas. The significant DOLP variations, as well as the FWHM of C and R, are attributed to variations in collagen organization in these tissues. It is known that periductal collagen consists of short concentric segments (61) and parenchymal collagen comprises of short segments, whereas lobular collagen appears to consist of long and straight segments. The measurements here indicate that parenchymal collagen may consist of the shortest segments within the laser focal volume, followed closely by periductal collagen. This distinction of collagen between the periductal, lobular and parenchymal regions is surprising and should be investigated further. An additional independent cancer marker in pathological tissues treated with H&E dyes is the quantification of nuclei size and shape by performing THG imaging. This information could be used by automated algorithms for differentiating cancer cells. By using a laser excitation of 1030 nm, the THG signals at 343 nm have sufficient transmission through glass for efficient detection, as compared with 800 nm laser excitation, commonly used in non-linear optical imaging, which results in THG signal at 267 nm, where glass absorbs the signals. Therefore, with 1030 nm lasers, SHG and THG can be performed simultaneously without severely affecting the imaging parameters and hence, it is beneficial to combine THG in the analysis, allowing correlations in collagen structure with nuclei size and shape for a more robust cancer diagnosis.
CONCLUSIONS
Human pancreas tissue samples were successfully imaged by polarization-resolved SHG, MPF, and THG microscopies. Using the several polarization SHG parameters (R, FWHM of C occurrence and DOLP), the normal tissue can be distinguished from tumor tissue. These parameters could be used together for automated cancer detection and as a research tool to understand how the extracellular matrix is formed and evolves with tumor initiation and progression. Further, variations between the collagen structure within normal tissue types were also observed, indicating that collagen structure and ultrastructure vary even within a single organ according to the tissue function. THG microscopy also revealed differences in the nuclear morphology. Combined SHG and THG imaging can be applied for automated cancer detection and can also be used to study the interaction of cancer cells with collagenous structures of the extracellular matrix.
ETHICS STATEMENT
This study was carried out in accordance with the recommendations of the Tri-Council Policy Statement and the University Health Network Research Ethics Board. Additional written consent for this study was not needed to be obtained because the tissues used in this study were collected from a biobank where consent had already been received. The protocol was approved by the University Health Network Research Ethics Board. | v3-fos-license |
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} | pes2o/s2orc | The synergic effect of vincristine and vorinostat in leukemia in vitro and in vivo
Background Combination therapy is a key strategy for minimizing drug resistance, a common problem in cancer therapy. The microtubule-depolymerizing agent vincristine is widely used in the treatment of acute leukemia. In order to decrease toxicity and chemoresistance of vincristine, this study will investigate the effects of combination vincristine and vorinostat (suberoylanilide hydroxamic acid (SAHA)), a pan-histone deacetylase inhibitor, on human acute T cell lymphoblastic leukemia cells. Methods Cell viability experiments were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distributions as well as mitochondria membrane potential were analyzed by flow cytometry. In vitro tubulin polymerization assay was used to test tubulin assembly, and immunofluorescence analysis was performed to detect microtubule distribution and morphology. In vivo effect of the combination was evaluated by a MOLT-4 xenograft model. Statistical analysis was assessed by Bonferroni’s t test. Results Cell viability showed that the combination of vincristine and SAHA exhibited greater cytotoxicity with an IC50 value of 0.88 nM, compared to each drug alone, 3.3 and 840 nM. This combination synergically induced G2/M arrest, followed by an increase in cell number at the sub-G1 phase and caspase activation. Moreover, the results of vincristine combined with an HDAC6 inhibitor (tubastatin A), which acetylated α-tubulin, were consistent with the effects of vincristine/SAHA co-treatment, thus suggesting that SAHA may alter microtubule dynamics through HDAC6 inhibition. Conclusion These findings indicate that the combination of vincristine and SAHA on T cell leukemic cells resulted in a change in microtubule dynamics contributing to M phase arrest followed by induction of the apoptotic pathway. These data suggest that the combination effect of vincristine/SAHA could have an important preclinical basis for future clinical trial testing. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0176-7) contains supplementary material, which is available to authorized users.
paclitaxel [4]. At lower concentrations, both types affect only microtubule dynamics, inducing abnormal mitotic spindle formation and causing cell arrest in the M phase and, subsequently, cell apoptosis [5]. Microtubule-binding agents have played critical roles in the history of chemotherapeutic drug development and remain a first choice in the treatment of solid tumors and hematological malignancies. As with other anticancer agents, however, the problems of drug resistance, neuropathy, immunosuppression, and poor solubility must be addressed. The current trends in tubulin-binding agent development are to change the dosage to improve solubility or a combination of these agents with other anticancer drugs to reduce toxicity and enhance activity [6].
The purpose of this study is to evaluate the combined effects of vincristine and SAHA on an acute T cell lymphoblastic leukemia (ALL) model. We determined the mechanism underlying cell arrest in the mitotic phase and subsequent apoptosis following combination treatment. We found that vincristine and SAHA have a synergic effect on microtubules through different mechanisms. Moreover, this combined influence was also observed in in vivo results. Consequently, we suggest that a vincristine and SAHA combination treatment could be used in the clinical setting.
Results
Cytotoxic effects of vincristine and SAHA, alone and in combination, on human leukemic MOLT-4 cells A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the cytotoxicity of the microtubule-destabilizing agent vincristine and the HDACi vorinostat (SAHA) on human ALL MOLT-4 cells. We first tested the cytotoxic effect of SAHA and vincristine alone and in combination. As shown in Fig. 1a, there was no significant cytotoxicity at concentrations up to 500 nM of SAHA. However, SAHA had an IC 50 of 840 nM for 48 h, when concentration reached the highest level (1000 nM). In addition, vincristine exhibited cytotoxicity against human leukemic MOLT-4 cells with an IC 50 of 3.3 nM at 48 h (Fig. 1b). To determine whether an interaction between SAHA and vincristine took place, the cytotoxic potency of a combination assay was measured. Cells treated with 500 nM SAHA and various concentrations of vincristine (0.3 to 3 nM) significantly inhibited cell survival compared to each treatment alone (Fig. 1c).
Effects of vincristine in combination with SAHA on human T cell leukemic cell survival
To further explore the synergistic cytotoxic effects, we determined the effects on cell cycle distribution. As compared with SAHA, treatment with vincristine induced an increase in the G 2 /M phase of the cell cycle. In particular, the combination of vincristine plus SAHA caused an almost complete arrest of cells in the G 2 /M phase following short-term treatment (24 h) and a subsequent induction in the sub-G 1 phase following long-term treatment (48 h) (Fig. 2a). Figure 2b shows the statistical results. Next, the combination index (CI) method was used to evaluate the synergistic combinations [25]. A CI value of >1.0, 1.0, and <1.0 indicates an antagonistic, additive, or synergistic interaction, respectively, between the drugs. In the G 2 /M phase, the CI values of vincristine (0.3, 1, and 3 nM) combined with 500 nM SAHA were 1.63, 0.72, and 0.32, respectively, and the CI values in the sub-G 1 phase were 0.97, 0.77, and 0.28, respectively (Fig. 2c). And this synergistic combination effect also was noted in the other T cell leukemic cell line, CCRF-CEM (Fig. 2d), rather than in acute myeloid leukemic cells (Additional file 1: Figure S2). Moreover, vincristine (1 or 3 nM) combined with various concentrations of SAHA also shows synergistic effect (Additional file 2: Figure S1). These data indicate that vincristine and SAHA synergistically induced cell arrest in the G 2 /M phase and subsequently in the sub-G 1 phase. MOLT-4 cells were treated with various concentrations of SAHA and vincristine alone for 24 and 48 h, respectively. c The combination of SAHA (S) and vincristine (V) was compared to the effect of vincristine alone. Data are expressed as mean values ± SD of at least three separate determinations. *P < 0.05, ** P < 0.02, *** P < 0.005
Effects of SAHA in combination with vincristine on mitotic arrest in human leukemic MOLT-4 cells
To further elucidate the synergistic effect mechanism on the G 2 /M phase of cell cycle progression, we investigated SAHA in combination with vincristine on tubulin polarization change and mitosis-related proteins. As shown in Fig. 3a, there were no obvious tubulin polarization changes following SAHA treatment under cell-free conditions. However, in combination with vincristine, a significant induction of microtubule depolymerization was observed (Fig. 3a). Additional file 3: Figure S3 shows a more comprehensive result, including various vincristine-and SAHA-alone in vitro tubulin polymerization assays. To understand the effects of microtubule dynamics on mitosis following drug treatment, the microtubule arrangement in human leukemic MOLT-4 cells was examined by βtubulin staining. As shown in Fig. 3b(b), there was no significant change in microtubule distribution and cell morphology after SAHA treatment. In addition, at low vincristine concentrations, cells had accumulated at the metaphase stage of mitosis with abnormal spindles (Fig. 3b(c)). In this study, spindles with bipolar and multipolar organization, which had abnormal long astral microtubules and chromosomes, were found to be unequally distributed. Nevertheless, at a high vincristine concentration, microtubule depolymerization was observed ( Fig. 3b(d)). In the present study, the vincristine and SAHA combination exerted more explicit effects than vincristine alone with regard to abnormal spindles and chromosomes ( Fig. 3b and Additional file 4: Figure S4). These results suggest that SAHA potentiated the effects of vincristine due to inhibition of microtubule dynamics. M phase-regulating proteins were also analyzed. The combination of various concentrations of vincristine (0.3, 1, and 3 nM) with SAHA (500 nM) for 24 h increased the phosphorylation of two mitotic markers (MPM2 and H3S10) in a concentrationdependent manner. These results proved that the combination of vincristine and SAHA induced M phase arrest. Moreover, vincristine in combination with SAHA also induced mitotic arrest by stimulating cyclin B1, aurora B, phospho-Cdc2 (Thr161), and phospho-PLK1 expression, and suppressing Cdc25c and phospho-Cdc2 (Tyr15) levels. The magnitude of these changes was more obvious than those observed with vincristine treatment alone (Fig. 3c). These findings demonstrate that SAHA enhanced vincristineinduced M phase arrest and that the combination therapy induced microtubule dynamics instability and mitotic activation.
Effects of SAHA in combination with vincristine on the apoptotic pathway and HDAC activity in human leukemic MOLT-4 cells Mitochondria play a crucial role both in the intrinsic and extrinsic apoptotic pathways. To test whether the vincristine/SAHA-mediated apoptotic pathway was associated with mitochondrial function, a change in mitochondrial transmembrane potential (Δψm) was assessed. As shown in Fig. 4a, treatment with SAHA or vincristine alone was insufficient to affect the mitochondrial membrane potential; however, this phenomenon was enhanced by co-treatment with SAHA in a timedependent manner. The Bcl-2 protein family plays a regulatory role in controlling the mitochondrial apoptotic pathway. The data showed that the combination treatment more effectively downregulated the expression of the pro-survival members of the Bcl-2 family, such as Bcl-2, Bcl-xl, and Mcl-1, than did either treatment alone (Fig. 4b).
Caspase activation plays an important role in the classic apoptotic pathways. In this study, it was also found that caspases 3, 6, 7, 8, and 9 and PARP were activated by the combination treatment for 48 h (Fig. 4c). Moreover, to confirm whether HDACs, both in the single or combination treatment, were involved in the apoptosis pathway, the expression of HDACs and H3 acetylation were evaluated. Vincristine (3 nM) in combination with SAHA effectively inhibited HDAC3 and HDAC6 expression and enhanced H3 acetylation, which represents an inhibition in HDAC activity, in a time-dependent manner (Fig. 4d).
HDAC6 inhibition was involved in vincristine/SAHAinduced apoptosis
Previous findings have shown HDAC6-induced tubulin acetylation to affect the dynamics and function of microtubules [9][10][11][12]. As shown in Fig. 5a, SAHA, a pan- a Tubulin assembly was determined by an in vitro tubulin polymerization kit and then detected by spectrophotometry. Paclitaxel (10 μM) and vincristine (10 μM) were used as positive controls. Paclitaxel is a polymerizing agent and vincristine is a depolymerizing agent. S 5 represents SAHA 5 μM, and S 50 + V 3 represents SAHA 50 μM combined with vincristine 3 μM. This experiment was examined in a cell-free condition. b MOLT-4 cells were treated with SAHA and vincristine alone or co-treatment for 24 h and then stained with β-tubulin and DAPI. The immunofluorescence images were captured by the ZEISS LSM 510 META confocal microscope. All images were under × 400 microscopic magnification. a control, b 500 nM SAHA, c 3 nM vincristine, d SAHA combined with vincristine, e 30 nM vincristine, f 30 nM paclitaxel. c Cells were treated with vincristine (0.3, 1, and 3 nM) alone or co-treated with SAHA (500 nM) for 24 h. Then, total cell lysates were obtained to evaluate the G 2 /M phase regulatory protein expression and the tubulin acetylation. Data are expressed as mean values ± SD of at least three separate determinations. S SAHA, V vincristine HDACi, induced tubulin acetylation; however, its combination with vincristine had no synergic effect. Tubastatin A, which is a specific HDAC6 inhibitor [26], was used to understand the role of HDAC6 in the vincristine/SAHA-treated cells. To evaluate the potential benefit of vincristine in combination with tubastatin A, the cytotoxicity of co-treatment was determined and the combination effects were analyzed. However, compared to tubastatin A alone, vincristine significantly enhanced the cytotoxicity of tubastatin A (Fig. 5b). Moreover, vincristine (1 and 3 nM) combined with various concentrations of tubastatin A induced cell accumulation at the G 2 /M phase followed by the sub-G 1 phase (Fig. 5c). The CI values were <1 in combination of vincristine and tubastatin at the G 2 /M phase and sub-G 1 phase (Fig. 5d). Co-treatment of vincristine and tubastatin revealed MPM2 and PARP activation consistent with the induction of apoptosis by western blot analysis (Fig. 5e). And vincristine and HDAC6 inhibitor combined synergism effect was further corroborated by the observation of vincristine and ACY1215 co-treatment in CCRF-CEM cells (Fig. 5f). These findings suggest that SAHA treatment may alter microtubule dynamics in cells through HDAC6 inhibition, even though the effect was insufficient to arrest cells in the G 2 /M phase. However, in combination with vincristine, which also had an effect on microtubules, SAHA caused extreme microtubule stress thus causing cell death.
The antitumor activity of vincristine and SAHA combination therapy in vivo
To evaluate whether the synergistic effect of vincristine plus SAHA could be clinically relevant, the antitumor activity of this co-treatment in severe combined immunodeficiency mice bearing established MOLT-4 tumor xenografts was investigated. Once a tumor was palpable (approximately 100 mm 3 ), mice were randomized into vehicle control and treatment groups (n = 6 per group). All mouse tumors were allowed to reach an endpoint volume of 2000 mm 3 , and in vivo antitumor efficacy was expressed as tumor growth delay (TGD; Fig. 6a). There were no improvements in TGD in mice treated with vincristine (0.1 mg/kg once weekly) or SAHA (50 mg/kg once daily) alone. However, log-rank analysis showed that the co-treatment exhibited significant antitumor activity in the MOLT-4 xenograft model (P = 0.0389). In addition, Kaplan-Meier curves displayed antitumor activity for the co-treatment group (vincristine, 0.025 mg/kg once weekly; SAHA, 200 mg/ kg once daily) (Fig. 6b). Notably, the mice tolerated all of the treatments without overt signs of toxicity; no significant body weight difference or other adverse side effects were observed ( Fig. 6d and Additional file 5: Figure S5). To correlate the in vivo antitumor effects with the mechanisms identified in vitro, intratumoral biomarkers were assessed by western blot analysis. Consistent with in vitro results, the combined treatment markedly induced caspase 3 activation and PARP cleavage in tumors, indicating elevated apoptosis (Fig. 6e). Taken together, these findings suggest that combination of vincristine and SAHA, both in vitro and in vivo, dramatically enhanced vincristine-induced cell death.
Discussion
Recent preclinical studies have reported that due to their broad anticancer potency and low toxicity, HDACis are often used in combination therapy to enhance conventional chemotherapeutic and molecular-targeted drugs. SAHA was the first HDACi approved by the FDA for T cell lymphoma. Moreover, vinca alkaloids have been extensively used in the clinical treatment of ALL. In spite of their usefulness, drug resistance and neuron toxicity remain serious clinical problems. Therefore, the purpose of this study was to investigate the anticancer activity of vincristine and SAHA in a T cell ALL cell line. Cytotoxic experiments have shown that the combination of vincristine and SAHA significantly induces cell death. Analyses of cell cycle and regulatory protein expressions have indicated that co-treatment with the two drugs has a synergistic effect on M phase arrest and is consistent with an increase of cell numbers in the sub-G 1 phase. Furthermore, combination treatment promotes cell apoptosis through intrinsic and extrinsic pathways. In vivo xenograft animal models demonstrated that, compared to treatment with either drug alone, vincristine in combination with SAHA prolongs survival time in mice, as suggested by the in vitro results. During these studies, the level of the effects of vincristine and SAHA on MOLT-4 cells was determined.
(See figure on previous page.) Fig. 4 Co-treatment with vincristine and SAHA induced mitochondrial membrane potential loss, caspase activation, and HDAC activity. a The mitochondrial membrane potential was measured by the flow cytometry analysis of rhodamine 123. The MOLT-4 cells were stained with 10 μM rhodamine 123 and incubated at 37°C for 30 min in the presence of SAHA alone (500 nM), vincristine alone (3 nM), or coexistence of both at different time intervals (12,24,36, and 48 h). The horizontal axis shows the relative fluorescence intensity and the vertical axis presents the cell number. b MOLT-4 cells were treated with different concentrations of vincristine (0.3, 1, and 3 nM) alone, a single concentration of SAHA (500 nM) alone, and a combination with both drugs for 24 and 48 h. Then, the cell lysates were collected for western blot analysis to detect the Bcl-2 family protein levels. c Cells were incubated with vincristine and SAHA for 48 h, and caspase 3, 6, 7, 8, and 9 activations and PARP cleavage were detected. d Similarly, the total cell lysates were analyzed to measure HDAC1, HDAC2, HDAC3, and HDAC6 expression and HDAC activity (H3 acetylation). Data are expressed as mean values ± SD of at least three separate determinations. S SAHA, V vincristine Combinations with various concentrations of both drugs were investigated to determine the lowest effective concentration of vincristine or SAHA that would provide the maximum cytotoxic effects. It was established that the cytotoxicity of 3 nM vincristine combined with 500 nM SAHA is much more potent in inhibiting cell survival ( Fig. 1) and altering cell cycle distribution (Fig. 2) compared to either treatment alone. A CI value smaller than 1 at the G 2 /M or sub-G 1 phases suggests that the combination of vincristine and SAHA had a synergistic action on T cell leukemic cells at certain concentrations (Fig. 2).
Prior studies have shown that α-tubulin acetylation is associated with microtubule dynamics and is regulated by HDAC6 [27]. Additional research has also found that HDACis induce microtubule acetylation and polymerization through HDAC6 inhibition [9][10][11][12]. Whether the polymerization or the depolarization of microtubules following the vincristine and SAHA combination treatment was responsible for G 2 /M cell cycle arrest needs to be determined for a complete understanding of this mechanism. In vitro tubulin polymerization assays showed that a high concentration of vincristine (10 μM) definitely caused microtubule depolymerization, but a high concentration of SAHA (50 μM) had no influence on microtubule polymerization (Fig. 3a). However, SAHA indeed causes the acetylation of tubulin (Figs. 4d and 5a). These results suggest that SAHA may induce tubulin acetylation, subsequently affecting microtubule polymerization by inhibiting HDAC6 activity. Nevertheless, SAHA-induced polymerization was not proven (Fig. 3a) since HDAC6 was not available in the in vitro tubulin polymerization kits, which contained only tubulin and GTP. Therefore, no SAHAinduced polymerization effects were observed in this assay. In addition, the literature indicates that tubulin acetylated by certain HDACis, such as trichostatin A, is associated with microtubule dynamics without affecting the polymerization of microtubules. Therefore, to further demonstrate the role of HDAC6, tubastatin A, a specific HDAC6 inhibitor, was used [26][27][28]. Tubastatin A and vincristine co-treatment was found to synergically induce cell death and G 2 /M phase cell arrest, proceeding to the sub-G 1 phase, compared with tubastatin A or vincristine alone (Fig. 5). These phenomena were in agreement with the results of vincristine/SAHA combination treatment. Consequently, an immunofluorescence assay was performed to demonstrate whether microtubules were affected by SAHA. Figure 3b shows that at low vincristine concentrations, abnormal spindles (star-like monopolar and multiple polars) and chromosome disorganization were observed, as has been previously reported by Jordan et al. [26]. However, the microtubule distribution remained unchanged following cell treatment with 500 nM SAHA. However, following SAHA/vincristine combination treatment, more cells were observed to have deteriorated spindles although without microtubule depolymerization or polymerization. Prior studies have shown that only higher concentrations (>10 nM) of microtubule agents, such as vinca alkaloids and taxol, affect microtubule mass, but microtubule dynamics are suppressed by low microtubule agent concentrations (<10 nM) [4]. Taken together, vincristine/SAHA-induced synergistic G 2 /M arrest may result from HDAC6 inhibition-induced microtubule dynamic alternation because of SAHA as well as vincristine. In brief, vincristine and SAHA exerted a synergic action on microtubule dynamics, albeit though different mechanisms.
A large amount of research shows that HDACis arrest cells in the G 1 phase mainly through p21 induction. Only a few studies mention the role of these compounds in G 2 /M, and the mechanism remains uncertain. For example, Blagosklonny et al. found that the HDACi trichostatin A causes tubulin acetylation that contributes to M phase arrest [28]. HDACis have also shown the capability to cause immature sister chromatid separation and slippage from the mitotic spindle assembly checkpoint (SAC) [29]. Moreover, cancer cells without wildtype p21 or p53 more easily become polyploid than wild-type cells [30]. These results indicate that HDACiinduced G 2 /M arrest is not due to an influence on transcriptional activity. In contrast, other studies point out that trichostatin A causes G 2 /M cell cycle arrest and SAC slippage through an increase in p21 transcriptional activity [31]. The mechanisms proposed above to explain (See figure on previous page.) Fig. 5 HDAC6 was involved in vincristine/SAHA-induced cell death. a MOLT-4 cells were treated with the indicated drugs for 12 and 24 h. The cell lysates were used to determine the HADC6 protein level and activity. b Cells were treated with tubastatin A alone, which is a specific HDAC6 inhibitor, or combined with 1 and 3 nM vincristine for 48 h. The cell viability was evaluated by MTT assay. c Cells incubated with tubastatin A alone or in combination with the indicated concentration of vincristine for 24 and 48 h. The cell cycle distribution was measured by flow cytometry. The figures are shown as quantitative data in the time course. d The CI value of combining tubastatin A with vincristine on G 2 /M and sub-G 1 phases. e Cells were treated with 10 or 20 μM tubastatin A (Tuba) alone and combined with 1 or 3 nM vincristine. The cell lysates were used for western bolt analysis. f CCRF-CEM cells were co-treated with vincristine and ACY1215, an HDAC6-specific inhibitor, for 48 h. The CI value of combining treatment on the sub-G 1 phase vincristine/SAHA-induced G 2 /M phase arrest should not be excluded. We found that p21 mRNA and protein level were elevated after treatment with vincristine or SAHA alone and was not enhanced by combined treatment (data not shown). It has been reported that all HDACis induced p21 but differentially caused tubulin acetylation, mitotic arrest, and cytotoxicity. Mitotic arrest rather than induction of p21 determined HDACi cytotoxicity [28]. Moreover, p21 is required for G 1 arrest, not for cell death, and is associated with resistance to HDACi-induced apoptosis [32]. Upregulated p21 was also found in vincristine-treated cells even though vincristine caused cell arrest in the G 2 /M phase, leading to apoptosis [33]. Therefore, we considered that in our study, increased p21 expression of vincristine-SAHA combined treatment was the cellular protection mechanism for repairing damaged DNA, and this phenomenon was not sufficient to cause apoptosis. Furthermore, vincristine and SAHA did not alter p53 protein and mRNA levels (data not shown). Consequently, the involvement of p53 and p21 in drug-induced G 2 /M arrest was eliminated.
The expression of related proteins in the M phase was evaluated to understand the exact phase at which the cells arrest (G 2 or M). Cdc25c is a tyrosine phosphatase that removes the inhibitory phosphorylation of Cdc2 at Tyr15, which has already been phosphorylated on Thr161 and contributes to Cdc2 activation. The Cdc2/ cyclin B complex then forms to move cells into the M phase [34]. Figure 3c shows that Cdc25c and p-Tyr15 Cdc2 protein levels declined and p-Thr161 Cdc2 increased following combination treatment. In addition, expression of the M phase markers pMPM2 and H3S10 was also induced [35]. These results demonstrate that combination treatment induces cell arrest in the M phase and not in G 2 .
From the above data, we speculate that the vincristine/ SAHA combination may alter microtubule dynamics, resulting in incorrect microtubule attachment to the centromere of chromosomes or loss of spindle tension across kinetochore pairs, subsequently causing SAC activation in the metaphase. An increased aurora B and PLK kinase protein expression and aurora B kinase activity, detected by H3S10 expression downstream of aurora B, are shown in Fig. 3c. SAC activation inhibits anaphasepromoting complex/cytochrome activity and decreases cyclin B degradation to inter-anaphase to accomplish cell division. Cells proceed to apoptosis owing to an inability to repair the dysfunction [36]. Therefore, we suggest that co-treatment-induced cell death was due to an inability to repair microtubule function even if the SAC was activated (Figs. 3c and 4c). In contrast, Dowling et al. have found that the synergistic effect of the HDACi trichostatin A and microtubule agents occurs through SAC inactivation [37]. In addition, studies have shown that HDACis induce aurora B kinase degradation and subsequently inactivate the SAC via HDAC3 inhibition [38]. Although HDAC3 is inhibited by 24-h vincristine and SAHA co-treatment (Fig. 4d), the protein level of aurora B kinase was increased (Fig. 3c). We believe that the arrest at the G 2 /M phase does not occur through the inhibition of HDAC3.
Caspase activation plays a vital role in the apoptosis pathways. Figure 4c shows that the activation of caspases 3 and 6 to 9 and PARP was synergistically induced by vincristine combined with SAHA. This result indicates that the combination treatment activates both the intrinsic and the extrinsic apoptosis pathways. Therefore, the expression of mitochondrial proteins and mitochondrial membrane potential were investigated. Bid is a key protein linking the extrinsic and intrinsic apoptosis pathways. As Fig. 4b shows, the protein level of pro-form Bid decreased in response to co-treatment with vincristine and SAHA. Decreases in pro-survival mitochondrial proteins Bcl-2, Bcl-xl, and Mcl-1 also occurred. A decrease in mitochondrial membrane potential was significantly induced by the combination in a time-dependent manner.
Taking all of the above findings into consideration, when vincristine and SAHA treatments were combined, MOLT-4 cell survival was significantly inhibited, mainly through the induction of M stage arrest and intrinsic and extrinsic apoptosis pathways. Moreover, the selective HDAC6 inhibitor exhibited similar synergism when combined with vincristine. In vivo xenograft animal models produced the same results as those of in vitro models. Pan-HDAC inhibitors influenced different types of HDACs, which caused side effects more than selective HDAC6 inhibitors. However, the most important and practical advantage to choose SAHA for this study is that SAHA has been approved for cancer treatment. Selective HDAC6 inhibitors such as tubastatin A and ACY1215 are only in phase I or phase II clinical trial. The finding of this paper not only provides another choice for clinical treatment but also offers an idea for the development and future application of HDAC6 inhibitors.
Cell lines
The human T cell acute lymphoblastic leukemic cell lines, MOLT-4 and CCRF-CEM, isolated from the relapsed and multiresistant patients, were obtained from Bioresource Collection and Research Center (Taiwan). Cells were maintained in RPMI-1640 medium supplemented with 10 % (v/v) fetal bovine serum (Gibco BRL Life Technologies, Grand Island, NY, USA) and 1 % of a mixture of penicillin (100 U/ml) and streptomycin (100 μg/ml, Biological Industries Ltd., Kibbutz Beit Haemek, Israel). All cells were cultured in an incubator in the presence of 5 % CO 2 at 37°C.
Cell viability assay
Cell viability was verified by MTT assay. Firstly, cells were seeded in a 24-well plate at a density of 4 × 10 5 cells/well in 1 ml culture medium and then treated with various concentrations of vincristine or SAHA alone or a combination of both for 24 and 48 h. After treatment with drugs, 100 μl MTT solution (0.5 mg/ml in phosphate-buffered saline (PBS)) per well was added to the 24-well plate in the dark and the plate was incubated at 37°C. The mitochondrial dehydrogenase of viable cells reduced MTT (yellow) to insoluble formazan dyes (purple). One hour later, the crystal formazan dyes were dissolved in the extraction buffer (0.1 M sodium acetate buffer, 100 μl/well). The absorbance was spectrophotometrically analyzed at 550 nm by an ELISA reader (Packard, Meriden, CT, USA).
Flow cytometry analysis
Evolution of the cell cycle histogram was performed by flow cytometry analysis to detect the changes in DNA content. Cells (1 × 10 6 ) were seeded in a 6-well plate in 2 ml fresh medium and treated with graded concentrations of vincristine, SAHA, or combination for the indicated time. Then, cells were collected, washed with PBS and fixed with 70 % (v/v) ice cold ethanol at −20°C for 30 min. The fixed cells were centrifuged to remove the ethanol, rinsed with PBS, resuspended in 0.1 ml DNA extraction buffer (0.2 M Na 2 HPO 4 -0.1 M citric buffer, pH 7.8) for 20 min, and subsequently stained with 500 μl PI solution (80 μg/ml propidium iodide, 100 μg/ml RNase A, and 1 % Triton X-100 in PBS) for 20 min at room temperature in the dark. Data were analyzed by FACScan Flow Cytometer and CellQuest software (Becton Dickinson).
In vitro tubulin polymerization assay
To determine the microtubule polymerization of the indicated drugs in a cell-free condition, CytoDYNAMIX Screen 03 Kit (Cytoskeleton Inc.) was performed. General tubulin buffer, GTP stock (100 mM), and tubulin protein (10 mg/ml) were all prepared well following the protocol. A 96-well plate was placed in the spectrophotometer to prewarm at 37°C for 30 min before detection. Then preparing the iced tubulin polymerization (TP) buffer, all mentioned processes were needed on the ice. Next, the drugs (2 μl) were added into each Eppendorf included with 85 μl TP buffer. The drugs must include DMSO (the control group), paclitaxel (10 μΜ), and vincristine (10 μΜ). Paclitaxel and vincristine were used as positive controls. Paclitaxel would induce the microtubule polymerization; in contrast, vincristine would depolymerize the microtubules. Finally, 30 μl tubulin proteins was added into the Eppendorf and transferred to the prewarmed 96-well plate. The absorbance was measured by a spectrophotometer and recorded every 1 min for 30 min at 340 nm and 37°C.
Immunofluorescence analysis
Microtubule distribution and morphology were detected by immunofluorescence. Cover slides were placed in the 24-well plate and coated with poly-D-lysine for 1 day at least to enhance the suspension cells attached to the cover slides. Cells were seeded into the 24-well plate (8 × 10 5 cells/well) and treated with vincristine, SAHA, or both drugs for 24 h. The following experiments were performed at room temperature. The cells were fixed with 8 % paraformaldehyde in PBS for 15 min. After washing with PBS for several times, the cells were permeabilized with 0.1 % Triton X-100 in PBS for 10 min. Then, the cells were rinsed with PBS for 10 min three times. For blocking, 3 % BSA in PBS was used. After 1 h, the cells were washed with PBS and incubated with a primary β-tubulin antibody (1:200) for 2 h and FITC-conjugated anti-mouse IgG antibody (1:200) for 2 h. The mounting medium, which contains DAPI stain, was dropped onto the slides, and cover slides were recovered to the slides. Images were detected and captured with the ZEISS LSM 510 META confocal microscope.
Western blot analysis
After the treatment, cells (10 6 cells/ml) were harvested. Whole cell pellets were washed twice with PBS, lysed in lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 % Triton X-100; 1 mM EDTA; 1 mM EGTA) supplemented with protease inhibitors (1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM NaF) for 30 min, and then centrifuged for 30 min at 13,000 rpm at 4°C. Total protein content was quantified by BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equivalent amounts of proteins were separated into diverse percentages by SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were blocked with 5 % nonfat milk in PBS for 1 h at room temperature and incubated with primary antibody in PBST buffer (0.1 % Tween 20 in PBS) at 4°C overnight. After washing the membranes with PBST, blots were developed with the corresponding HRP-conjugated secondary antibody diluted in 0.5 % nonfat milk in PBS for 1 h at room temperature. The membrane was washed frequently with PBST, and finally immunoreactive protein bands were displayed using an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK).
Mitochondrial membrane potential
Rhodamine 123 was used to evaluate mitochondrial membrane potential. Rhodamine 123 is a kind of cationic fluorescent dye, which localizes in the mitochondria. Loss of mitochondrial membrane potential is associated with a lack of rhodamine 123 retention and a decrease of fluorescence intensity. Cells were treated with vincristine, SAHA, or combination for the indicated time. Rhodamine 123 (final concentration 10 μM) was added and incubated for 30 min at 37°C in the dark. Then, cells were harvested and rinsed with PBS. The fluorescence intensity was measured by FACScan Flow Cytometer and CellQuest software (Becton Dickinson).
Tumor xenograft model
A tumor xenograft model was used to estimate the combination effect of vincristine and SAHA in vivo. MOLT-4 cells were implanted (10 7 cells/ml) into severe combined immunodeficiency (SCID) mice. When the average tumor size reached 100 mm 3 , mice were treated with an indicated dosage of vincristine or SAHA alone or a combination of both. Mice were scarified until the average tumor size was larger than 2500 mm 3 . Then, tumors were resected and frozen for the western blot analysis to evaluate the effect of vincristine/SAHA combination in vivo. All animal experiments followed ethical standards, and protocols have been reviewed and approved by the Animal Use and Management Committee of National Taiwan University (IACUC Approval No.: 20100225).
Statistical analysis
All experiments were done independently three times and presented as mean values ± SD, and then assessed by Bonferroni's t test. The animal experiments were determined by the Mann-Whitney test. P < 0.05 was considered statistically significant. | v3-fos-license |
2018-04-10T17:29:19.176Z | 2018-04-10T00:00:00.000 | 4698712 | {
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} | pes2o/s2orc | Food-Derived Hemorphins Cross Intestinal and Blood–Brain Barriers In Vitro
A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood–brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5–60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.
A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood-brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/ MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.
inTrODUcTiOn Hemorphins are a group of opioid peptides encrypted in the beta-chain of hemoglobin, in a conserved region between bovine and human hemoglobin. Hemorphins, from endogenous or food-derived hemoglobin, are found in many different tissues and species (1-3) and many bioactivities have been uncovered for these hemorphins, notably in blood pressure regulation (4)(5)(6) and cognitive functions (7). They were first shown to interact with opioid receptors (ORs) (8,9) that earned them the name "hemorphin, " and explain other activities like intestinal peristalsis (10,11), bladder contraction, inflammation, and pain modulation (12)(13)(14). Our laboratory is interested in the fate of alimentary or digested hemoglobin protein and particularly in the derived bioactive peptides thereof in relation to food intake regulation. We have previously identified five hemorphins from the simulated gastrointestinal digestion of hemoglobin. This simulated gastrointestinal digestion has been validated (15) and is now routinely used to identify and highlight bioactivities of food-derived peptides. These five hemorphins that were produced by and resistant to gastrointestinal digestion have been studied for their effects at the intestinal lumen level in relation to gut hormones synthesis and release and DPP-IV (CD26) regulation (16). Among these five hemorphins (Table 1), three have an N-terminal extension, LLVV-, LVV-, and VV-, one has a C-terminal extension, -QRF, and one has both an N-terminal, VV-and C-terminal -QRF, extension of the tetrapeptide core YPWT that has been demonstrated to bind to OR.
Opioid receptors are involved in many aspects of food intake regulation via central effects (17). Recently, peripheral ORs have also been involved in these regulations and a gut-brain loop has been described with a crucial role of the portal vein mu-OPs (18). It was thus proposed that opioid peptides originating from the digestion and absorption of dietary proteins would interact with the OR located in the portal vein and trigger a gut-brain loop mediating high-protein diet-induced satiety.
Given hemorphins size and evidence from the literature for other exorphins (19), it is likely that the passage of these peptides belongs to the paracellular transport mode involving effects on tight junction (TJ). Moreover, there is some literature implicating opioids in intestinal permeability disorders and TJ regulation. There are known deleterious effects of mu-OR agonists on the gut barrier and immune function in pain-treated patients or drug abusers (20,21). In mice, it was shown that morphine treatment is associated with disruption of TJ organization (22). TJ proteins are an ensemble of protein families that seal the paracellular space between epithelial cells. TJ proteins include transmembrane proteins, such as occludin and claudin families, and scaffolding proteins, such as zonula occludens-1 (ZO-1) and -2 (ZO-2).
Hence, we sought, by using two in vitro models, to demonstrate that food-derived hemorphins, intact or as fragments retaining OR-binding capacity, pass the intestinal and the BBBs. For the IB, Caco-2 was used as a well recognized and widely used model of human intestinal epithelium (23,24). Indeed, according to the Biopharmaceutics Classification System, there is a high correlation between Caco-2 cell permeability coefficients and fractional absorption values in humans (25). Moreover, especially interesting in the present study where the BBB passage has also been tested, the Caco-2 model revealed to be predictive of oral bioactivity and of BBB permeability (26), although another study concluded to a poor correlation between Caco-2 cell data and in vivo BBB transport (27). Several BBB models have been described over the past 40 years and are currently used in different research groups to analyze different aspects of BBB biology and drug targeting. The human brain-like endothelial cells (BLEC) co-culture model recently described (28) was chosen in this study. It expresses TJ and transporters typically observed in brain endothelium, displays most in vivo BBB properties and thus could be used for both mechanistic studies and as a screening tool for CNS-compound permeability studies in human (29,30).
We report here the results of both in vitro intestinal and BBB passage tests, analyzed by LC-MS/MS, of five food-derived hemorphins and preliminary results of hemorphins impact on TJ proteins of the IB.
cell culture
For the cAMP determination assay and the gene expression study, the cell line used was the Caco-2 cell line purchased from Sigma-Aldrich (Steinheim, Germany). Cells were grown at 37°C, 5% CO2 atmosphere in Dulbecco's modified Eagle's Medium (DMEM, 4.5 g L −1 glucose) supplemented with 10% FBS, 2 mM l-glutamine, 100 U mL −1 penicillin, and 100 µg mL −1 streptomycin (complete DMEM) until use. Caco-2 from passages 34-37 were used. For the transport studies, the cell lines used and the culture protocols conditions are described in detail in dedicated subsections below.
Transport studies
In Vitro Human IB Model The human intestinal cell line used for the transport experiments was the Caco-2/TC7 clone, a gift from Dr. Monique Rousset (UMRS 872, INSERM, Paris). Cells were routinely grown as previously described (31). For the experiment, they were seeded at the density of 60 × 10 3 cells cm −2 into cell culture inserts in 6-well culture plates and grown for 21 days. The day of the trans-intestinal transport test, growth media were removed, and cells were first rinsed with the transport medium, HBSS-Hepes buffer supplemented with CaCl2 (2.5 mM final) and MgCl2 (0.5 mM final) in the apical (1 mL) and basolateral chambers (2.5 mL), then pre-incubated for 30 min with the transport medium. All incubations were performed at 37°C, 10% CO2 atmosphere. To control the integrity of the Caco-2/TC7 cell barrier, the paracellular transport marker lucifer yellow (LY, 100 µM final) was applied apically into each well and its transport into the basolateral chamber was monitored by spectrofluorescence. Each hemorphin was diluted for a final concentration of 5 mM in transport medium-LY and applied to the apical chamber at t0. In parallel, control wells were incubated with transport medium-LY only and all conditions were tested in triplicate. At t5, 15, 30, and 60 min, 100 µL samples were collected in the basal chamber and replaced by the same volume of transport medium. At t120 min, all remaining media in basal and apical chambers were collected and all samples were stored at −80°C. The rate of LY transport was determined by fluorescence readings (ex 485/em 530 nm) at each time point on the spectrofluorometer Safas Xenius XC (Safas Monaco, Monaco, France) and calculating the apparent permeability coefficient (Papp) as follows: where ΔQ is the change in LY concentration in the basal chamber (μM); Δt is the transport duration (s); Vb is the volume in the basal chamber (mL); A is the surface area of the insert/transwell membrane (cm 2 ); and C0 is the initial concentration of LY applied to the apical chamber (μM). Papp is measured in cm s −1 and the Caco-2/TC7 cell barrier for each sample was considered intact if Papp <1 × 10 −6 cm s −1 .
In Vitro Human BBB Model: BLEC Model
The brain-like endothelial cell model has previously been described in detail (28). All the sample donors had given their written informed consent, in compliance with French legislation and the 2013 version of the Declaration of Helsinki. The sample collection was approved by the local investigational review board (Bethune Maternity Hospital Béthune, France). Briefly, this human model consists in isolating CD34+ cells from umbilical cord blood. These cells are then cultured in endothelial cell medium supplemented with 20% FCS for 15-20 days. After this period, cells are differentiated into endothelial cells. After a trypsinization step, they are seeded on matrigel coated-transwell inserts in 12-well culture plates, and subsequently cultured with brain pericytes, isolated from bovine brain capillaries. After 6 days of co-culture, human endothelial cells acquire the BBB phenotype, and are then named BLECs.
The day of the transport test, the same procedure as for the intestinal passage assay was applied except that the transwell inserts were transferred to a new 12-well culture plate at each time point, 30, 60, 90, and 120 min. All incubations were performed at 37°C, 5% CO2 atmosphere with slight agitations. The transport medium consisted in Ringer-Hepes solution (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2⋅6H2O, 6 mM NaHCO3, 5 mM HEPES, pH 7.4). Each hemorphin was diluted for a final concentration of 100 µM in transport medium-LY and applied to the apical chamber (0.5 mL; 1.5 mL transport medium in basal chamber) at t0. A test was performed without cells to insure that the filter membrane was not preventing the passage of the peptides. At each time point, 200 µL samples were collected in the basal chamber for the LY assay (measured with Synergy H1, BioTek) and the remaining 1.3 mL were collected at 30 min and frozen at −80°C for further LC-MS/MS analyses.
lc-Ms/Ms
LC-MS/MS analysis was performed on a UFLC-XR device (Shimadzu, Kyoto, Japan) coupled to a QTRAP ® 5500 MS/MS hybrid system triple quadrupole/linear ion trap mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a Turbo VTM ion source. Instrument control, data acquisition, and processing were performed using Analyst 1.5.2 software. The reverse phase liquid chromatography separation was carried out on a Kromasil C18 column (100 × 2.1 mm, 3.5 µm) with guard cartridge from AIT-France (Houilles, France). The injection volume was 2 µL. Elution was performed at a flow rate of 200 µL min −1 with waterformic acid 0.1% as eluent A and acetonitrile-formic acid 0.1% as eluent B. The injection duty cycle was 10 min, starting with a 1 min plateau with 25% B, followed by a linear gradient from 25 B to 60% B in 4 min, a 1 min plateau with 60% B, 1 min linear gradient from 60 to 25% B, and 3 min at 25% B to recondition the column. MS analysis was carried out in positive ionization mode. The ion source parameters were optimized and set as follows: ion spray voltage, 5,500 V; nebulizer gas (air) and curtain gas (nitrogen) flows, 50 and 25 psi, respectively; source temperature, 550°C with the auxiliary gas flow (air) set at 50 psi; declustering potential (DP), 100 V; and collision cell exit potential, 25 V. The mass spectrometer was operated at a unit resolution for both Q1 and Q3 with a dwell time of 90 ms in each transition.
The presence of each intact hemorphin and potential fragments were tracked by a combination of multiple reaction monitoring (MRM) and pseudo-MRM transitions. V-H4 and N-extended-H3 fragments were not searched because they probably present poor OR ligand capacity (32) and they have not been reported as present in body fluids or tissues.
radiobinding
The potential binding of each opioid peptide/hemorphin on OPs was assessed in a radiobinding competition test with H3-naloxone as the tritiated-specific ligand on rat brain membrane preparation. The protocol used follows the one described in Garreau et al. (9) with some modifications. Briefly, rat brains were homogenized in 50 mM Tris-HCl added with 240 mM sucrose, 5 mM MgCl2, and 2 mM EDTA and centrifuged first at 1,000 g for 5 min at 4°C. Supernatants were then centrifuged at 30 × 10 3 g for 30 min at 4°C and the resulting pellets were solubilized in the homogenization buffer. Protein dosage was performed with the Sigma QuantiPro BCA Assay Kit. Serial dilutions of each peptide or naloxone as positive control were realized in 50 mM Tris-HCl buffer pH 7.4 supplemented with 2% bovine serum albumin and incubated in 0.9 mg mL −1 of rat brain membrane preparation added with the proteases inhibitors bestatin (10 µM final) and thiorphan (0.1 µM final), and 1 nM final H3-naloxone for 30 min at 25°C. Nonspecific binding was determined by adding an excess of naloxone (5 × 10 −6 M final) instead of the peptide or naloxone dilutions. All conditions were run in duplicates. At the end of the incubation, separation of bound and unbound samples was realized by vacuum filtration through glass microfiber GF/B filters. Filters were inserted into scintillation vials, added with 3 mL Optiphase HiSafe 2 scintillation liquid, and radioactivity was counted on a beta-counter (Hidex 300 SL, Sciencetec, Villebon-sur-Yvette, France). Non-specific binding was subtracted from all values and specific binding was expressed as a percentage of total-specific binding (dpm). Competition binding curves were plotted in GraphPad Prism v6.01 and ED50 were determined by non-linear regression analysis.
caMP Determination assay
The effect of the five opioid peptides/hemorphins on the cAMP production was assessed in vitro in Caco-2 cells. Each hemorphin was applied on Caco-2 cells stimulated or not by FK, an activator of the cAMP pathway, in order to evaluate the ability of the peptides to decrease intracellular cAMP elevation induced by FK. Cells were seeded at 1 × 10 5 cells/well in 24-well plates and grown for 7 days in complete culture medium. The medium was renewed 24 h before the test and replaced by the incubation medium, DMEM supplemented with 1% l-glutamine (DMEMl-glu) 30 min prior to the test start. Each hemorphin was applied at 50, 500, or 5,000 µM (FK-stimulated condition) and 50 or 500 µM (non-stimulated condition) for 15 min at 37°C. The specific OR agonist DAMGO (1 and 100 nM) and the OR antagonist naloxone (1 µM) were tested in the same conditions. To verify that the hemorphin-induced effect on cAMP production was mediated through the activation of OPs, cells were pre-incubated for 30 min with 1 µM naloxone in DMEM-l-glu prior to the incubation with each opioid peptide (500 µM) or DAMGO (100 nM) in DMEM-l-Glu-FK in the same conditions as previously. All conditions were run in triplicate. At the end of the 15 min incubation, cells were immediately rinsed three times with PBS and plates were stored at −80°C until intracellular cAMP levels determination with the Mouse/Rat cAMP Parameter Assay Kit according to the manufacturer's guidelines. cAMP levels were normalized by the total protein concentration determined by the Pierce BCA Protein Assay Kit for each sample and expressed as a percentage of the reference condition FK only.
gene expression analysis
The impact of opioid peptides on claudin-4 gene expression in Caco-2 cells was evaluated by qPCR. Caco-2 were seeded at 80 × 10 3 cells cm −2 in cell culture inserts into 6-well culture plates and first cultured in complete DMEM for 3 weeks with media changes (basal and apical) every 2-3 days. 100 µM of each hemorphin in DMEM-l-glu or incubation buffer only were applied in triplicate in the apical compartment for 24 h at 37°C, 5% CO2. At the end of the incubation time, cells were rinsed two times in PBS and scrapped in TRI Reagent. Total RNA was further isolated and processed according to the protocol described in Caron et al. (15). qPCR analyses were performed with specific oligonucleotides for claudin-4: forward (F) 5′-CCACTCGGACAACTTCCCAA-3′ and reverse (R) 5′-ACT TCCGTCCCTCCCCAATA-3′, and peptidylprolyl isomerase (PpiA): (F) 5′-TGCTGACTGTGGACAACTCG-3′ and (R) 5′-TG CAGCGAGAGCACAAAGAT-3′ and the Power SYBR Green PCR Master Mix on a StepOne™ Plus system (Applied BioSystems, Life Technologies). mRNA fold induction was calculated according to the 2 −ΔΔCt method (ΔΔCt method, Applied Biosystems User Bull. #2 Dec. 97) using PpiA as internal control (reference gene) and expressed as a percentage of the control group induction.
statistical analysis
Data presented are mean ± SD. Statistical analysis was conducted in GraphPad prism v6.1. One-way ANOVA was applied with Tukey or Dunnett's test for post hoc analyses. For comparison of the cAMP levels, a two-way ANOVA was used to consider the different concentrations used, followed by a Dunnett's test for multiple comparisons. To assess the effect of the naloxone treatment in the complementary cAMP assay, a two-way ANOVA was used, and each hemorphin effect was compared to its corresponding naloxone treatment by a Sidak test. The Dunnett's post hoc test was used for DAMGO comparisons. Significance level was set at p < 0.05.
resUlTs iB Passage
In first analysis, the samples collected at each time point in the basal compartment below the Caco-2 cells layer modeling the IB were analyzed by LC-MS/MS with MRM to detect the whole peptide sequences. The analysis revealed that LVV-and VV-H4 as well as H7 were clearly able to cross intact the cell monolayer, whereas VV-H7 was found in traces and LLVV-H4 was not detected (Figure 1). H7 displayed the most rapid passage as it appeared basally as soon as 5 min incubation and its basal concentration increased faster. LVV-and VV-H4 appeared in the 60 min samples and behaved similarly. All peptides except the LLVV-H4 sequence were still found intact and abundant in the apical side after 120 min incubation (Figure 1).
Nevertheless, in addition to the intact peptides, potential fragments all comprising the tripeptide YPW were then searched in each basal sample by LC-MS/MS with pseudo-MRM. Expectedly, the peptides with an N-terminal extension were hydrolyzed and gave the other peptide sequences under investigation (e.g., in the well where LLVV-H4 was applied, LVV-and VV-H4 were found as well). However, although LVVand VV-H4 were able to cross the cell barrier when applied alone, they were not found in the basal side of the LLVV-H4 wells, where they were produced apically. Interestingly, for all tested peptides the most abundant fragment identified is the tetrapeptide core YPWT (Figure 2).
Results from the LY passage monitoring indicated that the integrity of the cell monolayer was not altered throughout the 120 min incubation (Figure 3). Papp values calculated were all below 1 × 10 −6 cm s −1 which is a commonly accepted threshold for barrier integrity. However, though not statistically significantly different from the control value (as assessed by Dunnett's post hoc analysis), peptides LY Papp values seemed to correlate with the ability of the hemorphins to cross the cell barrier. Indeed, peptides with Papp(LY) values similar or above the control one showed the most passage. This was the most striking for H7 for which Papp(LY) was almost six times higher than control one (7.632e−007 ± 6.504e−007 > 1.310e−007 ± 6.308e−008 cm s −1 , p = 0.0503), whereas LLVV-H4 and VV-H7 which did not or barely crossed the barrier displayed Papp(LY) decreased compared to control (non-significant).
BBB Passage
The same LC-MS/MS analyses were applied on the samples collected during the test of passage of the hemorphins across the co-culture layer modeling the BBB. The analyses revealed that all peptides were able to cross intact this cell barrier (Figure 4). In comparison to the IB model, the transport was faster and more efficient, since all hemorphins were detected basally already at 30 min of incubation and the passage occurred despite a much lower initial apical concentration, i.e., 100 µM in the BBB model vs 5 mM in the IB model.
The search for the peptides fragments yielded partially the same results. The same fragments were identified except the fragment YPWTQ which was only detected in the basal VV-H7 and H7 samples of the BBB co-culture model. Moreover, the fragment YPWT was also found basally in all samples. In contrast to the results of the IB test, YPWT was not the main fragment and longer ones were also found in the basal compartment.
With regards to the LY permeability, LLVV-H4 and VV-H4 significantly increased Papp(LY) compared to control (Dunnett's post hoc analysis: 133.2 ± 16.44%, p = 0.0446 and 142.2 ± 26.71%, p = 0.0106, respectively) without affecting the cell barrier integrity ( Figure 5). This was reflected by the Papp values that were all below 1 × 10 −6 cm s −1 which is a commonly accepted threshold for barrier integrity.
OP Binding
The determination of the OP-binding capacity of each hemorphin was performed on rat brain membrane preparation. The binding capacity of peptide LLVV-H4 could not be determined in the same conditions as for the other peptides, since its solubility was poor in the incubation buffer at concentrations higher than 5 × 10 −5 M. Especially because of the method of separation of the bound and unbound ligands on glass microfiber filters; we suspected that the peptide dilution clogged the filter. Nevertheless, at least for the 10 −10 to 10 −4 M range (data not shown) LLVV-H4 was not able to compete with the specific OR tritiated antagonist. In contrast, all other tested hemorphins were able to decrease the fixation of the 3 H-naloxone on OPs in a dose-dependent manner. Inhibition of the antagonist binding on OR was efficient in the 5 × 10 −7 to 5 × 10 −3 M range for H7 and in the 5 × 10 −5 to 5 × 10 −3 M range for the other hemorphins (Figure 6). Thus, except for H7, the binding curves display a steep decrease within 2 log units of hemorphins concentrations suggesting a competition for a single binding site. H7 binding curve displayed a shallower decrease on nearly 4 log units that could be indicative of a competition for more than one binding site. As illustrated by the competitive binding curve and the determination of IC50, the affinity of the hemorphins ranked as follows from the most affinity to the least: H7 > VV-H7 > LVV-H4 > VV-H4 (Figure 6 inset). The presence of the N-terminal extension impaired or reduced binding (compared to the C-terminal only extension), since peptides with an N-terminal extension only displayed the highest IC50 and the peptide VV-H7 with both N-and C-terminal extensions presented an intermediary IC50.
caMP Pathway activation
In order to precise the OR agonist, antagonist or mixed nature of the hemorphins, their effect on the accumulation of intracellular cAMP in Caco-2 cells upon stimulation with an activator of adenylate cyclase, FK, was investigated. OR agonists have a known inhibitory action on adenylate cyclase with a consecutive reduction of intracellular cAMP formation (33). Measurement FigUre 4 | Passage of the five hemorphins intact or their fragments across the brain-like endothelial cell blood-brain barrier (BLEC) model. The passage of the five hemorphins was tested in the BLEC model (co-culture of hematopoietic CD34+-derived endothelial cells with bovine pericytes). Each peptide was separately applied at 100 µM in the apical chamber ("blood" side) of a transwell and the presence of the intact peptides and potential fragments thereof in the basal chamber ("brain" side) was investigated by a combination of LC-MS/MS with multiple reaction monitoring (MRM) and pseudo-MRM analyses after 30 min incubation. Data are expressed as mean ± SD (n = 3). Notably, H7 was able to inhibit FK-induced cAMP elevation (of about 30%) at the two other lower concentrations of 500 and 50 µM (72.38 ± 13.25%, p = 0.0117 and 70.96 ± 14.58% of FK levels, p = 0.0075, respectively). Thus, 5 mM of LVV-H4 and H7 produced a decrease in the same size range, about 50%, as following the specific MOR agonist DAMGO incubation at 1 and 100 nM (50.30 ± 9.71%, p = 0.0001 and 55.56 ± 24.20% of FK levels, p = 0.0005, respectively). DAMGO did not present dosedependent effect either. Very surprisingly, naloxone, a specific, but non-selective OR antagonist did also significantly decrease FK-induced cAMP elevation (68.42 ± 6.87% of FK levels, p = 0.0394) when incubated alone at 1 µM with Caco-2 cells.
To confirm that the effect of hemorphins on cAMP production involves OR, we treated FK-stimulated cells with each hemorphin at 500 µM separately with or without naloxone at 1 µM. Unexpectedly, the naloxone treatment, when concomitant to hemorphins incubation, promoted a dramatically significant increase in FK-induced cAMP elevation (Figure 7B). It potentiated the FK effect on cAMP formation by at least 50% for each tested hemorphin as well as for DAMGO, and 38% for LVV-H4. Sidak post hoc analysis of each hemorphin or DAMGO treatment compared to its corresponding hemorphin or DAMGO and naloxone co-treatment yielded a statistically significant increase of cAMP of 93% for VV-H4 (p = 0.0006), 74% for VV-H7 (p = 0.0058), 93% for H7 (p = 0.0006), and 108% for DAMGO (p = 0.0184).
gene expression analysis (caco-2 Model of iB)
The potential effect of the five hemorphins on the TJ protein claudin-4 gene expression has been investigated in vitro on Caco-2 cells first grown for 3 weeks on cell culture membranes for full differentiation as epithelium. Quantitative PCR analyses showed that all hemorphins except LVV-H4 induced a significant decrease in claudin-4 mRNA levels of almost 50% or more of of intracellular cAMP under different concentrations of each hemorphin in FK-stimulated cells did not show a clear dose effect. The highest tested concentration of 5 mM did induce a significant decrease in FK-stimulated cAMP concentration compared to control levels for all sequences, except LLVV-H4, and is indicative of an agonist-like behavior of the hemorphins (Figure 7A). The highest reductions were achieved by LVV-H4, 50% (49.57 ± 14.79% of FK levels, p < 0.001) and H7, 47% (53.24 ± 17.41% of FK levels, p < 0.001), while VV-H4 induced a reduction of 35% (64.56 ± 7.67% of FK levels, p = 0.0009) and VV-H7 of 28% (72.01 ± 26.42% of FK levels, p = 0.0104).
FigUre 6 | Determination of hemorphins binding to rat brain membranes opioid receptors (OPs). The ability of the hemorphins/opioid peptides to bind to OPs was assessed in a radiobinding assay in vitro by competition with H3-naloxone (non-selective OP antagonist) on rat brain membrane preparation. Six concentrations ranging from 5 × 10 −8 to 5 × 10 −3 M were tested in duplicates and the results were expressed as a percentage of total binding without opioid peptides. Inset: dose-response curves were analyzed in GraphPad Prism 6 by non-linear regression from which IC50 were interpolated. Data presented are mean (average of two separate assays) ± SD.
DiscUssiOn
This study is a first report of the passage of food-derived hemorphins, i.e., exogenous hemorphins, through the intestinal as well as the BBB in vitro ( Table 1). Information on the in vitro absorption of alimentary proteins or of well-defined peptide sequences derived thereof has been mainly garnered from tests with milk proteins (34)(35)(36), much less from tests with whey proteins (19,37), or lately from egg albumin (38) but this information was unknown for hemorphins. The tested peptide the closest in terms of structure to hemorphins, is endomorphine-1 for which a low permeability through a caco-2 cell monolayer was reported (39). We found that all hemorphins except the LLVV-H4 sequence could cross intact the Caco-2 cell monolayer. A possible explanation for the failure of passage of this sequence is brush-border enzymes degradation. The presence of multiple fragments in both apical and basal compartments may suggest that the peptides interacted with brush-border, basolateral, or intracellular peptidases (Figure 2). Indeed, LVV-H4, VV-H4, and H4 fragments and no LLVV-H4 detection in the apical compartment at the end of the experiment could reflect that LLVV-H4 was hydrolyzed to such an extent that no passage to the basal compartment occurred. It is recognized that the primary factor limiting transepithelial transport of intact peptides and its bioavailability is the susceptibility to cellular peptidases (40). This can reflect that the transport is probably concentration-dependent, since this was not the case in the LVV-H4 wells, where the fragment VV-H4 was detected both in apical and basal compartments. This does not exclude that this latter fragment is produced from LVV-H4 in apical and basal. However, the Caco-2 model is acknowledged as quite a stringent test for in vitro IB passage, meaning that results obtained in this test rather underestimate in vivo human IB passage (41).
Regarding the passage of the BBB, though applied at a much lower concentration than in the IB/Caco-2 test (100 µM vs control levels (Figure 8). This effect was more pronounced in cells treated with hemorphins presenting a C-terminal extension than those harboring an N-terminal extension. The hemorphins presenting a C-terminal extension, VV-H7, and H7, reduced mRNA levels to 30.03 ± 10.99% (p = 0.0009) and 28.31 ± 2.52% (p = 0.0008) of control levels, respectively, VV-H4 to a similar extent with 24.74 ± 13.67% (p = 0.0005) and LVV-H4 to a lesser extent with 52.74 ± 19.37% (p = 0.0166) of control levels. The ability of the hemorphins/ opioid peptides to act on the cAMP pathway was evaluated in vitro. Caco-2 cells were incubated with or without increasing concentrations of each hemorphin, DAMGO, a known specific OP agonist and naloxone, a known OP antagonist, in FK-supplemented medium for 15 min at 37°C. Intracellular cAMP levels were determined by ELISA, normalized by the total protein concentration and expressed as a percentage of the reference group FK. Data presented are mean ± SD (average of two assays performed in triplicate; n = 6). *Statistically different from control; *p < 0.05, **p < 0.01, ***p < 0.005. (B) To determine whether the effect of the hemorphins was mediated through OPs, the same assay was performed with or without naloxone (1 µM) in the same incubation medium. Data presented are mean ± SD (n = 3). *Statistically different from control; **p < 0.01, ***p < 0.005. seeded on the filter of cell culture insert and cultured for 3 weeks until full differentiation as in the IB model. They were then incubated with 100 µM of each hemorphin/ opioid peptide in culture medium for 24 h. Total RNAs were isolated and processed for quantitative real-time PCR analyses. Claudin-4 transcript levels were normalized by reference gene peptidylprolyl isomerase transcript levels and expressed as % of control levels (without opioid peptides). Experiments were run in triplicate and data represented are mean ± SD. *Statistically different from control; *p < 0.05, ***p < 0.005. (42). However, a study comparing 18 peptides permeability through the BBB in rats in relation to their amino-acid structure and different physico-chemical properties (43) reported that N-terminal tyrosine constitutes a brake to BBB passage, yet H7 which presents such residue was clearly able to cross the in vitro BBB model in this study.
In addition, we show that fragments of the tested hemorphins were also able to pass the Caco-2 epithelium as well as the modeled BBB ( Table 1) and that is interesting inasmuch as these fragments retain a certain bioactivity, like the OP-binding capacity. Strikingly, the most frequent hemorphin fragment recovered is the core tetrapeptide YPWT (H4), common to all tested hemorphins. Three pieces of evidence would indicate that YPWT is produced in the apical compartment by hydrolysis of the hemorphins and is able to cross the cell barrier. First, the fragment YPWT was identified in the basal samples as soon as 5 min incubation either before the parent peptide was even detected (LVV-and VV-YPWT) or at the same time (VV-YPWT-QRF and YPWT-QRF). Second, for LLVV-H4 wells, the parent peptide was seemingly not able to cross the cell barrier and for VV-H7, only traces were detected basally. Finally, 120 min apical amounts of YPWT were notably lower than basal ones. H4 was the first hemoglobin-derived opioid peptide identified as such (8) and also has the feature to be very stable in plasma.
In this regard, we verified the OR-binding capacity of the hemorphins synthesized for this study on rat brain membrane preparation. Our results add the sequence LVV-H4 as OR ligand, which, to the best of our knowledge, had not been tested for its opioid activity. Also, our results are in agreement with the findings of Zhao et al. (32) showing that N-extensions on the H4 core tend to disfavor OP-binding, whereas C-extensions tend to increase it.
We further sought to characterize the agonist/antagonist nature of the hemorphins in the cAMP assay commonly used for OR-ligand investigation. Multiple studies realized mostly with mu-OR agonists in different cell lines found that OR agonism resulted in a decrease of intracellular cAMP formation (33,39,44). Here, however, unexpected results were found with the naloxone treatment which is a well-known non-selective OR antagonist. Naloxone alone yielded decreased cAMP levels in FK-stimulated Caco-2 cells and potentiated cAMP increase when applied with each hemorphin (Figure 7). A comparable result was reported with naloxone in a different cell line as Fukuda et al. (45) showed that naloxone can have a partial agonist activity in mu-and kappa-transfected CHO-cells. Also, H4 was found as agonist in the GPI bioassay, but antagonist when applied in the ileum from a morphine-tolerant rat (i.e., after a morphine pre-treatment) (11). Hence, our results regarding the effect of hemorphins on the cAMP pathway in Caco-2 cells are compatible with an interaction with OR, but may also be indicative of the involvement of other G-protein-coupled receptors. Considering that hemorphins and naloxone effects separately inhibited FK-induced cAMP elevation, these results suggest a synergistic effect of hemorphins and naloxone on the cAMP pathway. Although performance of the cAMP assay in Caco-2 cells is not classical, this choice was justified by the use of these cells in the IB model and would thus allow us to infer some evidence regarding the underlying mechanisms of the hemorphins effects on TJs.
Indeed, hemorphins were suspected of passing the IB epithelium by interaction with the TJ. There is some evidence that some substances are able to alter intestinal permeability via their action on TJ proteins (40,46,47). TJ may be regulated by phosphorylation that promotes or decreases permeability. For instance, in low resistance cells the TJ protein ZO-1 is significantly more phosphorylated than in high resistance monolayers (48). Also, it has been shown that the phosphorylation of claudin-4 enhanced paracellular permeability in HT29 colon cells (49). Hence, our results support the potential action of hemorphins via OR and the cAMP pathway to modulate claudin and other TJ proteins at least in intestinal epithelial cells.
However, hemorphins could also have a long-term effect on TJ proteins that is related to the implication of claudins in intestinal pathologies when their expression or localization is altered (50). We show here that concentrations as low as 100 µM of each hemorphin could decrease the gene expression of claudin-4 in intestinal Caco-2 cells after 24 h of contact (Figure 8).
The digestion of dietary proteins produces peptides that have biological activities, here hemorphins that present OP-binding capacity [see also their intestinal luminal effects in Domenger et al. (16)]. These biopeptides, that resist digestion, also proved to be able to cross the intestinal and BBBs in in vitro conditions, intact, and as fragments that retain opioid-binding capacity. As such they could interact with peripheral and central OPs and participate in appetite and food intake regulation. Further studies will also specify the mechanisms of passage of these hemorphins, especially their interaction with TJ proteins and their impact on barrier permeability. acKnOWleDgMenTs This work was supported by a grant of the Nord-Pas-de-Calais Region: "2nd appel à projet, Programme projets émergents. " It has also been carried out in the framework of Alibiotech project which is financed by the European Union, the French State, and the French Region of Hauts-de-France. The experiments were partly performed at IUT A which is gratefully acknowledged. | v3-fos-license |
2020-05-13T13:03:51.344Z | 2020-05-01T00:00:00.000 | 218598678 | {
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} | pes2o/s2orc | Flexibility and Preorganization of Fluorescent Nucleobase-Pyrene Conjugates Control DNA and RNA Recognition
We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase–fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase–fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson–Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.
Introduction
Over the past decades, fluorescent base analogues have received significantly increasing attention [1][2][3][4][5]. They can replace the natural bases in nucleic acids, and give them emissive properties that are often highly responsive to their immediate surroundings, and they have been used to monitor local environment or environmental changes upon nucleic acid conformational rearrangements [2,4,[6][7][8][9]. A few, on the other hand, have emissive properties that are virtually insensitive or have a low responsiveness to their local environment and hence, for example, are useful as bright nucleic acid labels or as donor and acceptor of FRET-pairs [1,5,[10][11][12]. One popular strategy to improve the emissive properties of fluorescent base analogues is to increase conjugation by extending the aromatic ring system of their natural counterparts. This often results in increased aromatic stacking-interactions with the neighboring nucleobases, which in turn is sometimes needed to compensate for a slightly decreased hydrogen-bonding ability. Recently, a family of quadracyclic fluorescent adenine analogues was developed [13][14][15], of which the qAN-family, derivatives with a nitrogen atom replacing one of the carbons in the outer ring [14], turned out to be highly fluorescent. These four-ring-system adenine Molecules 2020, 25, 2188 2 of 18 analogues preserve the hydrogen-bonding capacity but contain two additional ring systems that are facing the major groove of B-form DNA. The most promising member of these derivatives, qAN1, has been reported to be an excellent fluorescent base analogue inside DNA as well as a base-base FRET-donor [16]. qAN1 is a typical example of an enlarged aromatic system that compared to its natural counterpart, adenine, has an increased aromatic stacking-interaction inside base-stacks, giving rise to increased average melting temperature of duplexes containing qAN1 in place of A [16]. With the previously reported increased aromatic stacking and promising emissive properties we hypothesized that qAN1 could be an interesting DNA-intercalator in itself or as part of homo-or heterodimer intercalator complexes that could report on DNA binding events via a fluorescence response.
Pyrene derivatives are sensitive fluorescent probes widely used for the characterization of micro-heterogeneous systems [17][18][19] because of their long emission lifetime (>100 ns) and the ability to form excimers, as well as their pronounced hydrophobicity. Moreover, pyrene can only form a limited number of typical interactions with DNA/RNA: Aromatic stacking intercalation into DNA/RNA, binding within the DNA minor groove via a combination of hydrophobic and edge-to-face aromatic interactions, or by forming pyrene excimer within the DNA minor groove or RNA major groove [20]. Pyrene is also prone to form exciplex in combination with other chromophores. For instance, we recently reported the formation of a pyrene-quinoline exciplex with an emission maximum that was 60 nm bathochromically shifted compared to the common pyrene emission [21].
As a result of these recent finding, we decided to design hybrid compounds that combine two fluorophores; the fluorescent nucleobase analogue qAN1 and pyrene. Although both fluorophores are characterized by similar excitation and emission wavelength regimes, they significantly differ in hydrophobicity and in particular only qAN1 has the ability to form H-bonds with the target biomolecule (DNA or RNA).
In the design of a linker connecting the two fluorophores we opted for the preparation of a new amino acid analogue of qAN1 including a triazole unit (qAN1-AA, Scheme 1), coupling it with either a short and rigid pyrene analogue (qAN1-pyr1) or a longer and more flexible one (qAN1-pyr2). We expected the difference in linker length and flexibility to have an influence on the aromatic stacking interactions between the two large aromatic systems, pyrene and qAN1. Such aromatic interactions could result in intra-or inter-molecular stacked structures, formed either spontaneously in aqueous solution or upon binding to the particular DNA or RNA target of interest. Both types of interactions events could eventually result in a highly selective fluorimetric response to the DNA/RNA target.
Molecules 2020, 25, x FOR PEER REVIEW 2 of 20 to compensate for a slightly decreased hydrogen-bonding ability. Recently, a family of quadracyclic fluorescent adenine analogues was developed [13][14][15], of which the qAN-family, derivatives with a nitrogen atom replacing one of the carbons in the outer ring [Error! Bookmark not defined.], turned out to be highly fluorescent. These four-ring-system adenine analogues preserve the hydrogenbonding capacity but contain two additional ring systems that are facing the major groove of B-form DNA. The most promising member of these derivatives, qAN1, has been reported to be an excellent fluorescent base analogue inside DNA as well as a base-base FRET-donor [16]. qAN1 is a typical example of an enlarged aromatic system that compared to its natural counterpart, adenine, has an increased aromatic stacking-interaction inside base-stacks, giving rise to increased average melting temperature of duplexes containing qAN1 in place of A [Error! Bookmark not defined.]. With the previously reported increased aromatic stacking and promising emissive properties we hypothesized that qAN1 could be an interesting DNA-intercalator in itself or as part of homo-or heterodimer intercalator complexes that could report on DNA binding events via a fluorescence response. Pyrene derivatives are sensitive fluorescent probes widely used for the characterization of micro-heterogeneous systems [17][18][19] because of their long emission lifetime (˃100 ns) and the ability to form excimers, as well as their pronounced hydrophobicity. Moreover, pyrene can only form a limited number of typical interactions with DNA/RNA: Aromatic stacking intercalation into DNA/RNA, binding within the DNA minor groove via a combination of hydrophobic and edge-toface aromatic interactions, or by forming pyrene excimer within the DNA minor groove or RNA major groove [20]. Pyrene is also prone to form exciplex in combination with other chromophores. For instance, we recently reported the formation of a pyrene-quinoline exciplex with an emission maximum that was 60 nm bathochromically shifted compared to the common pyrene emission [21].
As a result of these recent finding, we decided to design hybrid compounds that combine two fluorophores; the fluorescent nucleobase analogue qAN1 and pyrene. Although both fluorophores are characterized by similar excitation and emission wavelength regimes, they significantly differ in hydrophobicity and in particular only qAN1 has the ability to form H-bonds with the target biomolecule (DNA or RNA).
In the design of a linker connecting the two fluorophores we opted for the preparation of a new amino acid analogue of qAN1 including a triazole unit (qAN1-AA, Scheme 1), coupling it with either a short and rigid pyrene analogue (qAN1-pyr1) or a longer and more flexible one (qAN1-pyr2). We expected the difference in linker length and flexibility to have an influence on the aromatic stacking interactions between the two large aromatic systems, pyrene and qAN1. Such aromatic interactions could result in intra-or inter-molecular stacked structures, formed either spontaneously in aqueous solution or upon binding to the particular DNA or RNA target of interest. Both types of interactions events could eventually result in a highly selective fluorimetric response to the DNA/RNA target. Novel qAN1-amino acid qAN1-AA and pyrene conjugates qAN1-pyr1 (shorter, more rigid linker) and qAN1-pyr2 (longer, more flexible linker).
Our synthetic approach included the preparation of a novel propargylated qAN1, and its application with an amino acid azido derivative, which, by the assistance of CuAAC "click" chemistry, provided the desired triazole-linked qAN1-amino acid conjugate.
Our synthetic approach included the preparation of a novel propargylated qAN1, and its application with an amino acid azido derivative, which, by the assistance of CuAAC "click" chemistry, provided the desired triazole-linked qAN1-amino acid conjugate.
For this purpose, compound 3 was treated with HCl/methanol to remove the acid-labile Boc protecting group and simultaneously esterify the carboxylic acid, affording the methyl ester 6 in quantitative yield. Using standard HCTU/HOBt coupling conditions with triethylamine in DMF [42] The reaction of qAN1 with propargyl bromide in DMF with K 2 CO 3 gave propargylated qAN1 (4) in 95% yield (Scheme 3). Alkyne 4 was coupled to azide 3 using standard CuAAC conditions in anhydrous DMF and DIPEA at room temperature overnight, resulting in conjugate 5 in good yield. Compound 5 was deprotected with TFA/CH 2 Cl 2 to afford the target triazole linked qAN1-amino acid conjugate qAN1-AA in quantitative yield.
Our synthetic approach included the preparation of a novel propargylated qAN1, and its application with an amino acid azido derivative, which, by the assistance of CuAAC "click" chemistry, provided the desired triazole-linked qAN1-amino acid conjugate.
For this purpose, compound 3 was treated with HCl/methanol to remove the acid-labile Boc protecting group and simultaneously esterify the carboxylic acid, affording the methyl ester 6 in quantitative yield. Using standard HCTU/HOBt coupling conditions with triethylamine in DMF [42] Scheme 3. Synthesis of qAN1-amino acid conjugate qAN1-AA.
The same "click" protocol was used for the synthesis of the qAN1-amino acid-pyrene conjugates qAN1-pyr1 and qAN1-pyr2 (Scheme 4).
For this purpose, compound 3 was treated with HCl/methanol to remove the acid-labile Boc protecting group and simultaneously esterify the carboxylic acid, affording the methyl ester 6 in quantitative yield. Using standard HCTU/HOBt coupling conditions with triethylamine in DMF [42] the derivative 6 was reacted with 1-pyrenecarboxylic or 1-pyrenebutyric acid, to give azido-pyrene ligands 7 (87%) and 8 (92%), respectively.
The protected qAN1-amino acid-pyrene conjugate 9 was readily synthesized from propargylated qAN1 4 and azido ligand 8 through CuAAC "click" conditions employing CuI and DIPEA in dichloromethane. Further evaporation of the solvent and the addition of MeOH gave the protected product 9 as a yellow powder in 92% yield. Interestingly, when compound 4 was coupled to the azido ligand 7 under identical conditions as for the preparation of compound 9, the Boc group spontaneously cleaved on the silica gel plates during elution, yielding a final conjugate qAN1-pyr1 in 76% yield. This procedure also proved useful in a Boc deprotection of pyrene conjugate 9, whereby instead of deprotection with TFA/CH2Cl2, compound 9 was spontaneously deprotected during elution on the silica gel plate. Using CH2Cl2/MeOH (20:1) as eluent, deprotected conjugate qAN1-pyr2 was isolated in 82% yield.
Characterization of qAN1-Derivatives in Aqueous Solutions
Upon examination of water solubility, we found the three final compounds (qAN1-AA, qAN1-pyr1, qAN1-pyr2) to be moderately soluble in water, and that their aqueous solutions were stable at least for a day at a room temperature, which was checked by excellent reproducibility of the UV/Vis and fluorimetric calibration experiments (Suppl. Info. Figures S1-S3) several times during the day. For easier handling in the experiments in this study, 5 mM stock solutions of the three compounds were prepared in DMSO. These stock solutions were further diluted in an aqueous buffer immediately prior to use. The total DMSO content in the experiments was kept below 0.2%.
The absorbance (UV/Vis spectra) of the studied compounds was proportional to their concentrations up to at least c = 20 µM (Suppl. Info. Figure S1-S3). The shape of the qAN1-AA UV/Vis spectrum (Suppl. Info. Figure S1) agreed well with data reported for qAN1 [Error! Bookmark not defined.] and significantly differed from UV/Vis spectra of pyrene conjugates qAN1-pyr1 (Suppl. Info. Figure S2) and qAN1-pyr2 (Suppl. Info. Figure S3) due to the pyrene absorbance contribution ( Figure 1). The UV/Vis spectra of qAN1-pyr1 and qAN1-pyr2 were similar but the changes induced by heating the aqueous solutions were significantly different (Suppl. Info. Figure S1-S3). The UV/Vis spectra of qAN1-AA and qAN1-pyr1 displayed no significant changes upon heating to 90 °C, whereas the corresponding spectrum of qAN1-pyr2 at temperatures > 40 °C strongly broadened and the baseline > 500 nm significantly raised above zero, indicating scattering effects (Suppl. Info. Figure S3C). Upon reaching 95 °C, the baseline of the UV/Vis spectrum in aqueous solution of qAN1-pyr2 The protected qAN1-amino acid-pyrene conjugate 9 was readily synthesized from propargylated qAN1 4 and azido ligand 8 through CuAAC "click" conditions employing CuI and DIPEA in dichloromethane. Further evaporation of the solvent and the addition of MeOH gave the protected product 9 as a yellow powder in 92% yield. Interestingly, when compound 4 was coupled to the azido ligand 7 under identical conditions as for the preparation of compound 9, the Boc group spontaneously cleaved on the silica gel plates during elution, yielding a final conjugate qAN1-pyr1 in 76% yield. This procedure also proved useful in a Boc deprotection of pyrene conjugate 9, whereby instead of deprotection with TFA/CH 2 Cl 2 , compound 9 was spontaneously deprotected during elution on the silica gel plate. Using CH 2 Cl 2 /MeOH (20:1) as eluent, deprotected conjugate qAN1-pyr2 was isolated in 82% yield.
Characterization of qAN1-Derivatives in Aqueous Solutions
Upon examination of water solubility, we found the three final compounds (qAN1-AA, qAN1-pyr1, qAN1-pyr2) to be moderately soluble in water, and that their aqueous solutions were stable at least for a day at a room temperature, which was checked by excellent reproducibility of the UV/Vis and fluorimetric calibration experiments (Suppl. Info. Figures S1-S3) several times during the day. For easier handling in the experiments in this study, 5 mM stock solutions of the three compounds were prepared in DMSO. These stock solutions were further diluted in an aqueous buffer immediately prior to use. The total DMSO content in the experiments was kept below 0.2%.
The absorbance (UV/Vis spectra) of the studied compounds was proportional to their concentrations up to at least c = 20 µM (Suppl. Info. Figure S1-S3). The shape of the qAN1-AA UV/Vis spectrum (Suppl. Info. Figure S1) agreed well with data reported for qAN1 [14] and significantly differed from UV/Vis spectra of pyrene conjugates qAN1-pyr1 (Suppl. Info. Figure S2) and qAN1-pyr2 (Suppl. Info. Figure S3) due to the pyrene absorbance contribution ( Figure 1). The UV/Vis spectra of qAN1-pyr1 and qAN1-pyr2 were similar but the changes induced by heating the aqueous solutions were significantly different (Suppl. Info. Figure S1-S3). The UV/Vis spectra of qAN1-AA and qAN1-pyr1 displayed no significant changes upon heating to 90 • C, whereas the corresponding spectrum of qAN1-pyr2 at temperatures > 40 • C strongly broadened and the baseline > 500 nm significantly raised above zero, indicating scattering effects (Suppl. Info. Figure S3C). Upon reaching 95 • C, the baseline of the UV/Vis spectrum in aqueous solution of qAN1-pyr2 returned to zero, indicating the dissolution of all colloids. Importantly, the UV/Vis spectrum of qAN1-pyr2 aqueous solution at 95 • C closely resembled to the corresponding spectrum in methanol (Suppl. Info. Figure S3), but with a bathochromic shift (∆λ ≈ +8 nm) and hypochromic effect for the former sample (Suppl. Info. Figure S3). These findings suggest a strong aromatic stacking of qAN1-pyr2 in aqueous solution at the room temperature, which is lost upon heating or changing the solvent to methanol.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 20 returned to zero, indicating the dissolution of all colloids. Importantly, the UV/Vis spectrum of qAN1-pyr2 aqueous solution at 95 °C closely resembled to the corresponding spectrum in methanol (Suppl. Info. Figure S3), but with a bathochromic shift (Δλ ≈ +8 nm) and hypochromic effect for the former sample (Suppl. Info. Figure S3). These findings suggest a strong aromatic stacking of qAN1-pyr2 in aqueous solution at the room temperature, which is lost upon heating or changing the solvent to methanol. However, the fluorescence of the pyrene conjugates qAN1-pyr1 and qAN1-pyr2 differed significantly from qAN1-AA ( Figure 2). Both compounds emitted with significantly lower intensity compared to qAN1-AA. From previous studies it is known that the quantum yield for the qAN1monomer chromophore in aqueous solution is 18% [Error! Bookmark not defined.] indicating quantum yields of qAN1-pyr1 and qAN1-pyr2 of approximately 1% and 3%, respectively. The emission maximum of qAN1-pyr1 at λmax = 400 nm suggests that the pyrene emission is dominant Similar to the absorption, the fluorescence of qAN1-AA ( Figure 2) closely resembled previously reported data for qAN1 [14], which suggests that the attachment of a triazole-amino acid tail does not influence absorption nor emission of the qAN1-chromophore.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 20 returned to zero, indicating the dissolution of all colloids. Importantly, the UV/Vis spectrum of qAN1-pyr2 aqueous solution at 95 °C closely resembled to the corresponding spectrum in methanol (Suppl. Info. Figure S3), but with a bathochromic shift (Δλ ≈ +8 nm) and hypochromic effect for the former sample (Suppl. Info. Figure S3). These findings suggest a strong aromatic stacking of qAN1-pyr2 in aqueous solution at the room temperature, which is lost upon heating or changing the solvent to methanol. However, the fluorescence of the pyrene conjugates qAN1-pyr1 and qAN1-pyr2 differed significantly from qAN1-AA ( Figure 2). Both compounds emitted with significantly lower intensity compared to qAN1-AA. From previous studies it is known that the quantum yield for the qAN1monomer chromophore in aqueous solution is 18% [Error! Bookmark not defined.] indicating quantum yields of qAN1-pyr1 and qAN1-pyr2 of approximately 1% and 3%, respectively. The emission maximum of qAN1-pyr1 at λmax = 400 nm suggests that the pyrene emission is dominant However, the fluorescence of the pyrene conjugates qAN1-pyr1 and qAN1-pyr2 differed significantly from qAN1-AA (Figure 2). Both compounds emitted with significantly lower intensity compared to qAN1-AA. From previous studies it is known that the quantum yield for the qAN1-monomer chromophore in aqueous solution is 18% [14] indicating quantum yields of qAN1-pyr1 and qAN1-pyr2 of approximately 1% and 3%, respectively. The emission maximum of qAN1-pyr1 at λ max = 400 nm suggests that the pyrene emission is dominant over qAN1 emission (λ max = 430 nm), which in this heterodimer appears to be almost completely quenched. Heating the solutions of both qAN1-AA (Suppl. Info. Figure S4) and qAN1-pyr1 ( Figure 3B left) induced only negligible emission change in accordance with the similarly negligible absorption changes observed (Suppl. Info. Figures S1 and S2).
Intriguingly, the fluorescence spectrum of qAN1-pyr2 strongly differed from both qAN1-AA and qAN1-pyr1 (Figure 2). The emission of qAN1-pyr2 is characterized by two maxima: One at λ max = 400-430 nm corresponding to the qAN1-pyr1 emission and another, significantly red-shifted maximum (λ max ≈ 500 nm) of similar intensity. The qAN1 fluorophore showed similar red-shifted emission (λ max ≈ 500 nm) when incorporated in some ds-DNA oligonucleotides (Wranne et al. [16] and Figure 2) and that effect was attributed to qAN1 tautomerization in the excited state, caused by aromatic stacking interactions of qAN1 with adjacent base pairs. If the effect can be attributed to the same mechanism here that would suggest intramolecular stacking interactions between pyrene and qAN1 within one qAN1-pyr2 molecule, resulting in exciplex-type emission. Another possible reason for the appearance of the λ max at approximately 500 nm is pyrene-excimer emission [18,19,21], which is caused by two or more aromatically stacked pyrenes, thus presuming intermolecular interactions of two or more qAN1-pyr2 molecules.
To further test both hypotheses, we studied the concentration dependent emission and noticed that the qAN1-AA and qAN1-pyr1 emission intensities were proportional to concentration in a wide range of concentrations (up to 10 µM; Figure 3A left and Suppl. Info. Figure S4), whereas the qAN1-pyr2 emission was proportional to concentration only up to 2 µM. At higher concentrations (c = 2-8 µM, Figure 3A right) the emission of qAN1-pyr2 shows distinct non-linearity, implying intermolecular interactions.
Moreover, the heating of the qAN1-pyr2 solution at high concentration (8 µM) resulted in a strong increase in the emission at λ max = 495 nm ( Figure 3B right). Returning to room temperature only partially restored the spectrum, even after four hours. This agrees well with the corresponding heating UV/Vis experiment (Suppl. Info. Figure S3); both UV/Vis and fluorescence results suggesting molecule aggregation/colloidization. The heating-induced aggregation is a well-known phenomenon for non-polar molecules and particularly hydrocarbons in aqueous solutions [43]. Thus, our results suggest that qAN1-pyr2 at concentrations < 2 µM is mostly present in the intramolecularly stacked form, whereas at higher concentrations (>2 µM) or upon heating intermolecular dimers or larger aggregates are formed by stacking of pyrene chromophores, resulting in additional pyrene excimer emission (Figure 3B right).
The extensive difference in emissive response of two close analogues, qAN1-pyr1 and qAN1-pyr2, could be correlated to and explained by the difference in linker length and rigidity, as schematically presented intramolecular aromatic stacking of a pyrene and qAN1 for the longer and more flexible linker of qAN1-pyr2 ( Figure 3C Figure S19).
Interactions with ds-DNA, ds-RNA and ss-RNA
With the insight from the investigations of the pure compounds regarding aggregation issues (vide supra), we decided to use compound concentrations < 2 µM when studying their interaction with various nucleic acid structures.
To study the interactions with DNA/RNA, several common types of DNA and RNA were chosen (Suppl. Info. Chart S1). Naturally occurring calf thymus (ct)-DNA represents a typical B-helix structure Figure S19).
Interactions with ds-DNA, ds-RNA and ss-RNA
With the insight from the investigations of the pure compounds regarding aggregation issues (vide supra), we decided to use compound concentrations < 2 µM when studying their interaction with various nucleic acid structures.
To study the interactions with DNA/RNA, several common types of DNA and RNA were chosen (Suppl. Info. Chart S1). Naturally occurring calf thymus (ct)-DNA represents a typical B-helix structure with a balanced ratio of GC-(48%) and AT-(52%) base pairs. Synthetic alternating polynucleotides poly (dGdC) 2 and poly (dAdT) 2 represent two extreme situations (only AT-or GC-basepairs, respectively), differing significantly in their secondary structures as well as in the availability of the minor groove for small molecule binding (the guanine amino group sterically hinders deep molecule penetration). For comparison between double stranded (ds) DNA and ds-RNA, poly rA-poly rU RNA was chosen as an A-helical structure characterized by a major groove available for the binding of bulky small molecules.
Furthermore, to study the ability of adenine-analogue qAN1 to recognize complementary nucleobases, we also studied the single stranded synthetic ss-RNA polynucleotides poly G, poly A, poly U and poly C, each of them characterized by different properties. Adenine ss-RNA mimics 50-250 adenine nucleotides at the 3 end of mRNA, poly G is related to guanine-rich sequences in both DNA and RNA, whereas poly C and poly U are significantly more flexible than purine-RNAs, and with less organized secondary structures.
Fluorimetric Titrations
Due to the sensitivity of the method, we started studying the interaction between our compounds and nucleic acids by fluorescence measurements. Addition of any ds-RNA/RNA or ss-RNA in the range r [qAN1-AA]/[polynucleotide base] = 1-0.01 induced small changes in the fluorescence spectrum of qAN1-AA, varying between 2-10% from the starting emission intensity (Suppl. Info. Figure S5). None of the titration experiments revealed any biologically relevant affinity even at 100-fold excess of any of polynucleotides, thus showing that qAN1-AA alone does not interact significantly with DNA or RNA.
Fluorimetric titrations of the conjugates of qAN1 and pyrene (qAN1-pyr1 and qAN1-pyr2) with DNA/RNA revealed significant binding (Figures 4 and 5, and Suppl. Info.). Moreover, the fluorimetric response was directly correlated to the length and rigidity of a linker between qAN1 and pyrene (cf. Figures 4 and 5). Fluorimetric titrations were processed by means of the Scatchard eq. (McGhee, vonHippel formalism) [44,45], to afford the binding constants and the density of binding sites (Table 1, log K s and ratio n), as well as the relative emissive properties of complex formed (I/I 0 ).
Fluorimetric Titrations
Due to the sensitivity of the method, we started studying the interaction between our compounds and nucleic acids by fluorescence measurements. Addition of any ds-RNA/RNA or ss-RNA in the range r[qAN1-AA]/[polynucleotide base] = 1-0.01 induced small changes in the fluorescence spectrum of qAN1-AA, varying between 2-10% from the starting emission intensity (Suppl. Info. Figure S5). None of the titration experiments revealed any biologically relevant affinity even at 100-fold excess of any of polynucleotides, thus showing that qAN1-AA alone does not interact significantly with DNA or RNA.
Fluorimetric titrations of the conjugates of qAN1 and pyrene (qAN1-pyr1 and qAN1-pyr2) with DNA/RNA revealed significant binding (Figures 4 and 5, and Suppl. Info.). Moreover, the fluorimetric response was directly correlated to the length and rigidity of a linker between qAN1 and pyrene (cf. Figures 4 and 5). Fluorimetric titrations were processed by means of the Scatchard eq. (McGhee, vonHippel formalism) [44,45], to afford the binding constants and the density of binding sites (Table 1, log Ks and ratio n), as well as the relative emissive properties of complex formed (I/I0). Due to the sensitivity of the method, we started studying the interaction between our compounds and nucleic acids by fluorescence measurements. Addition of any ds-RNA/RNA or ss-RNA in the range r[qAN1-AA]/[polynucleotide base] = 1-0.01 induced small changes in the fluorescence spectrum of qAN1-AA, varying between 2-10% from the starting emission intensity (Suppl. Info. Figure S5). None of the titration experiments revealed any biologically relevant affinity even at 100-fold excess of any of polynucleotides, thus showing that qAN1-AA alone does not interact significantly with DNA or RNA.
Fluorimetric titrations of the conjugates of qAN1 and pyrene (qAN1-pyr1 and qAN1-pyr2) with DNA/RNA revealed significant binding (Figures 4 and 5, and Suppl. Info.). Moreover, the fluorimetric response was directly correlated to the length and rigidity of a linker between qAN1 and pyrene (cf. Figures 4 and 5). Fluorimetric titrations were processed by means of the Scatchard eq. (McGhee, vonHippel formalism) [44,45], to afford the binding constants and the density of binding sites (Table 1, log Ks and ratio n), as well as the relative emissive properties of complex formed (I/I0). Detailed analysis of the fluorimetric results obtained for qAN1-pyr1 and qAN1-pyr2 binding to nucleic acids reveal that both analogues showed similar, moderate affinity toward ds-DNA and ds-RNA (except for qAN1-pyr2/ds-RNA, for which negligible emission change did not allow log K s determination). For both compounds the strongest emission change was observed for AT-dsDNA (Figures 4 and 5 and Table 1), suggesting that DNA minor groove is preferential binding site. Namely, GC-DNA minor groove is sterically hampered and ds-RNA-major groove is much deeper and allows presence of much more water molecules (Suppl. Info. Chart S1), both characteristics exposing molecules to water and diminishing fluorescence change effect. [44]. c Linear dependence of emission change to c polynucleotide allowed only estimation of logK s . d Too small emission changes for processing by Scatchard eq. e Estimate of log K s due to small emission changes.
To shed more light on the thermodynamic properties and binding mode of the interaction between the compounds and the ds-DNA/RNA we performed thermal denaturation experiments. It is well established that upon heating duplexes of polynucleotides they dissociate into two single-stranded polynucleotides at a well-defined temperature (melting temperature Tm). Non-covalent binding of small molecules to duplex nucleic acids usually increases the thermal stability of duplexes, leading to an increase in Tm values [46]. For instance, if a pyrene analogue would intercalate into ds-DNA, a melting temperature increase of 5 • C or more would be expected, whereas a pyrene moiety interacting within a groove of a nucleic acid structure should result in a negligible stabilization effect [47].
Our studied compounds revealed only negligible stabilization of DNA or RNA duplexes (∆Tm = 0-1 • C, Suppl. Info. Table S2 and Figures S13-S15), which suggests that the compounds do not interact with the nucleic acids through intercalation, but rather by binding in the DNA/RNA grooves (in agreement with fluorimetric titration results above), through a combination of hydrophobic, van der Waals and other non-specific interactions, resulting in a moderate binding affinity.
Further, addition of single stranded (ss)-RNAs induced different emission changes for the studied compounds ( Figure 6, Suppl. Info. Figures S8-S12). Also, the binding constants (Table 1) were about an order of magnitude lower than obtained for most ds-polynucleotides.
The emission of qAN1-pyr1 was only slightly changed by addition of any of ss-RNA (<15%) with no apparent selectivity between different RNA sequences (Supp. Info. Figure S8).
However, the fluorimetric response of qAN1-pyr2 is stronger and different in comparison to qAN1-pyr1 and the change in spectral shape is strongly dependent on the nature of the nucleobase for ss-RNA ( Figure 6).
(in agreement with fluorimetric titration results above), through a combination of hydrophobic, van der Waals and other non-specific interactions, resulting in a moderate binding affinity.
Further, addition of single stranded (ss)-RNAs induced different emission changes for the studied compounds (Figure 6, Suppl. Info. Figures S8-S12). Also, the binding constants (Table 1) were about an order of magnitude lower than obtained for most ds-polynucleotides. The emission of qAN1-pyr1 was only slightly changed by addition of any of ss-RNA (<15%) with no apparent selectivity between different RNA sequences (Supp. Info. Figure S8).
However, the fluorimetric response of qAN1-pyr2 is stronger and different in comparison to qAN1-pyr1 and the change in spectral shape is strongly dependent on the nature of the nucleobase for ss-RNA ( Figure 6).
Firstly, the addition of poly U (Watson-Crick complementary base to qAN1) resulted in emission quenching of the qAN1-pyr2 at λmax = 500 nm ( Figure S12, 25% quenching), significantly stronger in comparison to the W-C non-complementary poly C ( Figure S11, 5% quenching). Strong quenching was observed also upon addition of poly A (Figure 6 left, 25% quenching). Intriguingly, the change in qAN1-pyr2 emission at λ = 400-430 nm ( Figures S12 and 6 left) was minor, suggesting that the binding process between qAN1-pyr2 and poly U or poly A mainly involves an intramolecularly stacked pyrene-qAN1 fluorophore (λmax = 500 nm). Since qAN1 is an adenine analogue, it seems feasible that Uracil-qAN1 (Watson-Crick) or Adenine-qAN1 (reverse Hoogsteen [48]) pair is formed, additionally stabilized by aromatic stacking by adjacent pyrene moieties. Such a duplex-like structure would show slight resemblance with the previously studied duplex structure containing qAN1 (Wranne et al. [Error! Bookmark not defined.], Figure 2: TA-sequence), which also showed the emission at λmax = 500 nm. Accordingly, inability of poly C to form H-bonds with qAN1 and small aromatic surface of cytosine did not support binding and thus resulted in negligible fluorimetric change.
The most intriguingly, a strong emission increase of qAN1-pyr2 at λ = 400-430 nm is observed exclusively upon poly G addition, accompanied by negligible change at λmax = 500 nm (Figure 6 right). This suggested that structure of qAN1-pyr2/poly G complex is significantly different compared to the complex with poly A or poly U, likely because guanine bases of the poly G cannot form efficient H-bond pairing with qAN1 due to the unfavorable order of H-bond donors and H-bond acceptors. Taking into account that emission of reference compound qAN1-AA did not change upon any polynucleotide addition up to 100 µM range, strong increase of emission at λmax = 408 nm suggests at least partial release of either pyrene or qAN2 from self-folded (and self-quenched) qAN1-pyr2 form. Negligible change of emission at 500 nm could be result of pyrene stacking with guanines compensating the release of free qAN2. Firstly, the addition of poly U (Watson-Crick complementary base to qAN1) resulted in emission quenching of the qAN1-pyr2 at λ max = 500 nm ( Figure S12, 25% quenching), significantly stronger in comparison to the W-C non-complementary poly C ( Figure S11, 5% quenching). Strong quenching was observed also upon addition of poly A (Figure 6 left, 25% quenching). Intriguingly, the change in qAN1-pyr2 emission at λ = 400-430 nm ( Figure S12 and Figure 6 left) was minor, suggesting that the binding process between qAN1-pyr2 and poly U or poly A mainly involves an intramolecularly stacked pyrene-qAN1 fluorophore (λ max = 500 nm). Since qAN1 is an adenine analogue, it seems feasible that Uracil-qAN1 (Watson-Crick) or Adenine-qAN1 (reverse Hoogsteen [48]) pair is formed, additionally stabilized by aromatic stacking by adjacent pyrene moieties. Such a duplex-like structure would show slight resemblance with the previously studied duplex structure containing qAN1 (Wranne et al. [16], Figure 2: TA-sequence), which also showed the emission at λ max = 500 nm. Accordingly, inability of poly C to form H-bonds with qAN1 and small aromatic surface of cytosine did not support binding and thus resulted in negligible fluorimetric change.
The most intriguingly, a strong emission increase of qAN1-pyr2 at λ = 400-430 nm is observed exclusively upon poly G addition, accompanied by negligible change at λ max = 500 nm (Figure 6 right). This suggested that structure of qAN1-pyr2/poly G complex is significantly different compared to the complex with poly A or poly U, likely because guanine bases of the poly G cannot form efficient H-bond pairing with qAN1 due to the unfavorable order of H-bond donors and H-bond acceptors. Taking into account that emission of reference compound qAN1-AA did not change upon any polynucleotide addition up to 100 µM range, strong increase of emission at λ max = 408 nm suggests at least partial release of either pyrene or qAN2 from self-folded (and self-quenched) qAN1-pyr2 form. Negligible change of emission at 500 nm could be result of pyrene stacking with guanines compensating the release of free qAN2.
However, such an unstacking of qAN1-pyr2 would have a thermodynamic price, and also would not include H-bonding which could be present for both poly U and poly A. This would result in the significantly lower affinity of qAN1-pyr2 that we find towards poly G (log K s < 3) in comparison to complexes with poly U and poly A (log K s ≈ 5).
Circular Dichroism (CD) Experiments
In order to get insight into the changes of polynucleotide properties induced by the small molecule binding, we chose CD spectroscopy as a sensitive method for the study of secondary structure conformational changes [49]. In addition, achiral small molecules can acquire an induced CD spectrum (ICD) upon binding to polynucleotides, which could give useful information about modes of interaction [49,50].
In our investigations, however, the addition of our compounds did not induce any significant changes in CD spectrum of any ds-DNA/RNA. Neither was any measurable ICD effect > 300 nm observed (Suppl. Info. Figures S16-S18). These results along with the negligible thermal denaturation effect (Table S2) point in the direction that DNA/RNA double helices are not disturbed upon small molecule interaction, thus speaking against an intercalative binding mode and suggesting the grooves of ds-polynucleotides as binding sites. However, absence of any measurable ICD effect in the 300-350 nm region (at which both qAN1 and pyrene absorb) argues against typical, uniformly-oriented groove binding. Negligible ICD effect for the groove-binding compounds would imply either: (a) Chromophores are not homogeneously oriented within binding site with respect to a chiral axis of the polynucleotide, or (b) transition vectors of qAN1 and pyrene responsible for the absorbance at 300-350 nm yield opposite ICD signs upon binding to DNA/RNA, resulting in ICD bands of negligible intensity.
Conclusions
The here designed and prepared novel amino acid-fluorescent nucleobase derivative, qAN1-AA, allows several conjugation strategies within peptide-backboned systems (PNAs or similar), either by direct incorporation of qAN1-AA by peptide bond formation or via a "click"-related route: First introducing azide-amino acid at targeted peptide position and then "clicking" the nucleobase at any convenient reaction point. In addition, our novel propargyl-qAN1 precursor can also be "clicked" to any other azide-based building block.
We find that the amino acid-fluorescent nucleobase derivative (qAN1-AA) does not interact with DNA/RNA at biologically relevant conditions. However, linking the qAN1-AA with pyrene yielded conjugates (qAN1-pyr1, qAN1-pyr2) capable of interacting with various DNA/RNA (1-10 µM affinity), with a fluorescent response that senses differences in secondary structure and nucleobase composition of nucleic acid targets.
The difference in linker length between the pyrene conjugates strongly influenced their spectroscopic properties in aqueous solution. The longer and more flexible linker directed qAN1-pyr2 into a intramolecularly stacked qAN1/pyrene conformation, yielding strongly red-shifted emission (λ = 500 nm) in comparison to both free pyrene or qAN1, whereas the shorter and more rigid linker of qAN1-pyr1 resulted in a non-stacked conformation characterized by the emission of free pyrene at λ = 400-430 nm. This intrinsic conformational difference was the basis of different fluorimetric responses of the studied conjugates upon binding to various DNA or RNA. We find evidence that the conjugates bind to ds-DNA/RNA grooves with essentially no impact on polynucleotide double helix stability or chirality.
The most pronounced change in emission of both qAN1-pyr1 and qAN1-pyr2, was observed for AT-DNA addition. That is in accordance with the AT-DNA minor groove availability to small molecule binding [51]. It has previously been established that the AT-DNA minor groove has ideal dimensions for insertion of small molecules [52] (Suppl. Info. Chart S1). This seems also to be the case not only to flexible qAN1-pyr1 but also for the self-folded dual aromatic system as qAN1-pyr2. On the contrary, the GC-DNA minor groove is sterically hindered by protruding amino groups of guanines, leaving small molecule more exposed to water and thus its emission less changed [51,52]. The ds-RNA minor groove is too shallow and broad for efficient small molecule binding and RNA major groove can accommodate these small molecules but due to large depth (Suppl. Info. Chart S1). Cantor, C.R. et al. and Egli, M. et al., [52,53] does not exclude water molecules as efficiently as AT-DNA minor groove, resulting in less efficient fluorescence change.
Most importantly, the intramolecularly pre-oriented qAN1-pyr2 clearly differentiated between ss-RNAs by opposite emission response. This is most likely based on H-bonding complementarity: Watson-Crick complementary poly U or reverse Hoogsteen-complementary [48] poly A to the qAN1 moiety yielded efficient emission quenching at 500 nm, whereas non-complementary poly C did not influence emission and the non-complementary poly G yielded an strong emission increase at 408 nm (likely to bis-intercalation of qAN1-pyr2, see discussion under Figure 6). Unlike qAN1-pyr2, the non-intramolecularly folded qAN1-pyr1 emission did not change significantly upon addition of any ss-RNA, clearly stressing the importance of qAN1-pyr2 intramolecular pre-organization for variation in emission response upon interaction.
Chromophores and especially fluorophores which can exist in multiple conformational states with the possibility to change between them based on binding to various targets, have high potential for the use in molecular sensing applications. For that reason here presented qAN1-pyr2 shows promise as a specific fluorimetric probe for non-covalent sensing of poly G and also as a good lead compound for further development of probes targeting uracil-or adenine-DNA abasic sites, i.e., DNA abasic sites as products of chemotherapy or environmental impacts are promising targets for aryl-nucleobase conjugates [54] and particularly pre-organized, self-folded analogues has previously shown high selectivity [55][56][57][58]. Therefore, we envision that qAN1-pyr2 and its future derivatives could not only have sensor applications but also, simultaneously, display therapeutic properties.
Synthesis
Circular dichroism (CD) spectra were recorded on JASCO J-815 spectropolarimeter at room temperature using 1 cm path quartz cuvettes with a scanning speed of 200 nm/min (an average of 3 accumulations). A buffer background was subtracted from each spectrum. CD experiments were performed by adding portions of compound stock solution into the solution of the polynucleotide (c = 2 × 10 -5 M).
Thermal melting experiments were performed on a Varian Cary 100 Bio spectrometer in quartz cuvettes (1 cm). The measurements were done in aqueous buffer solution at pH 7.0 (sodium cacodylate buffer, I = 0.05 M). Thermal melting curves for ds-DNA, ds-RNA and their complexes with dyes were determined by monitoring the absorption change at 260 nm as a function of temperature [46]. Tm values are the midpoints of the transition curves determined from the maximum of the first derivative and checked graphically by the tangent method. The ∆Tm values were calculated subtracting Tm of the free nucleic acid from Tm of the complex. Every ∆Tm value here reported was the average of at least two measurements. The error in ∆Tm is ± 0.5 • C.
Funding:
The financial support of the Croatian Science Foundation project IP-2018-01-5475 is gratefully acknowledged.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
2018-06-21T13:03:54.736Z | 2018-06-01T00:00:00.000 | 46926692 | {
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} | pes2o/s2orc | Sterepinic Acids A–C, New Carboxylic Acids Produced by a Marine Alga-Derived Fungus
Sterepinic acids A–C (1–3), new carboxylic acids with two primary alcohols, have been isolated from a fungal strain of Stereum sp. OUPS-124D-1 attached to the marine alga Undaria pinnatifida. Dihydro-1,5-secovibralactone (4), a new vibralactone derivative, was isolated from the same fungal metabolites together with known vibralactone A (5), and 1,5-secovibralactone (6). The planar structures of these compounds have been elucidated by spectroscopic analyses using IR, HRFABMS, and NMR spectra. To determine the absolute configuration of the compounds, we used the phenylglycine methyl ester (PGME) method. These compounds exhibited less activity in the cytotoxicity assay against cancer cell lines.
Introduction
Our ongoing search for seeds of antitumor chemotherapy agents from marine microorganisms has led to the isolation of several antitumor and/or cytotoxic compounds [1][2][3][4][5][6][7][8]. In particular, we focused on the bioactive compounds with small molecular weight due to their advantages, such as easy synthesis and modification for increasing the activity. In addition, the synthesis of small bioactive compounds establishes a hypothetical biosynthesis mechanism of larger bioactive compounds. In this study, we isolated four new carboxylic acids with two primary alcohols, designated as sterepinic acids A-C (1-3) and dihydro-1,5-secovibralactone (4), together with the known vibralactone A (5) and 1,5-secovibralactone (6), from a strain of Stereum sp. OUPS-124D-1 derived from the marine alga Undaria pinnatifida. 5 was reported by Liu et al. [9], and many studies then followed this work, isolating the derivatives of 5 including 6 [10][11][12][13][14][15]. We report the determination of the absolute configurations of 1-4 by applying the phenylglycine methyl ester (PGME) method [16]. In addition, we report on the investigation of the cytotoxicity of these compounds against several cancer cell lines.
Results
Stereum sp., a microorganism from U. pinnatifida, was cultured at 27 • C for 5 weeks in a medium (50 L) containing 1% glucose, 1% malt extract, and 0.05% peptone in artificial seawater adjusted to pH 7.6. After the incubation, the culture was filtrated through DIAION HP-20, and its MeOH elution was purified employing a stepwise combination of silica gel column chromatography and reverse phase HPLC to afford sterepinic acids, A (1) (64.8 mg); B (2) (13.3 mg); C (3) (16.8 mg); and dihydro-1,5-secovibralactone (4) (12.4 mg), as a pale yellow oil, respectively ( Figure 1). The molecular formula of sterepinic acid A (1) has been determined as C12H20O4 from its molecular weight of 229.1443 [M + H] + in HRFABMS. Its IR spectrum exhibited bands at 3330 and 1710 cm −1 , that are characteristics of hydroxy and carbonyl groups, respectively. An analysis of the 1 H and 13 C NMR spectra of 1 (Tables 1 and S1), using DEPT and 1 H-13 C heteronuclear multiple quantum coherence spectroscopy (HMQC), showed the presence of two olefin methyls (C-11 and C-12); four sp 3 -hybridized methylenes (C-5, C-6, C-7, and C-8), including two oxygen-bearing sp 3 methylenes (C-6 and C-7); one sp 3 -methine (C-2); two sp 2 -methines (C-3 and C-9); two quaternary sp 2 -carbons (C-4 and C-10); and one carbonyl group (C-1). In the 1 H-1 H correlation spectroscopy (COSY) analysis, correlations were observed between H-5 and H-6; H-2 and H-3; and H-2 and H-8, as shown by the bold lines in Figure 2. In the HMBC spectrum (Figure 2), the correlations from H-11 and H-12 to C-9 and C-10; from H-2 to C-1 and C-4; from H-3 to C-1, C-5, and C-7; from H-5 to C-3; from H-6 to C-4; from H-7 to C-3, C-4, and C-5; from H-8 to C-1 and C-10; from H-6 to C-4; and from H-7 to C-4, and C-5 elucidated the planar structure of 1 as 6-hydroxy-4-(hydroxymethyl)-2-(3methylbut-2-en-1-yl) hex-3-enoic acid. The elucidation of the absolute stereostructure of 1 is described below, together with those of 2-4. The molecular formula of sterepinic acid A (1) has been determined as C 12 H 20 O 4 from its molecular weight of 229.1443 [M + H] + in HRFABMS. Its IR spectrum exhibited bands at 3330 and 1710 cm −1 , that are characteristics of hydroxy and carbonyl groups, respectively. An analysis of the 1 H and 13 C NMR spectra of 1 (Table 1 and Table S1), using DEPT and 1 H-13 C heteronuclear multiple quantum coherence spectroscopy (HMQC), showed the presence of two olefin methyls (C-11 and C-12); four sp 3 -hybridized methylenes (C-5, C-6, C-7, and C-8), including two oxygen-bearing sp 3 -methylenes (C-6 and C-7); one sp 3 -methine (C-2); two sp 2 -methines (C-3 and C-9); two quaternary sp 2 -carbons (C-4 and C-10); and one carbonyl group (C-1). In the 1 H-1 H correlation spectroscopy (COSY) analysis, correlations were observed between H-5 and H-6; H-2 and H-3; and H-2 and H-8, as shown by the bold lines in Figure 2. In the HMBC spectrum (Figure 2), the correlations from H-11 and H-12 to C-9 and C-10; from H-2 to C-1 and C-4; from H-3 to C-1, C-5, and C-7; from H-5 to C-3; from H-6 to C-4; from H-7 to C-3, C-4, and C-5; from H-8 to C-1 and C-10; from H-6 to C-4; and from H-7 to C-4, and C-5 elucidated the planar structure of 1 as 6-hydroxy-4-(hydroxymethyl)-2-(3-methylbut-2-en-1-yl) hex-3-enoic acid. The elucidation of the absolute stereostructure of 1 is described below, together with those of 2-4. Sterepinic acids, B (2) and C (3), were assigned the molecular formula of C24H38O7, with both compounds showing molecular weight almost twice as large as that of 1. While the general features of NMR spectra (Tables 1, S2 and S3) closely resembled those of 1, the 1 H and 13 C signals of 2 and 3 were observed in pairs or with the overlapping of two signals for each functional group (vide info.), except for the proton signal of the oxygen-bearing methylenes (C-7 (δH 4.48 d, and δH 4.62 d) in 2) and C-6 (δH 4.20 m) in 3). This phenomenon suggested that 2 and 3 were the dimers of 1. As expected, for the HMBC spectrum of 2 (Table S2), the correlations shown in Figure 3A were used to construct two carboxylic acids, both of which are identical to the planar structure of 1. In addition, the correlation from H-7 in one carboxylic acid to C-1′ in another carboxylic acid revealed that the two carboxylic acids were condensed to a dimer esterified between C-7 and C-1′ ( Figure 3A and Table S2). By contrast, the HMBC correlation from H-6 to C-1′observed in 3 demonstrated that the chemical structure of 3 was similar to that of the dimer esterified between C-6 and C-1′ ( Figure 3B and Table S3). Dihydro-1,5-secovibralactone (4) exhibited the molecular formula C12H20O4, containing two fewer hydrogen atoms, and one less oxygen atom than 1. Compared with the NMR spectra of 4 (Tables 2 and S4), those of 1 showed large differences in the proton signals of H-1 (δH 3.68 m) and H-5 (δH 4.68 ddd and 4.33 ddd), corresponding to H-2 and H-6 in 1, respectively, and the carbon signals of C-1 (δH 40.2), C-2 (δH 121.2), and C-7 (δH 174.3), corresponding to C-2, C-3, and C-1, respectively, in 1. The numbering of the carbon positions followed the numbering mentioned in a previous report [6]. 4 was observed to be the monomer with the same carboxylic acid unit as 1. In addition, HMBC correlations from H-5 to C-7 (Table S4 and Figure 4) elucidated the planar structure of 4 as a dihydroisomer of 1,5-secovibralactone (6) [10]. Sterepinic acids, B (2) and C (3), were assigned the molecular formula of C 24 H 38 O 7 , with both compounds showing molecular weight almost twice as large as that of 1. While the general features of NMR spectra (Table 1, Tables S2 and S3) closely resembled those of 1, the 1 H and 13 C signals of 2 and 3 were observed in pairs or with the overlapping of two signals for each functional group (vide info.), except for the proton signal of the oxygen-bearing methylenes (C-7 (δ H 4.48 d, and δ H 4.62 d) in 2) and C-6 (δ H 4.20 m) in 3). This phenomenon suggested that 2 and 3 were the dimers of 1. As expected, for the HMBC spectrum of 2 (Table S2), the correlations shown in Figure 3A were used to construct two carboxylic acids, both of which are identical to the planar structure of 1. In addition, the correlation from H-7 in one carboxylic acid to C-1 in another carboxylic acid revealed that the two carboxylic acids were condensed to a dimer esterified between C-7 and C-1 ( Figure 3A and Table S2). By contrast, the HMBC correlation from H-6 to C-1 observed in 3 demonstrated that the chemical structure of 3 was similar to that of the dimer esterified between C-6 and C-1 ( Figure 3B and Table S3). Sterepinic acids, B (2) and C (3), were assigned the molecular formula of C24H38O7, with both compounds showing molecular weight almost twice as large as that of 1. While the general features of NMR spectra (Tables 1, S2 and S3) closely resembled those of 1, the 1 H and 13 C signals of 2 and 3 were observed in pairs or with the overlapping of two signals for each functional group (vide info.), except for the proton signal of the oxygen-bearing methylenes (C-7 (δH 4.48 d, and δH 4.62 d) in 2) and C-6 (δH 4.20 m) in 3). This phenomenon suggested that 2 and 3 were the dimers of 1. As expected, for the HMBC spectrum of 2 (Table S2), the correlations shown in Figure 3A were used to construct two carboxylic acids, both of which are identical to the planar structure of 1. In addition, the correlation from H-7 in one carboxylic acid to C-1′ in another carboxylic acid revealed that the two carboxylic acids were condensed to a dimer esterified between C-7 and C-1′ ( Figure 3A and Table S2). By contrast, the HMBC correlation from H-6 to C-1′observed in 3 demonstrated that the chemical structure of 3 was similar to that of the dimer esterified between C-6 and C-1′ ( Figure 3B and Table S3). Dihydro-1,5-secovibralactone (4) exhibited the molecular formula C12H20O4, containing two fewer hydrogen atoms, and one less oxygen atom than 1. Compared with the NMR spectra of 4 (Tables 2 and S4), those of 1 showed large differences in the proton signals of H-1 (δH 3.68 m) and H-5 (δH 4.68 ddd and 4.33 ddd), corresponding to H-2 and H-6 in 1, respectively, and the carbon signals of C-1 (δH 40.2), C-2 (δH 121.2), and C-7 (δH 174.3), corresponding to C-2, C-3, and C-1, respectively, in 1. The numbering of the carbon positions followed the numbering mentioned in a previous report [6]. 4 was observed to be the monomer with the same carboxylic acid unit as 1. In addition, HMBC correlations from H-5 to C-7 (Table S4 and Figure 4) elucidated the planar structure of 4 as a dihydroisomer of 1,5-secovibralactone (6) [10]. Dihydro-1,5-secovibralactone (4) exhibited the molecular formula C 12 H 20 O 4 , containing two fewer hydrogen atoms, and one less oxygen atom than 1. Compared with the NMR spectra of 4 ( Table 2 and Table S4), those of 1 showed large differences in the proton signals of H-1 (δ H 3.68 m) and H-5 (δ H 4.68 ddd and 4.33 ddd), corresponding to H-2 and H-6 in 1, respectively, and the carbon signals of C-1 (δ H 40.2), C-2 (δ H 121.2), and C-7 (δ H 174.3), corresponding to C-2, C-3, and C-1, respectively, in 1. The numbering of the carbon positions followed the numbering mentioned in a previous report [6]. 4 was observed to be the monomer with the same carboxylic acid unit as 1.
In addition, HMBC correlations from H-5 to C-7 (Table S4 and Figure 4) elucidated the planar structure of 4 as a dihydro-isomer of 1,5-secovibralactone (6) [10]. For the determination of the absolute stereostructures of metabolites isolated in this study, we first examined the absolute configuration of 1, which is the common unit in all compounds of this study. 1 showed the presence of a secondary carboxy group at C-2, and we therefore used the PGME method [16]. The 1 H chemical-shift differences between the (S)-and (R)-PGME amides 1a and 1b revealed the S configuration at C-2 ( Figure 5). For the determination of the absolute stereostructures of metabolites isolated in this study, we first examined the absolute configuration of 1, which is the common unit in all compounds of this study. 1 showed the presence of a secondary carboxy group at C-2, and we therefore used the PGME method [16]. The 1 H chemical-shift differences between the (S)-and (R)-PGME amides 1a and 1b revealed the S configuration at C-2 ( Figure 5). Next, for the elucidation of the stereochemistry of 2-4, we attempted to perform hydrolysis to derive 1 from 2-4; however, due to the small volume of reaction, the carboxylic acid was not produced. We therefore tried methanolysis to facilitate the purification of the product resulting from the reaction. The treatment with concd H2SO4 of MeOH solution of 2 only gave a methyl carboxylate, the spectral data ( 1 H NMR spectrum and the optical rotation) for which were identical to those of the methyl ester Next, for the elucidation of the stereochemistry of 2-4, we attempted to perform hydrolysis to derive 1 from 2-4; however, due to the small volume of reaction, the carboxylic acid was not produced. We therefore tried methanolysis to facilitate the purification of the product resulting from the reaction. The treatment with concd H 2 SO 4 of MeOH solution of 2 only gave a methyl carboxylate, the spectral data ( 1 H NMR spectrum and the optical rotation) for which were identical to those of the methyl ester of 1; i.e., 2 is found to be in the 2S, 2 S absolute configuration. The same procedure applied to 3 and 4 revealed the S configuration at C-2 and C-2 in 3, and the S configuration at C-2 in 4, respectively. This evidence confirmed that 2-4 were composed of 1. A lone pair on the alcohol oxygen atom attacks a carboxy carbon atom by an intra-or intermolecular nucleophilic reaction, as shown by the arrows coded using three different colors (Scheme 1). The routes shown in red and blue, which are the dimerization routes, produce 2 and 3, respectively. On the other hand, the route shown in black leads to 4 followed by a dehydrogenation to 6. Meanwhile, Zhao et al., performed an in vitro enzymatic conversion, and verified biochemically the enzymatic production of 5 from 6 by the analyses of LC/MS/MS [17]. Next, for the elucidation of the stereochemistry of 2-4, we attempted to perform hydrolysis to derive 1 from 2-4; however, due to the small volume of reaction, the carboxylic acid was not produced. We therefore tried methanolysis to facilitate the purification of the product resulting from the reaction. The treatment with concd H2SO4 of MeOH solution of 2 only gave a methyl carboxylate, the spectral data ( 1 H NMR spectrum and the optical rotation) for which were identical to those of the methyl ester of 1; i.e., 2 is found to be in the 2S, 2′S absolute configuration. The same procedure applied to 3 and 4 revealed the S configuration at C-2 and C-2′ in 3, and the S configuration at C-2 in 4, respectively. This evidence confirmed that 2-4 were composed of 1. A lone pair on the alcohol oxygen atom attacks a carboxy carbon atom by an intra-or intermolecular nucleophilic reaction, as shown by the arrows coded using three different colors (Scheme 1). The routes shown in red and blue, which are the dimerization routes, produce 2 and 3, respectively. On the other hand, the route shown in black leads to 4 followed by a dehydrogenation to 6. Meanwhile, Zhao et al., performed an in vitro enzymatic conversion, and verified biochemically the enzymatic production of 5 from 6 by the analyses of LC/MS/MS [17]. Cancer cell growth-inhibitory properties of sterepinic acids A-C (1-3) and dihydro-1,5secovibralactone (4) were examined using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines; however, these metabolites did not exhibit significant activity against these cancer cells (Table 3). We therefore continue to investigate related compounds with more potent cytotoxicity from this fungal metabolite and examine another assay. Cancer cell growth-inhibitory properties of sterepinic acids A-C (1-3) and dihydro-1,5secovibralactone (4) were examined using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines; however, these metabolites did not exhibit significant activity against these cancer cells (Table 3). We therefore continue to investigate related compounds with more potent cytotoxicity from this fungal metabolite and examine another assay.
Fungal Material
A strain of Stereum sp. was initially isolated from a piece of the marine alga Undaria pinnatifida collected at collected in Osaka bay, Japan in May 2015. The fungal strain was identified by Techno Suruga Laboratory Co., Ltd. The surface of the marine alga was wiped with EtOH, and its snip applied to the surface of nutrient agar layered in a Petri dish. Serial transfers of one of the resulting colonies provided a pure strain of Stereum sp.
Culturing and Isolation of Metabolites
The fungal strain was cultured at 27 • C for 4 weeks in a liquid medium (50 L) containing 1% malt extract, 0.05% peptone, and 1% D-glucose in artificial seawater adjusted to pH 7.5. The culture was filtered under suction, and the culture filtrate was passed through to DIAION HP20, and washed with water to remove water-soluble component. The fraction eluted with MeOH were evaporated in vacuo to afford a mixture of crude metabolites (10.2 g) that exhibited cytotoxicity against the P388 cell line (IC 50 < 10 µg/mL). The mixture was chromatographed on a silica gel column with a CH 2 Cl 2 -MeOH gradient as the eluent to afford Fraction (Fr.) 1 (2% MeOH in CHCl 3
Formation of the (S)-and (R)-PGME Amides
To a solution of 1 (5.8 mg, 0.025 mmol) and (S)-PGME (0.054 mmol) in dry DMF (1 mL) was added to EDC-HCl (0.050 mmol), HOBt (0.050 mmol), and DMAP (catalysis volume). The reaction mixture was stirred at room temperature 2 hours. Water (1.0 mL) was added to the reaction mixture, and then extracted using CH 2 Cl 2 . The organic layer was evaporated under reduced pressure, and the residue was purified by HPLC using MeOH-H 2 O (50:50) as the eluent to afford (S)-PGME amide 1a (0.9 mg, 0.0024 mmol) as a pale yellow oil.
Methanolysis of 2-4
To a solution of 2 (3.2 mg) in MeOH (0,5 mg) was added concd H 2 SO 4 (0.01 mL), and the reaction mixture was left at room temperature for 1 hr. The mixture was diluted with water, and extracted with CH 2 Cl 2 , and the extract was evaporated under reduced pressure, and then the residue was purified by HPLC using MeOH-H 2 O (60:40) as the eluent to afford methyl ester (0.8 mg) as a pale yellow oil.
Using the same procedure as above with 2, a solution of 3 (3.3 mg) in MeOH (0.5 mL) was treated with concd H 2 SO 4 (0.01 mL), and purified by HPLC using MeOH-H 2 O (60:40) as the eluent to afford methyl ester (0.8 mg).
Using the same procedure as above with 2, a solution of 4 (2.4 mg) in MeOH (0.5 mL) was treated with concd H 2 SO 4 (0.01 mL), and purified by HPLC using MeOH-H 2 O (60:40) as the eluent to afford methyl ester (0.7 mg).
Conclusions
In this study, new carboxylic acids designated as sterepinic acids A-C (1-3) and dihydro-1,5secovibralactone (4), have been isolated from a strain of Stereum sp. derived from marine sponge.
Their absolute configurations were established by the application of the PGME method to 1 and the chemical transformation of 2-4.
In the screening for the search of the seeds of antitumor agents, these compounds did not exhibit significant cytotoxic activity against three cancer cell lines. | v3-fos-license |
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} | pes2o/s2orc | Microwave-Supported Leaching of Electric Arc Furnace ( EAF ) Slag by Ammonium Salts
The effect of microwave-supported leaching of electric arc furnace (EAF) slag by ammonium salts was investigated to improve calcium leaching ratio for CO2 mineral sequestration process. The results show that the calcium leaching ratio from EAF slag at the constant temperature in the microwave field increases about 10% than that under the water bath at the same leaching time. The greater the microwave power, the higher the impact of calcium leaching rate, which proves that microwave treatment can improve the leaching ratio of calcium. The rapid calcium leaching step (up to 5 min) is possibly due to the easy reaction of calcium silicate, while the slower calcium leaching step (after 5 min) is owing to the difficult reaction of calcium ferroaluminates for the hydrolysis of iron and aluminum. The leaching behaviors of magnesium and calcium ions affected by different leaching parameters are similar and the concentration of aluminum, iron and phosphorus can be neglected. Calcium ion is probably not precipitated in the real leaching solution from steel slag by ammonium chloride solution as its concentration is less than 0.32 mol/L. However, the concentration of magnesium ion starts to drop sharply when the pH value is higher than 10 and it has precipitated completely at pH value of 11.6.
Introduction
As fossil fuels will still be the main power source in the near future and its combustion would emit significant amounts of greenhouse gases, an economically viable approach to sequestrate CO 2 is becoming more and more necessary.Meanwhile, the worldwide production of crude steel has reached 1.63 × 10 6 Mt in 2016 [1], large amounts of steel slag generated simultaneously, which are estimated to about 10~15% by weight of the total produced crude steel [2].Furthermore, the iron and steel industry is recognized as one of the largest CO 2 emission industries with total amounts of 1500~1600 Mt CO 2 emission [3].Currently, the waste steelmaking slag are mainly used as fluxing agent of iron and steel smelting [4,5], fertilizer of agricultural production [6,7], construction materials (such as slag cement, brick, concrete and construction aggregate) [8], soil conditioner [9], as well as adsorbent of environmental protection [10].However, for these applications only a small part of the slag can be utilized, especially in China, where the utilization rate of steel slag is only 22%, which is far behind the developed countries [11].The uses of CO 2 in the waste off-gas and unused slag are limited for various causes and difficulties.Therefore, large amounts of slag are stockpiled, which are costly and seriously harmful to the environment, such as damaging soil and vegetation or polluting air and water.
In 1990, Seifritz proposed a safe and permanent method of CO 2 sequestration based on chemically combining CO 2 with abundant raw materials to form stable carbonate minerals [12].It was improved by using waste slag to sequestrate CO 2 due to significant levels of calcium, high alkalinity in the slag and in situ CO 2 mineral carbonation in iron and steel plants.An approach involving calcium ion leaching from the waste slag with the ammonium salts, and subsequently reacting with CO 2 to produce precipitation calcium carbonate (PCC), has been reported recently [13][14][15][16][17].The usage of ammonium chloride solution as an extraction reagent in the indirect aqueous processes of CO 2 sequestration is currently widely investigated for high efficiency and selective extraction of calcium and easy purification process.The process mainly consists of a two-step reaction as follows.2CaO•SiO 2 (s) + 4NH 4 Cl(aq)→2CaCl 2 (aq) + 2H 2 O + SiO 2 (s)↓ + 4NH 3 (aq) 4NH 3 (aq) + 2CO 2 (aq) + 2CaCl 2 (aq) + 4H 2 O(l)→2CaCO 3 (s)↓ + 4NH 4 Cl(aq) Ammonium chloride is consumed in the first step and regenerated in the second step, thus, the extraction agent can be recycled.Compared with ammonium salts leaching, the pH of the leached system needs to be improved after leaching for nitric acid and sulfuric acid to make the following carbonation reaction possible, while using acetic acid as a leaching solution would leach large amounts of other elements besides calcium, such as magnesium [18][19][20][21].Therefore, the whole process cost would be reduced by using ammonium chloride as a leaching agent, due to fewer requirements for the pH adjusting and purification process.However, the calcium extraction efficiency with ammonium salts is much lower than that with acidic extraction.In order to improve the calcium leaching ratio from electric arc furnace (EAF) slag, this paper proposes the methods of assisting the calcium leaching in a microwave field and proves the possibility of improving calcium extraction efficiency from EAF slag, and the optimization of the process was also studied.
Experimental Procedure
The as-received carbon steel EAF slag was provided by a local steelmaking plant, and the main chemical composition of EAF slag was analyzed with inductively coupled plasma emission spectroscopy (IRIS Advantage ER/S) as 39% CaO, 28% Fe 2 O 3 , 12% SiO 2 , 8% MgO and 4% Al 2 O 3 , which indicates that it is suitable for mineral CO 2 sequestration due to its high basicity (CaO/SiO 2 > 2.7) and calcium content.A batch of 2 kg EAF slag was crushed in a vibratory disk mill and ground in a planet-type ball mill.The ground slag was subsequently dried in an oven at 110 • C for 24 h.
In order to compare the calcium extraction efficiency in the microwave leaching process, a first batch of leaching experiments at the constant temperature with water bath and in a microwave field were carried out in a three-necked flask, respectively.In the experiment, the particle size of EAF slag, which is smaller than 54 µm, was selected.The leaching process was assisted by magnetic stirring at the maximum rate, and the other leaching process parameters were 20 mL/g liquid-solid ratio, 200 mL solution volume and 2 mol/L ammonium chloride solution.Moreover, the effect of various leaching parameters (stirring speed, leaching solution volume, liquid-solid ratio, leaching solution concentration) on calcium leaching ratio from EAF slag was also studied on a second experimental batch at fixed temperature (60 • C) under microwave field, on EAF slag particles grinded less than 100 µm.
In the conventional leaching experiment, the reaction temperature was conducted with water bath and the leachant was stirred magnetically.The microwave chemical reactor MCR-3 can provide two kinds of microwave outputs, i.e., a stable microwave power and a stable temperature with variable microwave heating output.The experiment temperature in the microwave field was monitored with a trifluoroethylene thermocouple.Both conventional and microwave leaching experiments were equipped with a condenser pipe system.
In the microwave leaching process of constant temperature, the leaching solution was heated to the aimed temperature, then the EAF slag was added into the leaching solution and the microwave chemical reactor was switched on immediately, while the leaching reaction in the leaching process of constant microwave power begins from room temperature.At a certain time, a part of the reactant was sampled and filtered.The calcium concentrations of leachate were analyzed with the ethylenediaminetetraacetic acid (EDTA) titration method of GB/T 15452-2009 [22].Moreover, the concentrations of magnesium, aluminum, iron and phosphorus at the final leaching solution were analyzed by inductively coupled plasma emission spectroscopy (IRIS Advantage ER/S, Thermo Jarrell Ash, Franklin, MA, USA).The filtered leached slag residue was washed with Reverse Osmosis water (RO water) three times and dried at 115 • C for 24 h.The mineralogical phases of the slag residue were determined by an X'Pert PRO MPD X-ray diffractometer with Cu Kα.A field emission scanning electron microscopy (Nova 400 NanoSEM, FEI, Hillsboro, OR, USA) coupled with energy dispersive spectrometer (INCAIE 350 PentaFET X-3 EDS, Oxford, UK) was used to analyse the as-received slag sample and the leached slag residue.
The leaching ratio of calcium ion was calculated by the following equation: where x Ca 2+ is the leaching ratio of calcium; c Ca 2+ is the concentration of Ca 2+ in the filtrate, µg/mL; V T is the total volume of the leaching solution, mL; m slag is the mass of the EAF slag, g; η Ca 2+ is the content of CaO in the EAF slag, %; M Ca , M CaO are the molecular mass of Ca and CaO, respectively.
Calcium Leaching by NH 4 Cl with Conventional and Microwave Leaching Processes
The calcium leaching ratio from the EAF slag by NH 4 Cl under different leaching environments are shown in Figure 1.It can be seen from Figure 1a that the calcium leaching ratio rose significantly at the initial period (5 min) and then tends to the maximum value, which attains ~87% with 120 min leaching at 100 • C. Figure 1b is the variation of calcium leaching ratio with leaching time at constant temperature in a microwave field.Comparing with Figure 1a,b, the tendencies of the calcium leaching ratios are similar, which attains the maximum leaching ratio within 5 min.However, the calcium leaching ratio at the constant temperature in the microwave field increases about 10% than that under the water bath at the same time.This is possibly due to the fact that the polar molecules in the leaching system vibrate rapidly in the microwave field, producing a large amount of energy, which heats the solution and increases the collision frequency between the slag materials and solution.Moreover, the fresh surfaces in the slag particles exposed on the crack caused by the microwave are beneficial for the extraction reaction [23][24][25].
Minerals 2017, 7, 119 3 of 13 ethylenediaminetetraacetic acid (EDTA) titration method of GB/T 15452-2009 [22].Moreover, the concentrations of magnesium, aluminum, iron and phosphorus at the final leaching solution were analyzed by inductively coupled plasma emission spectroscopy (IRIS Advantage ER/S, Thermo Jarrell Ash, Franklin, MA, USA).The filtered leached slag residue was washed with Reverse Osmosis water (RO water) three times and dried at 115 °C for 24 h.The mineralogical phases of the slag residue were determined by an X'Pert PRO MPD X-ray diffractometer with Cu Kα.A field emission scanning electron microscopy (Nova 400 NanoSEM, FEI, Hillsboro, OR, USA) coupled with energy dispersive spectrometer (INCAIE 350 PentaFET X-3 EDS, Oxford, UK) was used to analyse the as-received slag sample and the leached slag residue.
The leaching ratio of calcium ion was calculated by the following equation: where x is the leaching ratio of calcium; is the concentration of Ca 2+ in the filtrate, μg/mL; VT is the total volume of the leaching solution, mL; slag m is the mass of the EAF slag, g; η is the content of CaO in the EAF slag, %; MCa, MCaO are the molecular mass of Ca and CaO, respectively.
Calcium Leaching by NH4Cl with Conventional and Microwave Leaching Processes
The calcium leaching ratio from the EAF slag by NH4Cl under different leaching environments are shown in Figure 1.It can be seen from Figure 1a that the calcium leaching ratio rose significantly at the initial period (5 min) and then tends to the maximum value, which attains ~87% with 120 min leaching at 100 °C. Figure 1b is the variation of calcium leaching ratio with leaching time at constant temperature in a microwave field.Comparing with Figure 1a,b, the tendencies of the calcium leaching ratios are similar, which attains the maximum leaching ratio within 5 min.However, the calcium leaching ratio at the constant temperature in the microwave field increases about 10% than that under the water bath at the same time.This is possibly due to the fact that the polar molecules in the leaching system vibrate rapidly in the microwave field, producing a large amount of energy, which heats the solution and increases the collision frequency between the slag materials and solution.Moreover, the fresh surfaces in the slag particles exposed on the crack caused by the microwave are beneficial for the extraction reaction [23][24][25].
(a) It can be seen from Figure 1b,c that the greater the microwave power, the higher the impact of calcium leaching ratio, which proves that the thermal effect and non-thermal effect generated by the microwave field improve the leaching ratio.Meanwhile, the leaching solutions are boiling within several minutes at constant power in the microwave field.Therefore, the following experiments were further studied at the constant temperature in the microwave field.
XRD analysis of the as-received EAF slag and the leached slag residue after different leaching processes are shown in Figure 2. The crystalline phases of the as-received EAF slag, containing Ca(Fe,Al)2O5, CaFe(Si2O6), RO phase, CaCO3, CaMgSi2O6, CaSiO3, Ca2SiO4 and Ca3SiO5, are complex, while the crystalline phases of the leached slag residue at different leaching parameters are similar.Compared with the as-received EAF slag and the leached slag residue, it can be found that CaSiO3, Ca2SiO4 and Ca3SiO5 are disappeared and SiO2 is detected after the leaching process, which indicates that CaSiO3, Ca2SiO4 and Ca3SiO5 are dissolved in the ammonium chloride solution and SiO2 is produced.It can be seen from Figure 1b,c that the greater the microwave power, the higher the impact of calcium leaching ratio, which proves that the thermal effect and non-thermal effect generated by the microwave field improve the leaching ratio.Meanwhile, the leaching solutions are boiling within several minutes at constant power in the microwave field.Therefore, the following experiments were further studied at the constant temperature in the microwave field.
XRD analysis of the as-received EAF slag and the leached slag residue after different leaching processes are shown in Figure 2. The crystalline phases of the as-received EAF slag, containing Ca(Fe,Al Figures 3 and 4 are the SEM images of as-received EAF slag and the leached slag residue with microwave (85 °C) for 60 min, respectively.Table 1 shows the EDS results of the related compounds.Not only the phases detected in the XRD such as Ca2SiO4, Ca2(Fe,Al)2O5 and RO phase in Figure 3, but also the phases undetected in the XRD such as SiO2, f-CaO (free calcium oxide) and metal Fe are observed in the SEM images.This may be due to metallic iron and free lime being below the detection limit of conventional XRD.It can also be seen that the RO phase and calcium-ferrite solid solution often coexist with the calcium silicate [26,27].Besides containing iron, the RO phase, with poor hydration activity, contains high magnesium.Calcium-ferrite solid solution usually contains aluminum existing as calcium ferroaluminates.In Figure 4, it is found that the particles with high contents of iron mainly contain RO phase and calcium ferroaluminates.The RO phase contains low calcium element at original phase, while the calcium ferroaluminates contain relatively high calcium element.Given that the calcium ferroaluminates in the slag residue leached by ammonium chloride solution still contain relatively high contents of calcium, it shows that the calcium in the form of calcium ferroaluminates is difficult to extract.Moreover, as the phosphorus is mainly dissolved in C2S and C3S in the steelmaking slag [28,29], the leached slag residue particles containing phosphorus, probably the calcium silicates before leaching, have high levels of silicon and less amounts of iron and calcium, which indicates that most of the calcium has been leached from C2S and C3S phases by ammonium chloride solution.The SEM results are consistent with the XRD analysis.Figures 3 and 4 are the SEM images of as-received EAF slag and the leached slag residue with microwave (85 • C) for 60 min, respectively.Table 1 shows the EDS results of the related compounds.Not only the phases detected in the XRD such as Ca 2 SiO 4 , Ca 2 (Fe,Al) 2 O 5 and RO phase in Figure 3, but also the phases undetected in the XRD such as SiO 2 , f-CaO (free calcium oxide) and metal Fe are observed in the SEM images.This may be due to metallic iron and free lime being below the detection limit of conventional XRD.It can also be seen that the RO phase and calcium-ferrite solid solution often coexist with the calcium silicate [26,27].Besides containing iron, the RO phase, with poor hydration activity, contains high magnesium.Calcium-ferrite solid solution usually contains aluminum existing as calcium ferroaluminates.In Figure 4, it is found that the particles with high contents of iron mainly contain RO phase and calcium ferroaluminates.The RO phase contains low calcium element at original phase, while the calcium ferroaluminates contain relatively high calcium element.Given that the calcium ferroaluminates in the slag residue leached by ammonium chloride solution still contain relatively high contents of calcium, it shows that the calcium in the form of calcium ferroaluminates is difficult to extract.Moreover, as the phosphorus is mainly dissolved in C 2 S and C 3 S in the steelmaking slag [28,29], the leached slag residue particles containing phosphorus, probably the calcium silicates before leaching, have high levels of silicon and less amounts of iron and calcium, which indicates that most of the calcium has been leached from C 2 S and C 3 S phases by ammonium chloride solution.The SEM results are consistent with the XRD analysis.Figure 5 is the element mapping of typical leached slag residue with microwave power of 540 W for 10 min radiation.It shows that the particles with large amounts of calcium are rich in iron and aluminum, while those particles with less calcium are rich in silicon.Based on the thermodynamics analysis that calcium silicate and calcium ferroaluminates can react with ammonium chloride [30], as well as the fact that iron and aluminum will completely hydrolyze in the system of NH 4 Cl-NH 3 -H 2 O, it can predict that CaSiO 3 , Ca 2 SiO 4 and Ca 3 SiO 5 easily react in ammonium chloride solution and the hydrolysis of iron and aluminum on the surface of the particles hinders further calcium leaching from calcium ferroaluminates.Figure 5 is the element mapping of typical leached slag residue with microwave power of 540 W for 10 min radiation.It shows that the particles with large amounts of calcium are rich in iron and aluminum, while those particles with less calcium are rich in silicon.Based on the thermodynamics analysis that calcium silicate and calcium ferroaluminates can react with ammonium chloride [30], as well as the fact that iron and aluminum will completely hydrolyze in the system of NH4Cl-NH3-H2O, it can predict that CaSiO3, Ca2SiO4 and Ca3SiO5 easily react in ammonium chloride solution and the hydrolysis of iron and aluminum on the surface of the particles hinders further calcium leaching from calcium ferroaluminates.Since the principal phases of EAF slag are calcium silicate, RO phase and calcium ferroaluminates, and RO phase contains little calcium; the main calcium sources in the leaching reaction process are calcium silicate and calcium ferroaluminates.From Figure 1, the rapid calcium leaching step (up to 5 min) is possibly due to the easy reaction of calcium silicate, and the slower calcium leaching step (after 5 min) is owing to the difficult reaction of calcium ferroaluminates for the hydrolysis of iron and aluminum.Since the principal phases of EAF slag are calcium silicate, RO phase and calcium ferroaluminates, and RO phase contains little calcium; the main calcium sources in the leaching reaction process are calcium silicate and calcium ferroaluminates.From Figure 1, the rapid calcium leaching step (up to 5 min) is possibly due to the easy reaction of calcium silicate, and the slower calcium leaching step (after 5 min) is owing to the difficult reaction of calcium ferroaluminates for the hydrolysis of iron and aluminum.
Effect of Different Leaching Parameters on Calcium Leaching Ratio from EAF Slag
Although the leaching rate of calcium increases with the increasing of microwave power, the higher microwave power makes the solution temperature too high, even boiling and resulting in an overflow of ammonia, which goes against the absorption of carbon dioxide and the formation of calcium carbonate in the follow-up carbonate step.Therefore, the following calcium leaching experiments are studied at 60 • C under microwave fields.Figure 6 is the effect of different process parameters on the calcium leaching behavior.In Figure 6a, calcium leaching ratio increases significantly when the stirring rate is raised from 2/4 v m to 3/4 v m (v m is the maximum stirring rate of magnetic stirring); however, the leaching ratio does not increase much with the stirring rate increased from 3/4 v m to 4/4 v m .It indicates that the external diffusion is not the restrictive conditions of calcium leaching when the stirring is 3/4 v m .It is shown in Figure 6b that the calcium leaching ratio slightly increases with leaching solution volume decreasing, which may be due to the solution of the mixing intensity being relatively lower under a certain stirring rate when the reaction solution volume increased significantly.In Figure 6c,d, the calcium leaching ratio increases with the liquid-solid ratio increasing.Meanwhile, the concentration of calcium in the solution drops rapidly.Therefore, a suitable liquid-solid ratio is required considering the calcium leaching ratio and the concentration of calcium.With the concentration of ammonium chloride solution increasing, the concentration of calcium in the leachate rises (Figure 6e).It is possibly due to the fact that the lower pH value of the leaching system leaded by the higher concentration of ammonium chloride is favorable for the calcium leaching [2].However, the excessive concentration of ammonium chloride solution is also not good to avoid impacting the absorption and precipitation of CO 2 at the subsequent carbonate process.
As the extraction ratio of calcium in EAF slag is nearly 70% and reaction ratio of calcium in leaching solution with carbon dioxide almost reaches 100% [31], CO 2 uptakes can be calculated as 306 g CO 2 /kg slag for the EAF slag containing 39% calcium oxide, compared to the thin-film condition and slurry-phase route (176~280 g CO 2 /kg slag) [3].
Effect of Different Leaching Parameters on Calcium Leaching Ratio from EAF Slag
Although the leaching rate of calcium increases with the increasing of microwave power, the higher microwave power makes the solution temperature too high, even boiling and resulting in an overflow of ammonia, which goes against the absorption of carbon dioxide and the formation of calcium carbonate in the follow-up carbonate step.Therefore, the following calcium leaching experiments are studied at 60 °C under microwave fields.Figure 6 is the effect of different process parameters on the calcium leaching behavior.In Figure 6a, calcium leaching ratio increases significantly when the stirring rate is raised from 2/4 vm to 3/4 vm (vm is the maximum stirring rate of magnetic stirring); however, the leaching ratio does not increase much with the stirring rate increased from 3/4 vm to 4/4 vm.It indicates that the external diffusion is not the restrictive conditions of calcium leaching when the stirring is 3/4 vm.It is shown in Figure 6b that the calcium leaching ratio slightly increases with leaching solution volume decreasing, which may be due to the solution of the mixing intensity being relatively lower under a certain stirring rate when the reaction solution volume increased significantly.In Figure 6c,d, the calcium leaching ratio increases with the liquid-solid ratio increasing.Meanwhile, the concentration of calcium in the solution drops rapidly.Therefore, a suitable liquid-solid ratio is required considering the calcium leaching ratio and the concentration of calcium.With the concentration of ammonium chloride solution increasing, the concentration of calcium in the leachate rises (Figure 6e).It is possibly due to the fact that the lower pH value of the leaching system leaded by the higher concentration of ammonium chloride is favorable for the calcium leaching [2].However, the excessive concentration of ammonium chloride solution is also not good to avoid impacting the absorption and precipitation of CO2 at the subsequent carbonate process.
As the extraction ratio of calcium in EAF slag is nearly 70% and reaction ratio of calcium in leaching solution with carbon dioxide almost reaches 100% [31], CO2 uptakes can be calculated as 306 g CO2/kg slag for the EAF slag containing 39% calcium oxide, compared to the thin-film condition and slurry-phase route (176~280 g CO2/kg slag) [3].
Leaching Behaviors of Impurity Ions from EAF Slag
Figure 7 is the variation of concentration of calcium, magnesium, aluminum, iron and phosphorus ions in the leached solution under different leaching parameters for 120 min.The concentration of aluminum, iron and phosphorus ions are less than 10 −6 mol/L, which indicates that it can be neglected.In addition, the leaching behaviors of magnesium and calcium ions are similar.Although the concentration of magnesium is much less than that of calcium, it may be necessary to remove magnesium ion before the carbonate process [32,33].
Leaching Behaviors of Impurity Ions from EAF Slag
Figure 7 is the variation of concentration of calcium, magnesium, aluminum, iron and phosphorus ions in the leached solution under different leaching parameters for 120 min.The concentration of aluminum, iron and phosphorus ions are less than 10 −6 mol/L, which indicates that it can be neglected.In addition, the leaching behaviors of magnesium and calcium ions are similar.Although the concentration of magnesium is much less than that of calcium, it may be necessary to remove magnesium ion before the carbonate process [32,33].
Removal of Impurity Ions from the Leachate
As the concentrations of aluminum, iron and phosphorus ions can be neglected, and magnesium ion is the major impurity in the leached solution, the removal of magnesium ion by adjusting pH value was studied.A 500 mL experimental solution was set up containing 0.5 mol/L CaCl 2 , 0.2 mol/L MgCl 2 , 1 mol/L NH 3 •H 2 O and 2 mol/L NH 4 Cl, and then the configured solution was shaken up and left standing for 24 h, after which the configured solution was filtered, and the filtrate was selected as the solution for the removal experiment of magnesium ion by adding 15 mol/L sodium hydroxide solution.
In Figure 8, it can be seen that the required volume of sodium hydroxide solution to raise the pH value of the solution reduces gradually, which is due to the buffering effect caused by the system of NH 4 Cl-NH 4 -H 2 O.In addition, the concentration of calcium in the solution, 0.32 mol/L when the pH value is 11.6, is still high, which indicates that the calcium ion is probably not precipitated in the real leaching solution from steel slag by NH 4 Cl solution as the calcium concentration of the real solution is less than 0.32 mol/L (Figure 7).This is important to obtain high carbonation efficiency, since only dissolved Ca can be used for CO 2 sequestration.Indeed, Ca precipitation negatively affects CO 2 uptake.For this reason, is important to allow only Mg precipitation, maintaining Ca ions in the solution.However, the concentration of magnesium ion starts to drop sharply when the pH value is higher than 10 and it has precipitated completely at pH value of 11.6.This is due to the solubility of calcium being much bigger than that of magnesium.
Removal of Impurity Ions from the Leachate
As the concentrations of aluminum, iron and phosphorus ions can be neglected, and magnesium ion is the major impurity in the leached solution, the removal of magnesium ion by adjusting pH value was studied.A 500 mL experimental solution was set up containing 0.5 mol/L CaCl2, 0.2 mol/L MgCl2, 1 mol/L NH3·H2O and 2 mol/L NH4Cl, and then the configured solution was shaken up and left standing for 24 h, after which the configured solution was filtered, and the filtrate was selected as the solution for the removal experiment of magnesium ion by adding 15 mol/L sodium hydroxide solution.
In Figure 8, it can be seen that the required volume of sodium hydroxide solution to raise the pH value of the solution reduces gradually, which is due to the buffering effect caused by the system of NH4Cl-NH4-H2O.In addition, the concentration of calcium in the solution, 0.32 mol/L when the pH value is 11.6, is still high, which indicates that the calcium ion is probably not precipitated in the real leaching solution from steel slag by NH4Cl solution as the calcium concentration of the real solution is less than 0.32 mol/L (Figure 7).This is important to obtain high carbonation efficiency, since only dissolved Ca can be used for CO2 sequestration.Indeed, Ca precipitation negatively affects CO2 uptake.For this reason, is important to allow only Mg precipitation, maintaining Ca ions in the solution.However, the concentration of magnesium ion starts to drop sharply when the pH value is higher than 10 and it has precipitated completely at pH value of 11.6.This is due to the solubility of calcium being much bigger than that of magnesium.
Conclusions
(1) The calcium leaching ratio at the constant temperature in the microwave field increases about 10% than that under the water bath at the same time.The greater the microwave power, the higher the impact of calcium leaching ratio, which proves that microwave treatment can improve the leaching ratio.Meanwhile, the leaching solutions are boiling within several minutes at constant power in the microwave field.(2) The rapid calcium leaching step (up to 5 min) is possibly due to the easy reaction of calcium silicate and the slower calcium leaching step (after 5 min) is owing to the difficult reaction of calcium ferroaluminates for the hydrolysis of iron and aluminum.(3) The leaching behaviors of magnesium and calcium ions affected by different leaching parameters are similar and the concentration of aluminum, iron and phosphorus can be neglected.(4) Calcium ion is probably not precipitated in the real leaching solution from steel slag by NH4Cl solution as its concentration is less than 0.32 mol/L.However, the concentration of magnesium ion starts to drop sharply when the pH value is higher than 10 and it has precipitated completely at pH value of 11.6.
Conclusions
(1) The calcium leaching ratio at the constant temperature in the microwave field increases about 10% than that under the water bath at the same time.The greater the microwave power, the higher the impact of calcium leaching ratio, which proves that microwave treatment can improve the leaching ratio.Meanwhile, the leaching solutions are boiling within several minutes at constant power in the microwave field.(2) The rapid calcium leaching step (up to 5 min) is possibly due to the easy reaction of calcium silicate and the slower calcium leaching step (after 5 min) is owing to the difficult reaction of calcium ferroaluminates for the hydrolysis of iron and aluminum.(3) The leaching behaviors of magnesium and calcium ions affected by different leaching parameters are similar and the concentration of aluminum, iron and phosphorus can be neglected.(4) Calcium ion is probably not precipitated in the real leaching solution from steel slag by NH 4 Cl solution as its concentration is less than 0.32 mol/L.However, the concentration of magnesium ion starts to drop sharply when the pH value is higher than 10 and it has precipitated completely at pH value of 11.6.
Figure 1 .
Figure 1.Calcium leaching ratio from the electric arc furnace (EAF) slag by NH 4 Cl under the different environments: (a) at constant temperature under water bath; (b) at constant temperature in the microwave field; (c) at constant power in the microwave field.
) 2 O 5 , CaFe(Si 2 O 6 ), RO phase, CaCO 3 , CaMgSi 2 O 6 , CaSiO 3 , Ca 2 SiO 4 and Ca 3 SiO 5 , are complex, while the crystalline phases of the leached slag residue at different leaching parameters are similar.Compared with the as-received EAF slag and the leached slag residue, it can be found that CaSiO 3 , Ca 2 SiO 4 and Ca 3 SiO 5 are disappeared and SiO 2 is detected after the leaching process, which indicates that CaSiO 3 , Ca 2 SiO 4 and Ca 3 SiO 5 are dissolved in the ammonium chloride solution and SiO 2 is produced.
Figure 3 .
Figure 3.The SEM images of as-received EAF slag.
Figure 4 .
Figure 4.The SEM images of leached slag residue with microwave (85 °C) for 60 min.
Figure 3 .
Figure 3.The SEM images of as-received EAF slag.
Figure 3 .
Figure 3.The SEM images of as-received EAF slag.
Figure 4 .
Figure 4.The SEM images of leached slag residue with microwave (85 °C) for 60 min.Figure 4. The SEM images of leached slag residue with microwave (85 • C) for 60 min.
Figure 4 .
Figure 4.The SEM images of leached slag residue with microwave (85 °C) for 60 min.Figure 4. The SEM images of leached slag residue with microwave (85 • C) for 60 min.
Figure 6 .
Figure 6.Effect of different technological parameters on calcium leaching: (a) the stirring speeds; (b) the volume of leaching solution; (c,d) the liquid-solid ratio; (e) the leaching solution concentration.
Figure 7 .
Figure 7. Effect of different experimental parameters on Me n+ leaching: (a) the stirring speeds; (b) the volume of leaching solution; (c) the liquid-solid ratio; (d) the leaching solution concentration.
Figure 7 .
Figure 7. Effect of different experimental parameters on Me n+ leaching: (a) the stirring speeds; (b) the volume of leaching solution; (c) the liquid-solid ratio; (d) the leaching solution concentration.
Figure 8 .
Figure 8.The added volume of NaOH and the content of Mg 2+ /Ca 2+ versus pH.
Figure 8 .
Figure 8.The added volume of NaOH and the content of Mg 2+ /Ca 2+ versus pH. | v3-fos-license |
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} | pes2o/s2orc | Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates
Background Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. Results Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). Conclusions The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity. Electronic supplementary material The online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.
cleavage because of its high tolerance to organic solvents and remarkable bioconversion [14][15][16], involving the ∆ 1 -dehydrogenation of steroids. This dehydrogenation is a crucial modification in steroid synthesis because it increases the biological activity and economical value of the original steroidal substrate [17,18]. Both cortisone acetate and prednisone acetate are used as anti-inflammatory and anti-allergy drugs clinically [14,19,20]. The anti-inflammatory activity of prednisolone acetate increased three-to fourfold when introducing a C1-C2 double bond into ring A of hydrocortisone acetate by ∆ 1 -dehydrogenation, catalyzed by 3-ketosteroid-∆ 1dehydrogenase [14,21,22].
3-Ketosteroid-∆ 1 -dehydrogenase (KsdD, EC 1.3.99.4), which catalyzes the insertion of a double bond between the C1 and C2 atoms of the 3-ketosteroid A-ring ( Fig. 1a), has been found in several steroid-degrading bacteria, including A. simplex [22], Rhodococcus rhodochrous [23], Pseudomonas testosteroni [24], Nocardia coralline [25], and Mycobacterium sp. [26]. The enzyme KsdD is a flavoprotein dehydrogenating a wide variety of 3-ketosteroids. It is a key enzyme in microbial steroid catabolism needed for opening of the steroid B-ring. The catalytic mechanism of dehydrogenation of the C1-C2 bound of 3-ketosteroids has been elucidated: the two hydrogen atoms on the respective C1 and C2 atoms of the substrate undergo direct elimination without the formation of an hydroxyl intermediate or H 2 O [27][28][29]. A two-step mechanism has been proposed that indicates that the catalytic process proceeds via trans-diaxial elimination of the 1α,2β hydrogen atoms from a 3-ketosteroid substrate [30]. Interaction of the C3 carbonyl group of a steroidal substrate with an electrophile promotes labilization of the hydrogen atoms at the C2 position. A general base has been proposed to abstract a proton from C2 atom resulting in an enolate or a carbanionic intermediate. After hydride ion transfer from the C1 atom to flavin adenine dinucleotide (FAD) [31,32], a double bond is then formed between the C1-C2 atoms. The crystal Structure of KstD1 from Rhodococcus erythropolis revealed that Tyr487 and Gly491 promoted tautomerization, and Tyr318/Tyr119 and FAD abstracted a proton and a hydride ion, respectively [33,34]. These researches provided a fundamental understanding of KstD family enzyme, and the theoretical support for rational design of A. simplex KsdD3.
Recently, a novel putative 3-ketosteroid-∆ 1dehydrogenase gene from A. simplex (KsdD3, also called Pimelobacter simplex) has been identified. KsdD3 contained 1548 bp of a complete open reading frame encoding a protein (516 amino acids, 54.3 kDa) without signal peptide. Efforts have been made on A. simplex 156 to improve the ∆ 1 -dehydrogenation efficiency of this dehydrogenase by genetic manipulation, in which the control of the cat promoter was transferred into the strain and integrated into the 16S rDNA sites [14]. In our preparing experiment, we found that KsdD3 was largely overexpressed in solubility, and it showed the high catalytic activity. However, the enzymatic characterization and catalytic activity of KsdD3 remain unclear, and the mechanism of substrate recognition and selectivity are poorly understood. This has hampered its further application as an industrial catalyst. Therefore, the rational design of functional KsdD3 based on structural modelling analysis would strengthen our understanding of KsdD family enzymes [35]. To engineer an enzyme with a higher catalytic activity and broader selectivity, structure-based sitedirected saturation mutagenesis and biochemical assays were used to obtain KsdD3 mutants.
Cloning, expression and purification
The genomic DNA of A. simplex 156 was used as a template. The KsdD3 gene (GenBank accession No. AIY19527.1; Protein ID: WP_038682818.1) was amplified using PrimeSTAR MAX as DNA polymerase. The KsdD3 gene was subsequently inserted into pET28a(+) (Novagen, Madison, WI, USA) between the Nde I and EcoR I sites with a His 6 tag (HHHHHH) at the N-terminus. E. coli BL21(DE3) harboring pET28a-KsdD3 was grown in Luria-Bertani (LB) medium at 37 °C and 160 rpm. When the OD 600 reached 0.6-0.8, 0.5 mM isopropyl β-d-1thiogalactopyranoside (IPTG) was added into the cultures, and the medium was further incubated at 16 °C for 20 h. The cells were harvested by centrifugation at 5000×g and 4 °C for 15 min, and were then resuspended in lysis buffer [20 mM Tris-HCl (pH 8.0), 20 mM imidazole, 0.5 M NaCl, and 1 mM dithiothreitol (DTT)]. The pellets were disrupted by sonication, and centrifuged at 40,000×g for 30 min at 4 °C for the removal of cell debris. KsdD3 proteins in the supernatant were trapped on Ni-NTA superflow resin (Qiagen, Hilden, Germany). After washing, KsdD3 was eluted with elution buffer [20 mM Tris-HCl (pH 8.0), 400 mM imidazole, 0.3 M NaCl, and 1 mM DTT]. The KsdD3 was identified with 98% purity on SDS-PAGE with Image Lab ™ Software version 5.2.1 (Bio-Rad, Hercules, CA, USA) (Additional file 1: Fig. S1). The yellow-coloured KsdD3 was exchanged into buffer [20 mM Tris-HCl (pH 8.0), 0.2 M NaCl and 20 µM FAD] via ultrafiltration (10 kDa molecular weight cutoff ), finally diluted with the same buffer to 5 mg L −1 for enzyme activity assay. Protein concentrations were determined using the BCA protein assay kit (Solarbio, Beijing), with bovine serum albumin as a standard [36,37].
Activity assay
The enzyme activities of KsdD3 and its mutants were determined spectrophotometrically at 30 °C using PMS and DCPIP as electron acceptors [34,38]. The reaction mixture consists of 50 mM Tris-HCl, pH 7.5, 1.5 mM PMS, 40 μM DCPIP and 500 μM AD in methanol (2%) in 1 mL volume. The reaction was initiated by adding purified KsdD3 to a final concentration of 3.1 μM. The control was prepared by using the reaction system without the KsdD3 enzyme. In the dehydrogenation reaction, two protons were transferred from FADH 2 to form PMSH 2 , and the reduction of DCPIP was performed by transferring 2H of PMSH 2 to form DCPIPH 2 , which caused a decrease in the absorbance at 600 nm. The decreased absorbance at 600 nm (ε 600 nm = 18.7 × 10 3 cm −1 M −1 ) was monitored using a microplate reader [Infinite M200 Pro (Tecan Austria GmbH, Austria)] every 20 s for 3 min. One unit of activity was defined as the amount of enzyme giving a reduction of 1 µmol min −1 DCPIP. Specific activities are defined as nmol mg −1 min −1 (mU mg −1 ).
Characterization of KsdD3
AD was used as the substrate for the characterization of KsdD3. The optimal pH for purified KsdD3 was determined at 30 °C using different reaction buffers, which contained 50 mM each of MES, PBS, HEPES, Tris-HCl and Gly-NaOH in the pH rang of 5.5-9.5. KsdD3 activity at 30 °C, Tris-HCl buffer pH 7.5 was defined as 100%.
The effect of pH on the stability of KsdD3 was measured by determination of the residual activity in the standard assay, after buffer exchange of KsdD3 and pre-incubation for 2 h at 4 °C. Buffer exchange was performed by ultrafiltration (Millipore, Merck, German) at 4 °C and 3000×g. The maximal KsdD3 activity at 30 °C, Tris-HCl buffer pH 8.0 was defined as 100%.
To determine the optimal temperature, the activity assay was performed at various temperatures from 25 to 55 °C in 50 mM Tris-HCl buffer (pH 7.5). KsdD3 activity at 30 °C, Tris-HCl buffer pH 7.5 was defined as 100%.
The thermostability was determined at 30 °C by measuring the residual activity, after the incubation of the enzyme in Tris-HCl buffer (50 mM, pH 8.0) at different temperatures (25-55 °C) for 30, 60 and 120 min, and then cooling the mixture on ice for 10 min. The maximal KsdD3 activity at 25 °C, Tris-HCl buffer pH 8.0 was defined as 100%.
The effect of the organic solvents (dissolving the same amount of AD) on KsdD3 activity was determined by adding different volumes (0.8-35%, v/v) of methanol into the reaction system. The maximal KsdD3 activity at 30 °C, Tris-HCl buffer pH 7.5 and 2% methanol was defined as 100%.
To measure the organic-solvent tolerance, KsdD3 was pre-incubated in various solvents (methanol, ethanol, isopropyl alcohol, DMSO, DMF, acetone and acetonitrile) at different concentrations (10-50%, v/v) for 2 h in Tris-HCl buffer (50 mM, pH 8.0) at 4 °C. Before determination, the mixture was cooled on ice for 10 min, and the activity was then measured under standard reaction conditions of 30 °C and Tris-HCl buffer pH 8.0. The maximal KsdD3 activity under standard reaction conditions was defined as 100%.
Kinetic studies of KsdD3 wild-type (WT) and mutants were performed in the same reaction system using seven different concentrations of steroidal substrates, i.e., CA and 11α-OH-P: 0-300 μM; 21-DC: 0-400 μM; others: 0-500 μM. The fitting curves of KsdD3 WT and mutants toward seven steroidal substrates were plotted, and the kinetic parameters, K m and k cat were calculated using the Michaelis-Menten equation with GraphPad Prism 7.0 (GraphPad software, La Jolla, CA) by employing nonlinear regression.
where V is the substrate consumption rate in µmol s −1 L −1 ; V max is the maximum substrate consumption rate; [S] is the substrate concentration in µM; C is the concentration of KsdD3 in the reaction mixture; K m is the Michaelis-Menten constant in µM, and is equal to the concentration of the substrate when the reaction rate is half of the maximum velocity; k cat represents the turnover number in s −1 , and k cat /K m is also a constant on behalf of the catalytic efficiency of KsdD3 WT and mutants.
Product analysis of steroidal substrates using HPLC, GC-MS and NMR
The reaction system contains 10 mg of KsdD3, Tris-HCl buffer (50 mM, pH 8.0), 2 mM DTT, 500 µM steroids in DMSO (2%), and 10 µM FAD. Reactions were incubated at 30 °C overnight and then terminated by boiling water at 100 °C for 5 min. Denatured protein was removed by centrifugation at 12,000 rpm for 10 min. The reaction products were extracted with 0.5 mL of ethyl acetate, twice. After drying under a stream of nitrogen gas, the steroidal products were resuspended in 20 μL methanol for HPLC and GC-MS analysis, or dissolved in deuterated chloroform/DMSO for NMR analysis. The products of 1,4-androstadiene-3,17-dione, boldenone, methandienone, prednisone, prednisone acetate and 1-dehydroprogesterone were analyzed by GC-MS (VARIAN 4000 GC/MS) using a HP-5MS column (30 m × 0.25 mm × 0.25 mm, Agilent Technologies) in electron ionization (70 eV) mode. The temperature programming for GC column is the same as published before, and helium was used as the carrier gas at a flow rate of 1 ml min −1 [37]. The retention times and mass spectra of all peaks obtained were compared with those of standards available in the NIST library (nistmasspeclibrary.com).
Site-directed mutagenesis
Site-directed mutagenesis of KsdD3 was performed by reverse PCR with pET28a-KsdD3 as a template using the KOD-Plus-Mutagenesis kit (Toyobo, Japan) [39]. The primers used for mutagenesis are summarized in Additional file 1: Table S1. PCR was conducted using temperature settings of 94 °C for 2 min followed by 8 cycles of 98 °C for 10 s, 68 °C for 8 min. The template plasmid was digested using Dpn I after PCR, and product was further cyclized by T4 polynucleotide kinase and ligation high (a DNA ligase). All KsdD3 mutants were expressed and purified using the same procedures as for the WT enzyme.
Structure modeling of KsdD3
The three-dimensional (3D) model of KsdD3 was generated using Modeller 9.9.2 [40]. The crystal structure of 3-ketosteroid-∆ 1 -dehydrogenase from R. erythropolis SQ1 (PDB ID: 4C3×, 2.0 Å, 46% sequence identity with KsdD3) was chosen as the template [34]. Homology modelling was performed by the automodel command. Thereafter, each model was optimized by the variable target function method with conjugate gradients. Simulated annealing MD was then used to refine the structure [41]. The best model was chosen based on the values of the Modeller objective function and the DOPE assessment scores.
Steered molecular dynamics simulations and PMF calculations
Steered molecular dynamics (SMD) simulations apply an external force to the substrates' center of mass that is pulled out from the KsdD3 active site along a predefined direction. The substrate-protein complexes were used in Gromacs 5.1.2 software using the Gromos 96 53A6 force field. The GROMOS96 53a6 force field parameters of the substrate were obtained from the Automated Topology Builder and Repository 2.0 webserver (https ://atb. uq.edu.au/). The energy minimizations of the simulation systems including water molecules with and without substrate AD were optimized (Additional file 1: Table S2), and the potential energies of the two systems are shown in Table 2. The constant-velocity SMD simulations were performed in the present simulations. The pulling velocity was set to 0.01 Å ps −1 . A spring constant of 1000 kcal mol −1 Å −2 was applied to the substrate's centre of mass. Based on the above SMD trajectories, snapshots were taken to generate the starting configurations for the umbrella sampling windows. An asymmetric distribution of sampling windows was used to calculate the potential of mean force profiles of the WT and mutants over the distance along the substrate channel.
Bioconversion of steroids with purified KsdD3 enzyme and resting cells of E. coli
Enzymatic conversion of steroids was carried out in a 20 mL reaction mixture consisting of 50 mM Tris-HCl (pH 8.0), 25 µg purified KsdD3, 25 µM FAD, 1 g L −1 substrates (dissolved in 3% v/v DMSO) and 0.1 mM PMS at 30 °C. Recombinant E. coli BL21(DE3) harboring KsdD3 were cultivated as described above. Cells were harvested and washed with 50 mM Tris-HCl (pH 8.0). The biotransformation was performed in 10-mL reaction volume (30 mg of cell wet weight mL −1 ) containing 50 mM Tris-HCl (pH 8.0), 0.5 mM PMS and 5 g L −1 substrates (dissolved in 5% v/v DMSO). 1.0 mL samples of resting cells were acquired at intervals. Cells were disrupted by sonication, and the products were extracted twice with ethyl acetate. After drying under a stream of nitrogen gas, the samples were resuspended in 1.0 mL methanol for HPLC analysis. All reactions with selected steroids were performed in triplicate with three independent measurements, and controls were prepared with E. coli BL21(DE3) not harboring KsdD3. The theoretical conversion of WT was defined as 100% for calculating the relative activity of the mutants.
HPLC (Agilent Technologies, Waldbronn, Germany) was used to separate the substrates and products using a Diamensil C18 column (5 μm, 4.6 × 250 mm) at 30 °C. The solvent systems and UV absorbance wavelengths used depended on the substrates: the mobile phase consisted of 70% methanol and 30% water (v/v) with a flow rate of 0.6 mL min −1 at 241 nm for the substrates AD, testosterone and 17-MT; 30% acetonitrile and 70% water (v/v) with a flow rate of 0.6 mL/min at 254 nm for cortisone [17]; 58% methanol and 42% water (v/v) with a flow rate of 1 mL min −1 at 240 nm for CA. The quantitative analysis of the target compounds was based on the standard curve (correlation coefficients were > 0.999). The yields of KsdD3 WT and mutants towards different steroids were calculated according to the equation: where C 0 and C 1 are the concentration of the experimental and theoretical products, respectively.
Enzyme characteristics of KsdD3
KsdD3 is a FAD-dependent enzyme that catalyzes the formation of a double bond at the C1-2 position of 3-ketosteroid substrates (Fig. 1a). The catalytic activity of KsdDs is highly dependent on the assay conditions [17,27], and a comparison of buffer composition, pH, temperature and surfactant used to solubilize the substrate was investigated. The purified WT KsdD3 was active at pH 6.0-9.5 and the maximal activity was observed in Tris-HCl buffer pH 7.5. The KsdD3 exhibited good pH stability over the pH range 7.0-9.0 and more than 85% of the maximal activity was retained over this range (Fig. 2a). The optimal temperature for KsdD3 activity was 30 °C. The activity began to decrease dramatically when the temperature was over 40 °C. KsdD3 showed a better thermostability after incubating the enzyme in Tris-HCl buffer at different temperatures (25-55 °C) for 0.5 h, which retained over 65% residual activity until 40 °C. However, there was only 14% residual activity after preincubation at 40 °C for 1-2 h (Fig. 2b). KsdD3 lost almost all the activity at 45 °C. 3-ketosteroid-∆ 1 -dehydrogenase catalyzes the reaction of substrates in organic solvents and detergents using hydrophobic interactions because of the negligible solubility of steroidal substrates in buffers. KsdD3 retained over 80% activity in reaction systems including up to 7.5% (v/v) methanol, but lost almost all catalytic ability in 35% methanol. The catalytic activity of KsdD3 in various micelles was determined to investigate organic solvent resistance. All micelle solutions improved enzyme activity when KsdD3 was treated with 10-30% solutions, indicating that KsdD3 from A. simplex exhibited a higher tolerance to other organic solvents than for methanol (Fig. 2c). This is consistent with the previous reports that A. simplex has been widely used in steroidal transformations for its high tolerance to organic solvents [42].
Structural analysis of the KsdD3 homology model
A homology model of KsdD3 was generated using R. erythropolis SQ1 ∆1-KSTD1 as template (PDB ID: 4C3×, sequence identity 46%), which possessed a traditional Rossmann fold (nucleotide-binding fold) (Fig. 4a). KsdD3 contains a FAD binding domain and a catalytic domain. FAD is bound non-covalently to KsdD3 through a variety of interactions. The adenine moiety is located in a hydrophobic pocket formed mainly by the side chains of A12, V35, A37, A264, A197, L198 and A232. E36 has a bidentate hydrogen bonding interaction between the carboxylate group with the diol O2 and O3 atoms of ribose. The pyrophosphate oxygen atoms are hydrogen bonded to T44, T45, N260 and N478 of KsdD3. The isoalloxazine ring is surrounded by Y47, G49, G51, L156, M255, F296 and V359, which are almost all hydrophobic residues (Fig. 4b). The catalytic residues included Y120, Y320, Y488 and G492, which are conserved in the KsdD family (Fig. 4c). On the basis of the conserved catalytic mechanism of flavoenzymes [34], Y488 and G492 promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With the assistance of Y120, the general base Y320 abstracts the hydrogen from C2 as a proton, whereas FAD accepts the hydrogen from the C1 atom of the substrate as a hydride ion.
The substrate-binding domain shows a variety of different topologies in the KsdD family (Additional file 1: Fig. S5). The internal cavity is occupied by partially conserved hydrophobic residues: W117, F296, L298, W299, T357, V359 and G491 (Fig. 4d), which are predicted to interact with the buried substrate in this cavity.
Comparison of KsdD3 with family enzymes
Sequence alignment shows that KsdD3 shares at most 46% sequence identity with R. erythropolis SQ1-KsdD1, which gives us only a traditional Rossmann fold (nucleotide-binding fold), a FAD binding domain and a catalytic domain. The mechanism of substrate recognition and selectivity are poor understood. Xie reported that S138 played an important role in maintaining the active center via hydrogen bond network of Mycobacterium neoaurum KsdD [43]. However, this residue is partial conserved in this family enzyme, which is H134 in A. simplex and S131 in R. erythropolis SQ1 (Additional file 1: Fig. S5). Shao found that V366S stabilized the active center and enhancing the interaction of AD in M. neoaurum KsdD [44]. V366 was positioned to D321 of A. simplex KsdD3. Both H134S and D321S in A. simplex KsdD3 abolished the catalytic activity, which implied that H134 and D321 placed the irreplaceable role in substrate recognition. E140 and Y472 of M. neoaurum KsdD were located at the same position as I136 and F423 in A. simplex KsdD3 [45]. They were reported to form a hydrophobic pocket near the active site, and were crucial for catalysis because I136E destroyed the activity toward AD (Additional file 1: Fig. S6). Two mutants of S325F and T503I from R. erythropolis SQ1-KsdD2 have also been proven to play important role in substrate recognition or catalytic reaction [46]. T503 (T450 in A. simplex KsdD3) are conserved while S325 (A281 in A. simplex KsdD3) are partial conserved. However, both residues were located far away from substrate-binding site, and were not considered to react with substrate.
The structural model of KsdD3 showed that F296, L298 and W299 are positioned at the entrance of substratebinding cavity, and W117 is located at the inner cavity of substrate-binding cavity. Neither Trp is conserved in KsdD family. Furthermore, W299 and W117 form hydrophobic recognition wall between indolyl group and AD ring. This interaction relieves the steric interference between substrate and KsdD3. Hence, W299 and W117 were selected as the mutation sites for rational design.
Structure-based rational design of KsdD3
A single point mutagenesis group containing 20 single mutants at 11 residues discussed above was constructed and evaluated to identify the role of specific amino acids in substrate selectivity and structure-function relationships. The mutation of residues Y115, W117, H134, I136, F296, L298 D321 and M361 inactivated the enzyme towards AD (Additional file 1: Fig. S6), indicating that these residues play a crucial role in substrate recognition. P139A/D showed a similar catalytic level as WT KsdD3. Interestingly, W299A mutant improved the catalytic activity toward AD, while W117 mutants inactivated KsdD3. The W299A mutant enlarged the substratebinding cavity and relieved the steric interference with substrates, facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates (Fig. 5). Therefore, the rational design was performed beginning with site-directed saturation mutagenesis of W299 to investigate its catalytic activity under guidance of structural model of KsdD3 homologue.
The results revealed that substitution by a few amino acids, i.e. Ala, Glu, Gly, His, Asn, Gln, Ser and Tyr, resulted in a higher activity towards AD than was shown by WT KsdD3 (Additional file 1: Fig. S7). W299D/K/M/R showed a similar catalytic activity as the WT enzyme.
However, substitution with Cys, Phe, Ile, Leu, Pro or Val yielded a low activity. The biochemical data may imply that hydrophilic or hydrophobic residues with a smaller side chain (Ala or Gly) play a crucial role in substrate recognition at this position. W299A and W299G showed higher catalytic efficiency (k cat /K m ) and specific activity towards the selected substrate, i.e. testosterone, AD and cortisone, compared with the WT enzyme (Table 1 and Additional file 1: Figs. S8 and S9).
Additional file 1: Fig. S10 shows the PMF profiles of KsdD3 WT and mutants dissociating along the substrate channel. The analysis revealed that the values of ∆G off are − 6.14 kcal/mol for W199A mutant and − 7.10 kcal/mol for W299G, which is lower than that of WT (− 11.45 kcal/mol) ( Table 2). Therefore, as for the two mutants W299A and W299G, the association of substrate and the dissociation of product are easier than for WT. This is in agreement with the experimental values of the substrates ( Table 1).
Bioconversion of steroids by KsdD3 enzyme and resting cells
The bioconversion of steroidal substrates with purified KsdD3 enzyme and E. coli resting cells (Fig. 6) over 48 h was investigated. The ADD productivity gradually increased, and reached a plateau at 30 h. WT, W299A and W299G showed maximum ADD yields of 38, 71 and 58%, respectively, catalyzed by the KsdD3 enzyme at 48 h.
Conclusions
In summary, we have characterized KsdD3, showing that this enzyme exhibits organic solvent tolerance. Residues related to substrate recognition around the active site were investigated and mutation on W299 improved the substrate selectivity and activity based on rational design and protein engineering technologies. The SMD results and PMF calculations show that KsdD3 mutants need to overcome a lower potential energy barrier than the WT. Furthermore, both the KsdD3 enzyme mutants and E. coli strains harbouring KsdD3 genes improved the yields observed with several steroidal substrates.
The relative catalytic activity of KsdD WT and mutants toward various steroidal substrates. Fig. S9. Michaelis-Menten plots of KsdD3 WT and W299A, W299G mutants toward nine steroidal substrates. Fig. S10. Potential of mean force (PMF) profiles of the KsdD3 wild type and mutants over the distance along the substrate channel. | v3-fos-license |
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} | pes2o/s2orc | Aspen Soils Retain More Dissolved Organic Carbon Than Conifer Soils in a Sorption Experiment
The effect tree species have on soil organic carbon (SOC) has been hotly debated but, so far, few clear patterns have emerged. One example of a differing tree species effect on SOC are aspen forests in North America, which have been found to have more stable SOC than adjacent conifer forest stands. An important source for the formation of stable organo-mineral complexes in soil is dissolved organic carbon (DOC). DOC concentrations in mineral soil are often higher under the thick O-horizons of conifer forests than under aspen forests, but this does not correspond to more stable mineral SOC. This suggests that, instead of DOC concentration, DOC quality could be driving the observed differences in SOC. Therefore, we quantified the retention of contrasting forest detritus DOC in soils. Using a batch sorption experiment approach, we compared the retention of detritus leachates from four sources – aspen leaves (AL), aspen roots (AR), conifer (subalpine fir) needles (CN), and conifer (subalpine fir) roots (CR) – on soils sampled under aspen and conifer (subalpine fir and Douglas fir) overstories. The calculated sorption isotherms showed a higher retention of AL DOC than AR DOC, as indicated by all four sorption parameters – k and n (curve-fitting parameters), null point concentration (NPC; net sorption = net desorption), and endpoint (EP, retention at the highest initial DOC concentration). Leachates from CN and CR showed very similar retention behavior, and between the two species the retention of root leachates was more similar than the retention of foliage leachates. Soils sampled from aspen forests showed higher affinity for new DOC than conifer soils [higher sorption rate (n), lower NPC, and higher EP] regardless of the DOC source. The findings suggest that the higher DOC sorption on aspen soils might be a major driver for more stable SOC under aspen stands in North America.
INTRODUCTION
As forest soils store as much carbon as aboveground biomass (Pan et al., 2011), information on tree species' effects on soil organic carbon (SOC) storage is of interest to ecologists, ecosystem modelers, and forest managers. Most synthesis studies on this topic have not found globally consistent patterns (Vesterdal et al., 2013;Boča et al., 2014;Lin et al., 2017), but some species or functional groups stand out in terms of SOC storage and stabilization.
In a literature review, Laganière et al. (2017) reported that, in North America, SOC under quaking aspen (Populus tremuloides Michx.) is consistently more stable than under adjacent conifer stands. This is an important finding considering that quaking aspen is the most widely distributed tree species in North America (Little, 1971), and its current decline (Rogers, 2002;Di Orio et al., 2005) is often accompanied by conifer encroachment (Potter, 1998). While no consistent difference in SOC pools was detected by Laganière et al. (2017), in several areas in the state of Utah, United States, higher SOC stability was also associated with higher total SOC pools compared to nearby conifer stands (Woldeselassie et al., 2012;Boča and Van Miegroet, 2017). An analysis of C fluxes in these ecosystems (higher litterfall under aspen, higher DOC concentrations under conifers, higher fine root biomass under conifers, similar root turnover under both overstories), however, could not explain the differences in SOC pools under both overstory types . This leaves two mechanisms as potential drivers: (i) difference in dissolved organic matter (DOM) quality and sorption, and/or (ii) difference in root exudation. In this region, as in most of the Intermountain Western US, the majority (>75%) of the precipitation falls as snow. Spring snowmelt creates the largest soil water fluxes that affect the whole soil profile (fall rains mostly affect only topsoil) (LaMalfa and Ryle, 2008). This makes spring snowmelt water fluxes a likely pathway for C redistribution in soil, as soil macrofauna have not been observed in these areas (Ayres et al., 2009;Boča and Van Miegroet, 2017). We are not aware of a study that has compared root exudation under aspen and conifer trees. Higher specific root length of aspen compared to conifers (e.g., Steele et al., 1997;Bauhus and Messier, 1999) could, however, suggest more dynamic belowground processes under aspen. In this study, we examined the first mechanism: DOM quality and sorption.
In its dissolved form, organic matter can be transported through the soil profile and sorbed to mineral surfaces or incorporated into microbial biomass attached to these surfaces, thus participating in the formation of stable mineral-bound organic matter (Qualls, 2000;Kalbitz et al., 2005;Kalbitz and Kaiser, 2008). Factors affecting sorption are: (i) Fe and Al oxyhydroxide concentrations in soil (e.g., Moore et al., 1992;Lilienfein et al., 2004;Heckman et al., 2011); (ii) native SOC concentration, which affects the potential of soils to retain more C (Hassink, 1997;Six et al., 2002;Stewart et al., 2007); and (iii) the quantity and quality of dissolved organic carbon (DOC). Higher concentrations of DOC are known to result in higher total retention of C in laboratory sorption experiments, but DOC fluxes in the field do not correlate with SOC pools (as reviewed by Michalzik et al., 2001). Field measurements in Utah revealed higher DOC concentrations in soils under conifers . Considering that the examined plots were less than 50 m apart, and had similar soil characteristics (Van Miegroet et al., 2005;Olsen and Van Miegroet, 2010;Boča and Van Miegroet, 2017), the higher DOC under conifers should have resulted in higher C concentrations and higher mineral-associated SOC. Yet the opposite pattern was observed (Román Dobarco and Van Miegroet, 2014;Boča and Van Miegroet, 2017). This again suggests that other factors, such as DOC quality, are potentially more important drivers for sorption in this case.
The litter of quaking aspen is considered more labile than conifer litter due to differences in nutrient and lignin concentrations (Moore et al., 2006). In a litter decomposition study, Prescott et al. (2000) also suggested leaching losses as a major reason for faster aspen vs. conifer litter degradation. In the first year of decomposition, the mass loss of 35% from aspen litter (Prescott et al., 2000) was similar to the 32% leachable content from aspen litter observed by Taylor et al. (1989). DOM quality has been proposed as a major factor affecting organic matter sorption in soil with hydrophobic and more aromatic compounds being preferentially sorbed to mineral surfaces compared to more labile polysaccharide-derived hydrophilic DOM (Kaiser and Guggenberger, 2000;Kalbitz et al., 2005). Recently, Cotrufo et al. (2015) showed that SOC can be formed with high efficiency through microbial processing of DOM produced during the early stages of litter decomposition (labile non-structural compounds).
Root detritus is considerably less examined as a source of DOM, prohibiting researchers from calculating estimates of root DOC contribution to SOC (Kalbitz and Kaiser, 2008). Based on a soil column experiment, Uselman et al. (2007) suggested that root DOC could contribute to the accumulation of SOC, and later reported that fine root DOM was less labile than foliage DOM (Uselman et al., 2012). Hansson et al. (2010) reported no differences in aromaticity and sorption rates between root and needle DOM, but did find lower DOC production rates from roots. Both studies examined root and foliage DOM from coniferous species. We are not aware of any published data on root DOM quality from contrasting tree species. Finér et al. (1997) reported faster aspen root decomposition compared to adjacent conifers, but, as no data on root quality were recorded, it is unclear whether faster decomposition reflects differences in root DOM quality. Overall, the sorption of root DOM is a knowledge gap that needs to be filled.
The objective of this study was to investigate the retention and release (sorption and desorption) by forest soils of foliage-and root-derived DOC from two contrasting tree species -quaking aspen and subalpine fir [Abies lasiocarpa (Hook.) Nutt.] -in the montane regions of Utah in the western US. The proximity of the aspen and conifer forest stands in Utah's mountains, and their contrasting litter quality, make them ideal study systems to answer questions regarding the effects of substrate quality on DOC sorption. We used a batch sorption approach to quantify DOC retention based on: (i) the source and quality of leachate derived from aspen and fir, and (ii) soil properties at different sites and soil depths. We hypothesized that: (i) aspen foliage DOC will be more labile in its chemical composition than root DOC and fir needle DOC, and thus will be more effectively retained in the soil; (ii) native DOC will sorb better on native soil; (iii) topsoils will experience lower sorption due to higher initial SOC concentrations than subsoils; and (iv) soils with higher Fe and Al oxyhydroxide concentrations will experience higher sorption.
Soil Sampling and Analysis
Soils for the experiment were collected from adjacent aspen and conifer forest stands at T. W. Daniels Experimental Forest (TWDEF) in northern Utah and at Cedar Mountain (CM, specifically plot CM17) in southern Utah. A detailed description of the sampling sites and the sampling procedure is provided in Boča and Van Miegroet (2017). In brief, TWDEF is located at 2600 m elevation with an average annual temperature of 3.1 • C and mean annual precipitation of 1031 mm, with about 70% accumulating as snow (NRCS 1 ; USU Doc Daniel SNOTEL station). Cedar Mountain is a high−elevation plateau (1800-3200 m) with an average annual precipitation of 823 mm and a mean annual temperature of 4.7 • C [NRCS (see footnote 1); Kolob and Webster flat SNOTEL stations). Forests at both sites most likely originated from natural regeneration about a century ago (Wadleigh and Jenkins, 1996;DeRose and Long, 2007). Soils at both sites have been classified as Mollisols and Alfisols under aspen stands and as Alfisols under conifer stands, according to USDA soil taxonomy (McNab and Avers, 1994;Van Miegroet et al., 2005;Olsen and Van Miegroet, 2010).
The CM soils had two to three times higher total Fe and Al oxyhydroxide and SOC concentrations than those at TWDEF ( Table 1; Boča and Van Miegroet, 2017). As oxyhydroxides affect sorption behavior (e.g., Heckman et al., 2011;Kramer et al., 2012), we considered site as a factor in further analyses. The soils were collected from the top 10 cm (topsoil) and 40-50 cm (subsoil) of the soil profile to capture differences in native SOC concentration within a given overstory type. The lower sampling depths correspond to the ABt and BAt horizons under aspen and Bt horizons under conifers.
Soil texture was determined by particle size analysis with the hydrometer method at Utah State University's Analytical Lab. pH was measured by mixing 10 mL soil with 10 mL water on the ATI Orion 950 Ross FASTQC Titrator. Soils were extracted in triplicate with sodium pyrophosphate (NaPP), acid ammonium oxalate (AAO), and citrate-dithionite (CD) to estimate Fe and Al that were organically bound, or present in short range ordered (non-crystalline) and crystalline hydrous mineral phases (Sparks et al., 1996). The extracts were analyzed with an Atomic Absorption Spectrometer (Varian AA240 flame atomization, Australia). Concentrations of non-crystalline Fe and Al oxyhydroxides were calculated by subtracting NaPP values from AAO values, and concentration of crystalline Fe oxides was calculated by subtracting AAO from CD. Clay mineralogy was determined with an X-Ray diffraction spectrometer (Panalytical X'Pert Pro with monochromatic Cu K-alpha radiation). The soil was ground to <250 µm and analyzed for total organic carbon and inorganic C with Skalar Primacs SLC Analyzer (Skalar, Inc., Breda, Netherlands).
Leachate Preparation and Analyses
The plant material used in the experiment was collected at TWDEF and CM at the end of the 2015 growing season, and consisted of senesced aspen leaves, subalpine fir needles, and fine roots (<2 mm diameter) obtained from soil cores in both forest types at both sampling sites. The needles used were older, and collected from the Oi layer of the O-horizon. They were mixed with freshly senesced needles based on calculations of annual litterfall additions to the O-horizon. This was done to ensure that we are comparing similar material (i.e., Oi layer) for aspen and fir. We used a mix of older and fresh fir needles because, in contrast to aspen stands, most of the DOC under conifers originates from an O-horizon, which is mostly dominated by older foliage material in various stages of decomposition (Fröberg et al., 2003). The material was ground with a Wiley mill (20 mesh; Thomas Scientific, NJ, United States), analyzed for C with Skalar Primacs SLC Analyzer (Skalar, Inc., Breda, Netherlands), and for total nitrogen with PDZ Europa ANCA GSL IRMS elemental analyzer (Sercon Ltd., Cheshire, United Kingdom).
DOC stock solutions were obtained following a method developed prior to the experiment. In brief, 20 g of ground foliage or root material were saturated with ultrapure water and subjected to two freeze-thaw cycles for a week to facilitate the release of DOC from substrates. Freezing and thawing are common processes in the field sites during fall when air temperatures drop below 0 • C during night, and rise above freezing during the day 2 (NRCS SNOTEL -Kolob station). The thawing temperature was set at 5 • C to reduce microbial decomposition of the material. After thawing the material a second time, the substrates were leached with 2 L of a 0.08 millimolar KCl solution, which corresponded to an electrical conductivity (EC) of around 10 µS cm −1 , similar to the EC detected in snow sampled from the TWDEF site during spring 2014 and 2015 (Boča, unpublished data). The leachates were created by vacuum-filtering a litter-water slurry through a glass fiber filter (Sterlitech 0.4 µ m).
The stock solution of each leachate was analyzed for DOC immediately after the leaching, so that four working concentrations of around 10, 20, 40, and 80 mg L −1 could be prepared on the same day as the stock solution. The DOC concentrations used were within the range of DOC concentrations observed in soil pore water at TWDEF . The working solutions were adjusted with KCl to have a constant EC of around 150 µS cm −1 (1 millimolar KCl), similar to the highest values detected in soil pore water at TWDEF, and analyzed for DOC with the wet oxidation persulfate UV method using a Phoenix 8000 Carbon Analyzer (Tekmar-Dohrmann, OH, United States). The pH of leachates was measured in stock solutions, which had DOC concentrations of around 150 mg L −1 . The only exception was the stock solution derived from aspen leaves, which had DOC concentrations close to 1000 mg L −1 , and, hence, had to be diluted prior to pH measurements.
Experimental Setup
The experimental setup is depicted in Figure 1. In brief, the experiment had four leachate treatments -aspen leaves (AL), aspen roots (AR), conifer (subalpine fir) needles (CN), and conifer (subalpine fir) roots (CR) -and eight soil types -TWDEF aspen (TA), TWDEF conifer (TC), CM17 aspen (CMA), CM17 conifer (CMC), from 0-10 and 40-50 cm soil depths. The conifer stands, from which soils were sampled, were dominated by subalpine fir at TWDEF, and by Douglas fir (Pseudotsuga menziesii Mirb.) at CM. In this experiment, the two depths represent differences in initial C concentration under the same overstory, which is thought to affect soil C saturation/deficiency. Considering that the forests investigated had not been managed for timber production for at least a century, we assumed that SOC levels were at steady-state. Following the same assumptions as studies that developed the C saturation capacity concept (Hassink, 1997;Six et al., 2002;Stewart et al., 2007), we assumed that the upper soil was closer to SOC saturation and thus had lower C retention capacity while the soil at greater depth had a higher C deficiency.
The study was a full factorial experiment (32 combinations of leachate and soil), such that every soil was mixed with every concentration of every leachate (1:10 soil to solution w/v ratio), and a pure KCl solution (DI water with no DOC) with an EC of 150 µS cm −1 was included to measure the desorption of native SOC (Figure 1). The experiment was done in triplicate for concentrations of 0, 10, and 80 mg DOC L −1 , and in duplicate for concentrations of 20 and 40 mg DOC L −1 . The mixing of soil and solution was done in glass jars with septa caps to allow for measurements of CO 2 evolution from heterotrophic activity after shaking. The jars were shaken in the dark on an orbital shaker for 24 h (100 rpm) at room temperature. Due to the FIGURE 1 | Experimental design of the sorption experiment. Leachates from four plant substrates -aspen leaves (AL), aspen roots (AR), fir needles (CN), and fir roots (CR) -were added to aspen (A) and conifer (C) soils at four concentrations plus blank (0, 10, 20, 40, and 80 mg L −1 ). The two depths (0-10 and 40-50 cm) represented differences in native SOC concentration, and the T (for TWDEF) and CM sites represented differences in oxyhydroxide concentration. All measurements were done in triplicate for 0, 10, and 80 mg L −1 treatments, and in duplicate for 20 and 40 mg L −1 treatments. sample size, the shaking (equilibration) had to be split in 2 days. The first round of samples were prepared on the same day as the leachates themselves, and the second round was prepared on the next day. After shaking, CO 2 within the jars was measured by inserting needle extensions through the septa and analyzing the gas with a LICOR-8100 gas analyzer (LI-COR, Inc., NE, United States). Afterward, all samples were filtered through a 0.4 µm glass fiber filter (Sterlitech) and analyzed for DOC as described in subsection "Leachate Preparation and Analyses."
Fluorescence Analysis
Leachate (pre-sorption) and post-sorption solution quality was assessed with fluorescence and absorbance spectroscopy using an Aqualog fluorometer (Horiba Jobin Yvon, Japan). Fluorescence excitation wavelengths ranged from 248 to 800 nm, at an increment of 6 nm, while the emission range was 249 -828 nm at an increment of approximately 4.5 nm. As the Aqualog measures fluorescence and absorbance simultaneously, so absorbance was measured at the same wavelengths as excitation. Each sample was diluted to not exceed 0.3 cm −1 absorbance at 254 nm (Miller et al., 2010) to minimize inner-filter effects; this corresponded to approximately 7-10 mg DOC L −1 . The samples were measured at their natural pH as we were interested in the characterization of the natural DOM (see Cuss et al., 2014, for more information on pH effects on plant leachate fluorescence).
The fluorescence spectra were Raman normalized, corrected for the inner-filter effect, and blank-subtracted before calculating several spectroscopic indices and building a parallel factor analysis (PARAFAC) model (Murphy et al., 2013). All corrections and calculations were performed using the MATLAB (version R2017a) software. We calculated the humification index (HIXat ex 254 nm, area of peak under em 435-480 nm divided by peak area under em 300-345 nm), the fluorescence index (FIem 470 nm/em 520 nm at ex 370 nm) (Gabor et al., 2014a), and, by using UV-Vis data, specific ultraviolet absorbance at 254 nm (SUVA = abs @ 254 nm cm −1 × 100/DOC mg L −1 ; units = L mg C −1 m −1 ) as these have been utilized in other studies to characterize soil derived DOM (Gabor et al., 2014b;Strid et al., 2016). A higher value of the humification index (HIX) corresponds to lower hydrogen to carbon (H:C) ratios and indicates a greater degree of humification (Gabor et al., 2014a). The FI is used as an indicator of precursor material, with lower values indicative of DOM that is plant-dominated in origin, and higher values indicative of DOM that is predominately from microbial (originally algal) sources, with a difference in value of 0.1 considered to be significant (McKnight et al., 2001). SUVA has been used as a proxy for DOC aromaticity (Weishaar et al., 2003), hydrophobicity (Dilling and Kaiser, 2002), and microbial stability (Kalbitz et al., 2003).
To better characterize the quality of the DOM solutions, we analyzed the fluorescing compounds by building a PARAllel FACtor analysis (PARAFAC) model following the guidelines described by Murphy et al. (2013). PARAFAC uses all of the data contained in excitation-emission matrices (EEMs) to identify and quantify independent underlying spectral features, termed "components." A component represents a single fluorophore (compound that absorbs or re-emits light) or a group of highly related fluorophores (Fellman et al., 2010;Murphy et al., 2013;Aiken, 2014). A PARAFAC model was built using the drEEM toolbox (version 4.0). As input for the model we used all sorption and desorption (see subsection "Desorption") data, as well as data from pure leachates. After validation, all components were compared with information from a global model database using the OPENFluor plugin (Murphy et al., 2014) in OPENChrom R (Wenig and Odermatt, 2010;version Dalton) to estimate the chemical identity of each component. More detailed information regarding fluorescence measurements and model building is provided in Supplementary Material 1.
Desorption
At the end of the adsorption experiment, soils were dried in glass jars (same jars as used for the sorption process) for 7 days at 5 • C. Drying, a natural process in the examined soils, can potentially affect OM retention in soil (Borken and Matzner, 2009). We chose the drying temperature to reflect the natural soil temperature observed in the field (Scott Jones, unpublished data; raw data available at twdef.usu.edu). After drying, soils were extracted once with 40 mL of 1 millimolar KCl solution to determine desorption of the sorbed material. The desorption solutions underwent the same preparation procedure and measurements as the sorption solutions described in subsection "Experimental Setup" and "Fluorescence Analysis."
Data Analyses
Before fitting a sorption equation, we adjusted the data for CO 2 -C lost through mineralization. We calculated the CO 2 concentration in the headspace using the ideal gas law, and by taking into account the pH of the solution we calculated the CO 2 in the liquid phase. First, to correct for the "Birch effect" (Jarvis et al., 2007), and for the mineralization of native SOC, the CO 2 measured for each sample was corrected by the CO 2 measured for the same soil when only the pure KCl solution was added. The corrected values were then used to calculate sorption/desorption using the following equation: where iDOC is the initial DOC concentration added, sDOC is the DOC concentration measured in the solution after 24 h of shaking, and CO 2 -C is the amount of C lost via mineralization. The resulting rDOC is the DOC retained in soil or released from soil in the cases where no sorption occurred, which is why we further refer to DOC retention/release in the text. Finally, rDOC was converted to mg C retained/released per kg of soil.
To describe the sorption behavior of leachate DOC on soil, we fitted a non-linear function, loosely based on the Freundlich isotherm, using initial DOC concentrations and rDOC values. Following suggestions by Lilienfein et al. (2004) and Vandenbruwane et al. (2007) we fit the non-linear curves by subtracting the native organic C released as DOC (from extraction with the KCl solution) from the original equation, meaning, the parameter "a" was added, representing a non-zero intercept: In equation [2], rDOC is the mass of DOC (mg) released/retained per mass of soil (kg), C i is the DOC concentration added [initial DOC; as per Kothawala et al. (2008) who used it for the Langmuir equation], and "a" is the y intercept, which describes the native DOC released with the pure KCl solution (Vandenbruwane et al., 2007). Parameters k and n are curve-fitting parameters that together describe the shape of the curve. We used non-linear regression to estimate the parameters k and n using the function nls in the R package Stats (R Development Core Team, 2015, version 3.1.2).
We tested differences between leachate type and soil properties in regard to null point concentration (NPC; initial DOC concentration at which net sorption equals net desorption), endpoint (EP; C sorbed at the highest concentration of DOC added), and parameters k and n with a factorial analysis of variance (ANOVA) testing for main effects and two-way interactions with α = 0.05. When significant, we performed post hoc Tukey HSD tests to determine differences between individual leachate types. The soil properties considered were: soil type, which represented differences in soils affected by aspen and conifer overstories; site, which was representative of differences in Fe and Al oxyhydroxide levels as well as site SOC concentrations ; and depth, which represented differences relative to effective C saturation levels (topsoils closer to C saturation, and subsoils further away). Data were transformed where necessary to ensure equal variances and normal distribution of the residuals. We further tested the relationship between initial SOC concentration and the four retention response variables with a multivariate regression.
To compare PARAFAC results, we calculated the proportion that each PARAFAC component explained from total fluorescence. This approach allowed us to compare shifts in component dominance (solution quality) without having to consider the non-linear effect of concentration on fluorescence intensities. A high heterogeneity of variances in residuals among several factors (mostly leachate type and soil type) prohibited the use of an ANOVA, and, therefore, differences in components were analyzed for main effects only using the non-parametric Kruskal-Wallis test. If leachate type was found to have significant differences, we used the Wilcoxon signed rank test for pairwise comparisons with a Holm adjustment for the p-value to compare between the leachates. All statistical analyses were performed with the software R version 3.1.2 (R Development Core Team, 2015). The values depicting results are reported as mean ± standard deviation, unless noted otherwise.
Soil Characterization
As seen in Table 1, at each site, soils under both overstory types were fairly similar. All soils were loams with some soils at 40-50 cm depth being clay loams. The clay concentration was lowest in the CMC 40-50 cm soils at 18%, and varied from 21 to 29% in the other soils. The clays at TWDEF were dominated by a mixture of 1:1 and 2:1 clays (kaolinite, illite, vermiculite with smaller peaks of dickite and muscovite). Similar XRD spectral peaks were also detected for CM soils, but due to the high oxyhydroxide concentration, which interfered with the XRD measurements, the clay mineralogy could not be fully described. At CM CD extractable "free" (sum of all three fractions) Fe and AAO extractable Al (sum of organically bound and non-crystalline fractions) oxyhydroxide concentrations were very similar among overstories and averaged around 16 mg g −1 for Fe and around 4.9 mg g −1 for Al oxyhydroxides. At TWDEF, conifer soils had lower non-crystalline and crystalline Fe oxide concentrations (around 2.2 mg g −1 non-crystalline Fe oxides for aspen vs. 1.2 mg g −1 for conifers, and around 3.7 mg g −1 crystalline Fe for aspen vs. 3.1 mg g −1 for conifers), while the Al concentrations were similar ( Table 1). The largest measured differences between aspen and conifer soils were in terms of C concentration and pH, which were always lower under conifers, with differences ranging from 0.3 -0.6 percentage points for C, and 0.1 -0.7 units for pH ( Table 1). The main soil difference between sites was in the concentrations of non-crystalline and crystalline Fe and Al oxyhydroxides, which were three to four times higher at CM. The higher oxyhydroxide concentration also corresponded to higher C concentrations at CM compared to TWDEF with differences ranging from 1.9 to 2.3 percentage points. At both sites, C concentrations in the topsoils were approximately 2 percentage points higher than in the corresponding subsoils ( Table 1).
Leachate Characterization
Aspen leaves (AL) yielded the highest DOC concentration among leachates (136 mg DOC g −1 substrate), while the other three substrates released ten times less DOC per gram of material ( Table 2). Leachates from foliage had approximately two to three times higher total N values than corresponding root leachates, even though root biomass itself had higher (0.95% vs. 0.58% for AR and AL) or similar (0.5% vs. 0.45% for CR and CN) N concentrations. The leachates had similar SUVA values -0.8 to 1.1. AL had the lowest HIX value (0.06) suggesting higher H:C ratios and a more aliphatic nature of the solution compared to the other leachates, and the highest FI index (2.01). Foliar leachates had higher FI than those derived from roots ( Table 2).
We created a 4-component PARAFAC model, which validated via split-half analysis, and after normalization of the input data, accounted for 98.7% of the observed variation in DOC fluorescence. From the four components of the PARAFAC model the first two (C1 and C2) were identified as humic-like (similar to C3 and C2 in Stedmon et al., 2007) while C3 and C4 were protein-like (C4 similar to peak in panel X8 in Murphy et al., 2011;C3 similar to C4 in Gueguen et al., 2014). Intensities reported in Table 2 for each component (C1 -C4) indicate that the fluorescence signal of AL was almost entirely explained by C4 (86.5%), while the remaining components explained only 13.5%. By contrast, C4 had very small intensities in the AR and CN leachates and was entirely missing from the CR leachate. For AR and CN, the protein-like C3 explained most of the fluorescence (67 and 54%, respectively), followed by the humiclike components C1 and C2. For CR, C1 and C3 explained similar proportions of fluorescence (40 and 37%), and C2 explained the rest.
After 24 h of shaking, the raw intensities of the leachate solutions decreased (data not shown) due to a decrease in DOC concentration, but the relative contribution of each component did not change with mineralization. For the AL leachate, HIX did not change but it increased for the other three leachates to an end-value of 1.17 for AR, 1.34 for CN, and 2.99 for CR. Conversely, the FI decreased significantly for AL (1.87), but remained fairly constant for the other three leachates (end-value
DOC Retention/Release
The sorption isotherms depicted in Figures 2, 3 were adjusted for the amount of DOC mineralized and released as CO 2 (exact values are reported in the Supplementary Materials 2, 3). On average, more DOC was lost through mineralization in the root leachate treatments than foliage treatments −13% of added C mineralized for AL treatment vs. 18% for AR, 12% for CN vs. 18% for CR. Overall, similar proportions of DOC were mineralized in aspen and conifer soils (16 and 15%, respectively). The sorption behavior of leachate types was significantly different based on all four sorption metrics analyzed -the curve parameters (n and k) and NPC and EP (Table 3). Post hoc Tukey's HSD test indicated statistically significant differences between AL and AR in regard to all four parameters analyzed, but no significant differences were detected between CN and CR ( Table 4). Figures 2, 3 show the similarities between CN and CR on almost all soils, while the sorption isotherms diverge much more strongly between AL and AR.
Based on the sorption isotherm parameters k and n, AL had the steepest sorption isotherms, suggesting the highest retention. This was followed by CN > CR > AR (Table 4), with the latter two being significantly different from AL. Overall, AL had the lowest NPC (DOC concentration where net retention = net release) and the highest EP (C retained at highest DOC concentration added) values. For NPC, the only significant difference was between AL and AR, while for EP, AL differed significantly from AR and CN ( Table 4).
One of the most interesting findings of this study was the consistently higher DOC retention in aspen soils compared to conifer soils, irrespective of the source of DOC (Table 4). For a given leachate type, aspen soils reached NPC at lower DOC concentrations, i.e., they started to retain C at lower DOC concentrations and had overall higher EP values. Conifer soils often did not reach NPC with the DOC concentrations used in this study, especially for topsoils. The ANOVA on curve parameters corroborated this observation for NPC, EP, and n (Tables 3, 4). The lower n values for aspen soils indicated steeper retention curves than for conifer soils, i.e., greater sorption.
Null point concentration and curve shape (parameter n) differed significantly between top-and subsoil ( Table 3). The lower n values for topsoils were associated with similar k values (Table 4), indicating steeper curves for topsoils, i.e., higher retention rates. However, the steeper curves did not result in lower NPC, as topsoils on average had significantly higher NPC values than subsoils (Tables 3, 4).
The ANOVA results showed that the only significant difference between sites was for parameter k ( Table 3). It was larger for CM than TWDEF (Table 4), again indicating higher DOC retention rates in CM.
We found statistically significant interactions between leachate type and soil type for parameter k, and between depth and soil type for EP ( Table 3). The interaction between leachate type and soil type for parameter k was due to the fact that root-derived DOC had higher k in aspen soils (20.9 for AR and 27.4 for CR on aspen soils, and 11.5 for AR and 25.4 for CR on conifer soils), and foliage DOC had higher k in conifer soils (60.2 for AL and 47.6 for CN on conifer soils and 45.4 for AL and 36 for CN on aspen soils). The interaction between depth and soil type for EP indicates that maximum retention was higher in aspen topsoils than in aspen subsoils (126.5 mg C kg −1 soil and 80 mg C kg −1 soil, respectively), while in conifer soils the depth pattern was the opposite. No statistically significant relationships were found between native concentration of SOC and any of the different sorption parameters.
Post-sorption DOC Quality
We calculated fluorescence indices and PARAFAC components to evaluate the effect of leachate quality on DOM sorption patterns. FI values of the sorption solutions did not change in relation to initial DOC concentration and overall ranged from 1.39 to 1.6 for all soils irrespective of the leachate treatment. HIX values (high values mean greater degree of humification and a low H:C ratio) at the lowest initial DOC concentrations (10, 20 mg DOC L −1 ) reflected a soil signature (expressed as HIX at 0 mg DOC L −1 in Figure 4) of around 7. Values at these concentrations were also distinctly different from the preand post-sorption leachate baseline (<1 for AL, AR and CN, and <3 for CR; Figure 4). At the initial DOC concentration of around 40 mg L −1 the average HIX decreased to 3 for AL, 2.8 for AR, 3 for CN, and 4.4 for CR. We found no statistically significant differences between HIX values from solutions of aspen and conifer soils.
SUVA values stayed relatively constant for all concentrations of AR (2.3 ± 0.13), and decreased slightly for CN and CR (from 2.3 in the KCl treatment to 1.8 at 80 mg DOC L −1 ). For AL, SUVA values initially increased from 2.3 to 2.9 at concentrations FIGURE 2 | Non-linear isotherms representing release/retention of dissolved organic carbon (DOC) from aspen leaves (AL) and aspen roots (AR) on aspen soils (upper two graphs), and of fir needles (CN) and fir roots (CR) on conifer soils (lower two graphs) from TWDEF and CM sites. The y-axis indicates DOC retention in the area above zero, and DOC release in the area below the zero-line. The SE of laboratory replicates was mostly < 5%.
FIGURE 3 | Non-linear isotherms representing release/retention of dissolved organic carbon (DOC) from fir needles (CN) and fir roots (CR) on aspen soils (upper two graphs), and of aspen leaves (AL) and aspen roots (AR) on conifer soils (lower two graphs) from TWDEF and CM sites. The y-axis indicates DOC retention in the area above zero, and DOC release in the area below the zero-line. The SE of laboratory replicates was mostly < 5%. 0, 10, and 20 mg L −1 , and decreased to 2.4 and 1.8 at higher concentrations (40 and 80 mg L −1 , respectively). The non-parametric comparison of fluorescence components for all main effects (Table 5) showed that the humic C1 peak and protein-like C3 and C4 peaks differed significantly by leachate type. C1 proportion was highest for CR and C3 was highest for CN, while both constituted the smallest proportion of total fluorescence in samples treated with AL (Table 6). Most samples treated with AL were dominated by C4, which was much less abundant or completely absent in samples treated with the other leachates ( Table 6). Overall C4 was absent in 40% of all samples, mostly from the CR treatment (missing in 80%). The proportion of C4 was highest in samples treated with the highest AL DOC concentrations and was missing in a few topsoil samples treated with the lowest AL DOC concentrations. On average, AL treated subsoils had 13 percentage-point higher C4 proportions than topsoils. Overall, AL treated samples showed a high variability between individual component proportions due to different responses of top-and subsoils (Supplementary Figure 11).
The proportions of all components differed significantly by depth (Tables 5, 6). Topsoils had higher proportions of C1 and C2 than subsoils. In contrast the proportion of C3 was higher for subsoils than topsoils. C2 also differed significantly between sites, with CM soils having higher proportions compared to TWDEF soils ( Table 6).
The proportions of the first two components showed a similar trend with initial DOC, as did HIX (Supplementary Figures 3, 4). This means that for C1 and C2 the proportions decreased with increasing initial DOC concentration by 12 and 10 percentagepoints, respectively. In contrast, for the protein-like C3 and C4 the proportions increased by, on average, 17 (for AR, CN, CR) and 30 (for AL) percentage-points, respectively.
DOC Desorption
In the final step we evaluated whether and to what extent there was a difference in the strength with which the sorbed SOC was held in the soils. As seen by the y-axis intercepts in Figure 2, aspen SOC was generally less water soluble than conifer SOC, despite higher intrinsic SOC levels in aspen soils than conifer soils ( Table 1). On average, the desorption of this intrinsic SOC with the KCl solution yielded 9.2 ± 3.1 mg DOC g −1 C for aspen soils vs. 14 ± 5.6 mg DOC g −1 C for conifer soils.
In the single-step desorption following the sorption experiment (after 7 days of drying), aspen and conifer soils released similar DOC concentrations (3 ± 1.8 mg DOC L −1 from aspen soils and 3.6 ± 3.5 mg L −1 from conifer soils). However, as sorption on conifer soils was much weaker than on aspen soils, absolute retention was still almost twice as high for aspen soils as it was for conifer soils (10 mg DOC L −1 vs. 6.6 mg DOC L −1 ). Among the leachate treatments, soils that had been treated with AL showed the lowest desorption with 2.2 ± 2 mg L −1 , while the other treatments released higher DOC concentrations with 3.6 ± 2.6 mg L −1 for AR, 3.4 ± 2.7 mg L −1 for CN, and 4.1 ± 3 mg L −1 for CR (p = 0.04, F 3 , 112 = 2.84).
The soil solutions after the desorption process had changed qualitatively, and had increased HIX values -AL 18.01 ± 2.6, AR 12.3 ± 1.8, CN 10.8 ± 2.1, CR 12.5 ± 3.8 -compared to any of the sorption solutions shown in Figure 4. Similarly to HIX, SUVA values also increased from an average of 2.2 ± 0.18 to 4.1 ± 0.37 for all leachate treatments, substantiating a shift to a more aromatic composition. The FI values of the solution did not change from the ones found after the sorption process (on average 1.5).
In contrast to the sorption samples the fluorescence indices in the desorption samples were overall similar among all soils and treatments, suggesting a similar quality of the SOM in solution. The proportion of the first two fluorescence components (C1 and C2) was higher in desorption samples (49 ± 2%; 43 ± 5%, respectively) than sorption samples at various initial concentrations of DOC (44 ± 8%; 30 ± 9%, respectively). In Leachate AL 37 ± 2.7 ab 24 ± 2.9 8 ± 0.8 a 31 ± 5.8 a contrast, there was a decline in C3 from the sorption samples (16 ± 7%) to desorption solutions (6 ± 4%). The average proportion of C4 in the desorption samples ranged from 1.2 to 2.6%, with almost half of the samples completely lacking C4 (even the soils treated with AL), and the other half showing extremely small intensities (<5% of total fluorescence). None of the components in the desorption samples were significantly different as a function of leachate, soil type, and site. The proportion of C1 was significantly higher for topsoil than subsoil (50 ± 1.5% vs. 48 ± 2%; χ 2 = 11.25, p < 0.01).
DISCUSSION
Understanding how tree species affect SOC pools is crucial for building better C models and reaching various ecosystemservice goals. This is true especially now, when the distribution of tree species is changing at local and global scales due to climate change and forest management practices (McKenney et al., 2007;IPCC, 2019 -2.2.4). SOC under aspen forests has been shown to be more stable compared to adjacent conifer forests in various studies in North America (as reviewed by Laganière et al., 2017). The results of our study indicate that this stability might be due to enhanced sorption of DOC from aspen foliage. Furthermore, aspen DOM seems to help create mineral soil conditions that are more favorable to the sorption of incoming DOC, irrespective of the source.
Most sorption parameters in this study indicated that DOC derived from aboveground litter (foliage and needles) showed a higher retention than DOC of root leachates. The magnitude of this was, however, different for the two species. While the retention of AL differed significantly from the retention of AR, CN (subalpine fir needles) had only a slightly higher sorption than CR (subalpine fir roots). This, along with similar results for conifers reported by Hansson et al. (2010), suggests that the relative contribution of foliage and root DOC to mineralassociated organic matter can differ based on the tree species that dominate a forest stand. For aspen soils, the DOC contribution to mineral-associated SOC is most likely dominated by foliage leachates, while in conifer soils the contribution is represented by an equal mix of both.
The soils sampled from aspen and conifer stands in this study had similar soil mineral properties, and the soil horizons sampled (ABt and BAt in aspen and Bt in conifer soils at TWDEF) differed mostly in regard to the amount and type (aspen vs. conifer) of organic matter. Thus, the higher sorption and lower desorption in aspen soils, irrespective of the leachate type added, indicates that this effect is likely caused by SOM properties. The effect occurred even though aspen soils had higher SOC concentrations and was also more pronounced in the topsoils (steeper sorption slopes and higher maximum retention), which are more C-rich than subsoils.
Detritus and Soil DOM Quality
To test the hypothesis that DOM quality drives the sorption of DOM in mineral soil, we calculated various spectroscopic indices -SUVA, HIX, FI, and fluorophores from a PARAFAC model -and measured DOC and TN concentrations, and CO 2 release during the shaking process in the batch sorption experiment. The results did not always point in the same direction. In general, SUVA and HIX values indicated an aliphatic and labile nature of all plant leachates. FI suggested a more microbially derived nature of the foliage than root leachates. The leachate from aspen leaves stood out from the others in terms of HIX, FI, and the fluorescence components. They suggested that AL was of a more aliphatic nature than the other leachates. The DOC/TN ratio was, however, much higher for AL (145), and the mineralization of AL (not mixed with soil) was similar to the mineralization of CN leachates (similar proportion of DOC mineralized).
The high proportion of the protein-like C4 fluorescence component in AL clearly distinguished this leachate type from the others. The high C:N ratio (145) of the AL solution makes it unlikely, however, that this component was dominated by protein-like compounds. Other compounds, like tannins and lignin phenols, have been found to fluoresce with similar spectral signatures to proteins and amino acids (Aiken, 2014). Even though aspen foliage leachate has been found to contain much higher amounts of phenolic compounds compared to conifer needles (Startsev et al., 2008), the low HIX and high FI values indicate a highly aliphatic nature of AL leachates, which in turn would rule out high aromatic compound concentrations. Dominance of protein-like components in leachates of senesced litter together with low HIX and high FI has also been reported by Beggs and Summers (2011). Similar to our study, Beggs and Summers (2011) found that a similar protein-like peak to C4 was lost due to biodegradation from leachate formed from dead needles that had not been shed by the tree. Meanwhile, needles collected from the O-horizon in their study did not loose this component with biodegradation, suggesting different chemistry of the fluorophores representing this component. Overall, while a few studies have examined the fluorescence spectra of plant leachates with PARAFAC models (Beggs and Summers, 2011;Cuss et al., 2014;Wheeler et al., 2017), any interpretation of the protein-like peaks is still based on aquatic research findings in fresh or marine waters. Thus, there is a need to evaluate the assumption of amino acid contribution to the proteinlike peaks with more detailed chemical composition data from terrestrial DOM.
Fluorescence indices for the description of leachate-soil exchange processes were only of limited value. The HIX values at small initial-DOC concentrations were similar to the soil-HIX signature (KCl solution treatment), which indicated that the soilleachate mixture was mostly dominated by the desorbed SOM (Figure 3 and Supplementary Figures 3-6). The high proportion of C1 and C2 components in these solutions, which suggest a highly processed DOM nature, also confirmed this. At higher initial-DOC concentrations, the plant signature (solid horizontal line in Figure 4) became more dominant, and the solutions had high proportions of the protein-peaks C3 for AR, CN, CR, and C4 for AL. The soil-leachate mixture, however, never acquired a purely plant-like signature, suggesting that even at high DOC concentrations, where sorption should be favored, desorption of native SOM occurred.
Leachate and Soil Interactions
In a field study conducted at TWDEF , we measured higher DOC concentrations and losses between 5 and 45 cm depths under conifers. The SOC pools under conifer stands were, however, much smaller than under aspen stands. This experiment elucidated why field DOC levels might not be good indicators for DOC sorption and SOC stocks in the aspen-conifer forests in Utah. The lower NPC of AL in aspen soils (25.8 and 19.9 mg·L −1 in top-and subsoil, respectively) suggests that high concentrations are not necessary for the retention of AL DOC to occur in these soils. Conversely, the high NPC of subalpine fir leachates on conifer soils (102.4 and 52.1 mg·L −1 for CN and 503.7 and 83.6 mg·L −1 for CR in topand subsoil) suggests that the conifer field DOC concentrations (28.4-45.5 mg·L −1 under conifers and 7.3-23.8 mg·L −1 under aspen) might not be sufficient for DOC retention to commence in these soils. The lower sorption of subalpine fir DOC to conifer soils might suggest a lower stability of conifer SOC due to fewer mineral-organic associations. This is in agreement with results from long-term incubation experiments that have shown conifer soils having higher heterotrophic respiration than aspen soils (Giardina et al., 2001;Woldeselassie et al., 2012;Laganiére et al., 2013).
Our study does not allow us to elucidate the mechanisms behind higher DOC sorption in aspen soils. Based on other published literature we can hypothesize that the higher sorption was either due to an unmeasured property of the mineral soil or due to the properties of SOM. In regard to unmeasured properties we can only speculate that there could be differences in the very fine clays between soils that might have developed due to differing mineral weathering trajectories caused by the overlaying vegetation (Taylor et al., 2009). In regards to organic matter, one explanation could be that aspen, with their more nutrient rich foliage, facilitate a more rapid formation of mineral-associated organic matter via microbial pathways (Craig et al., 2018;Lavallee et al., 2018). Considering that aspen soils were more receptive to DOC irrespective of the source, the microbial biomass attached to minerals in patches already containing old C (Vogel et al., 2014) could be more active and/or efficient in aspen than in conifer soils. The similar proportion of mineralized DOC on both soils, found in this study, does not necessarily indicate similar carbon use efficiency, especially for substrates of contrasting quality (Manzoni et al., 2012), and, therefore, cannot exclude a difference in microbial activity in both soils. Indeed, as mentioned above, long-term laboratory incubations have shown that more SOC is mineralized in conifer soils than aspen soils (Giardina et al., 2001;Woldeselassie et al., 2012;Laganiére et al., 2013), suggesting a difference in microbial functioning. Furthermore, communitylevel physiological profiling data showed that microbial biomass increases with increasing aspen basal area (Román Dobarco et al., 2020). Finally, a second organic pathway could be organic matter precipitation by forming supramolecular organic-organic associations (Sutton and Sposito, 2005). Further studies with more detailed methods are needed to test these hypotheses.
Differences in pH have been commonly observed under angiosperm and gymnosperm overstories (Augusto et al., 2014), and soils in our study were no exception. Most laboratory findings assume a reduction of the sorption capacity with increasing pH (as reviewed by Michalzik et al., 2001). In our study, pH was slightly higher in aspen soils, which should lead to lower sorption. This, however, was not the case, suggesting that either the difference in pH was not big enough to cause measurable differences in sorption or the effect of DOM chemistry was stronger. In terms of potential impacts from differences in Fe oxihydroxide concentrations between the two overstory soils at TWDEF (Table 1), any effect of this parameter should remain smaller than its significance in causing differences between sampling locations. Large Fe and Al oxyhydroxide differences were observed between CM and TWDEF but sorption differed only minimally between these sites.
While in this experiment we used needles and roots only from subalpine fir, the results could still be representative of other conifers. The higher SOC pools under aspen compared to various conifer species in Utah (Woldeselassie et al., 2012;Román Dobarco and Van Miegroet, 2014;Boča and Van Miegroet, 2017), and higher SOC stability under aspen compared to various conifers in North America (Laganière et al., 2017), certainly point in that direction. Concomitantly, the absence of a consistently higher aspen effect on SOC pools in North America might indicate that other factors, like climate, might also control the species' effect. This could happen by climate causing variations in plant functional traits (Reich et al., 2003) or by differently affecting the strength of mechanisms for SOC accumulation.
Limitations
The initial DOC concentration in the aspen foliage leachate was ten times higher than the other leachates, indicating that freshly senesced aspen leaves contained more water-soluble compounds. These concentrations obtained under laboratory conditions were much higher than those observed in the field . While freezing of plant material can increase concentrations of dissolved organic C, N, phenolic compounds, and proteins (Kiikkilä et al., 2012), such conditions have also been observed in the natural environment, where the material was collected . We believe that grinding the biomass resulted in elevated concentrations of DOM in the leachates, but this was necessary to perform the experiment. While not completely representative of field conditions, the chosen approach for leachate generation still allows for a relative comparison of sorption between leachate types.
The NPC values from the laboratory experiment are unlikely to represent NPC values in the field due to the experimental setup (sieved soils, shaking process, and filtration). These values, however, allowed us to do a relative comparison between the sorption behavior of both soils, and they clearly showed fundamental differences between them.
CONCLUSION
In this study, we compared DOC sorption in soils of two contrasting forest overstory types -quaking aspen and conifers (subalpine fir and Douglas fir). We found that aspen soils retained more DOC than conifer soils, irrespective of the leachate type. Furthermore, aspen soils retained aspen foliage DOC especially well. While higher DOC concentrations overall increased sorption, this depended on the quality of the leachate source and the native SOC already present in soil. The findings suggest that while aspen forests have lower DOC concentrations in soil pore water measured in the field than conifers, sorption in aspen forest soils can commence at these lower concentrations. Furthermore, the study provides a foundation on which to build further investigations for understanding the exact mechanisms that allow for more efficient incorporation of labile DOM into SOC.
The results also indicate that the presence and maintenance of aspen forests in the landscape is favorable to the belowground C storage function of ecosystems. Encroachment by conifers into aspen stands, however, will not necessarily lead to immediate or quick soil C losses, as aspen SOC currently present in the soil is also receptive to sorption of DOC from conifer leachates. This dynamic could change in the long term if aspen SOC is replaced by conifer SOC.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
AUTHOR CONTRIBUTIONS
AJ and HV conceived the experiment. AB, AJ, and HV designed the experiment. AB executed the experiment, conceptualized, and wrote the manuscript as lead author. All the authors contributed to the interpretation of the findings and the final manuscript. | v3-fos-license |
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} | pes2o/s2orc | Characterization of an Asymmetric Occluded State of P-glycoprotein with Two Bound Nucleotides
P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5′-(γ-thio)triphosphate (ATPγS), one of low affinity (Kd 0.74 mm), and one “occluded” nucleotide of 120-fold higher affinity (Kd 6 μm). ATPγS also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATPγS (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATPγS-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.
The ATP-binding cassette (ABC) 2 protein superfamily is a very large and diverse group of (primarily) active membrane transporters found in all organisms from bacteria to humans (1)(2)(3). Proteins in this superfamily share a similar domain organization, with two membrane-embedded transmembrane domains and two cytoplasmic nucleotide binding domains (NBDs). These four domains are often expressed as separate subunits in prokaryotic ABC proteins. In eukaryotes ABC proteins consist of either single fused polypeptides comprising all four domains or "half-transporters" with a single transmembrane domain and NBD that operate as homo-or heterodimers. Binding and hydrolysis of ATP at the NBDs induce conformational changes that drive transport of substrate across the membrane. The NBDs of ABC proteins comprise three highly conserved sequences; the Walker A and B motifs, commonly found in nucleotide-binding proteins, and the Signature C motif, which is unique to this protein family (4). P-Glycoprotein (Pgp, ABCB1), the best studied eukaryotic ABC transporter, is a 180-kDa plasma membrane protein that functions as an efflux pump for amphipathic natural products, drugs, and peptides (5,6). Pgp has been implicated in the phenomenon of multidrug resistance, which is often observed in human cancers and represents a major impediment to the successful treatment of tumors using chemotherapy (7). Both NBDs of Pgp are capable of ATP binding and hydrolysis (8). Based on work showing trapping of ortho-vanadate in only one of the catalytic sites of Pgp (9), Senior et al. (10,11) proposed an alternating sites mechanism for the transporter in which only one active site is able to hydrolyze ATP at any point in time, with the two sites taking turns at catalysis. An alternating sites mechanism requires that all reaction intermediates are asymmetric, thus providing "memory" of which active site last turned over. The mechanism of Pgp-mediated drug transport has been the focus of intensive study (12,13), but how ATP hydrolysis during the catalytic cycle is coordinated between the two NBDs at the molecular level and how this is coupled to drug transport are still not understood.
An emerging consensus in the ABC protein field over the past few years has been that dimerization of the NBDs, which is driven by nucleotide binding, appears to be an essential step in the transport cycle. High resolution x-ray crystal structures of isolated bacterial NBD subunits and entire bacterial ABC proteins have revealed interdigitated "head-to-tail" dimers, where two molecules of ATP are bound at the dimer interface by the Walker A and B motifs of one NBD and the C motif of the opposing NBD (see for example, see Refs. 14 -18), an arrangement that was previously predicted (19). However, such stable nucleotide sandwich dimers have been found only when ATP hydrolysis is blocked either by the absence of Mg 2ϩ or by mutation of an essential catalytic residue, and they have not yet been observed in a catalytically active protein. Biochemical studies and simulations on bacterial ABC proteins have demonstrated that binding of ATP, but not ADP, induces dimerization of the NBDs (20 -24). It is now clear that an ABC protein conformation with two bound nucleotides is required to initiate the catalytic cycle; however, the symmetrical nature of the crystallographic sandwich dimers suggests that they probably do not represent a true catalytic intermediate (25). The recent x-ray crystal structure of Pgp with bound peptide substrate molecules was determined in the absence of nucleotide (26). Although the two NBDs appear to be located close to one another, they do not appear to be tightly associated, and thus, this structure does not provide any additional information on their mode of interaction during catalysis.
Combined mutation of the two "catalytic carboxylates" (Glu-556/1201; human Pgp) in the NBD Walker B motifs of Pgp resulted in a protein that displayed tight binding of 8-azido-ATP (27). Tombline et al. (28,29) were the first to report the isolation of an occluded state with ATP tightly bound at a maximal 1:1 stoichiometry in the catalytically inactive Pgp doublemutant (E552A/E1197A; mouse Pgp). The enzyme appears to be arrested in an occluded nucleotide conformation similar to that of a stabilized NBD dimer, representing a (normally) transient, asymmetric, catalytic intermediate (25). More recently, it was reported that a single molecule of the non-hydrolysable ATP analogue ATP␥S was occluded by wild-type Pgp (30), suggesting that this resembles the E⅐S intermediate in the catalytic cycle of the native transporter.
We seek to understand how NBD dimerization drives ATP hydrolysis by Pgp and determine the biochemical nature of the E⅐S intermediate that immediately precedes the ATP hydrolysis step. In the present work we report the biochemical and spectroscopic characterization of an asymmetric state of wild-type catalytically active Pgp with two bound molecules of ATP␥S. One nucleotide is bound with low affinity, and the other is an occluded nucleotide that binds with 120-fold higher affinity. Binding of two other non-hydrolyzable ATP analogues did not induce an asymmetric state. ATP␥S was also found to interact with Pgp with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of Cys residues in the NBDs. Binding of ATP␥S (but not ATP) was found to induce Trp residue heterogeneity, as indicated by collisional quenching, suggesting the existence of conformational asymmetry in the Pgp molecule. This asymmetric occluded state containing two bound nucleotides still displays high affinity binding of several drug substrates and modulators, which has important implications for the catalytic and transport cycle of Pgp. We propose a site-switching mechanism that leads to alternation of catalysis between the two active sites.
Pgp Purification-Plasma membrane vesicles were isolated from multidrug -resistant CH R B30 Chinese hamster ovary cells and stored at Ϫ80°C. Pgp was purified from CH R B30 plasma membrane as described previously (32) by initial extraction with 25 mM CHAPS buffer followed by solubilization of the S 1 pellet in 15 mM CHAPS buffer. The protein was further purified by affinity chromatography on a concanavalin A-Sepharose column. The final product consisted of 90 -95% pure Pgp in 2 mM CHAPS, 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MgCl 2 , pH 7.5. The purified Pgp preparation was kept on ice and used within 24 h. Plasma membrane protein was quantitated by the method of Bradford (33), and a modified Lowry assay was used to determine the concentration of purified Pgp (34) using bovine serum albumin (crystallized and lyophilized, Sigma) as a standard. Highly stable vanadate-trapped Pgp was prepared using Co 2ϩ ions as previously described (35,36).
Inhibition of Pgp ATPase Activity by Nucleotides and Nucleotide Analogues-The Mg 2ϩ -ATPase activity of Pgp was determined by measuring the release of inorganic phosphate from ATP. Purified Pgp was incubated with assay buffer (50 mM Tris-HCl, 5 mM MgCl 2 , pH 7.5) in the presence of 2 mM ATP at 37°C for 20 min as described earlier (37). To determine IC 50 values for the inhibition of ATPase activity as a function of concentration and incubation time, purified Pgp was preincubated for 5 min with increasing concentrations of ATP␥S or other ATP analogues, ATP was added, and activity was determined as described above. Details for each experiment are given in the figure legends. To determine the K i values for the inhibition of Pgp, ATPase activity was measured as a function of ATP concentration for several different nucleotide analogue concentrations. For classical competitive inhibition, the K i value was estimated by fitting the rate of ATP hydrolysis at increasing ATP concentrations to kinetic equations (using SigmaPlot, Systat Software, Chicago, IL). For noncompetitive inhibition, IC 50 values were estimated instead.
Protection of Pgp ATPase Activity from MIANS Inactivation by Nucleotides and Nucleotide Analogues-Covalent labeling of Pgp at Cys-428 and Cys-1071 within the NBD Walker A motifs was achieved using the thiol-specific probe, MIANS, as described previously (32). After incubation of Pgp with increasing concentrations of nucleotide or nucleotide analogue for 5 min, 20 M MIANS was added (from a 2.5 mM stock solution in methanol), and the protein was further incubated for 10 min followed by the addition of 2 mM dithioerythritol to quench the reaction. After 2 min, 2 mM ATP was added, and the ATPase activity was determined as described above. No MIANS was added to the control samples. IC 50 values for the protection of Pgp ATPase activity from MIANS inactivation were estimated from the difference in ATPase activity between Pgp incubated with and without MIANS as a function of nucleotide concentration.
Hydrolysis of ATP␥S by Pgp-Purified Pgp in assay buffer was incubated with 0.1 mM ATP␥S and 1 Ci of ATP␥[ 35 S] (50 l total volume) for times ranging from 0 to 4 h at 37°C. A 5-l aliquot of each sample was spotted on activated silica gel 60 F254 aluminum-backed TLC sheets (Merck) and separated as described (38). The radioactivity corresponding to ATP␥[ 35 S] and 35 S-labeled thiophosphate was quantitated using a storage phosphor screen with a Bio-Rad Personal Molecular Imager. Purified Pgp was incubated with unlabeled ATP under identical conditions for 0 -4 h, the products were separated by TLC, and the extent of hydrolysis was determined by imaging under UV illumination.
Stoichiometry of ATP␥[ 35 S] Binding to Pgp Using Equilibrium Dialysis-Equilibrium dialysis measurements were performed using several 250-l Micro-Equilibrium Dialyzer 2-Chamber Systems, using membranes with a molecular mass cutoff of 100 kDa between the two chambers (Harvard Apparatus, St. Laurent, QC, Canada). All the samples were prepared in 2 mM CHAPS, 2 mM dithioerythritol, 20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 buffer, pH 7.4. For binding experiments, 125 l of purified Pgp (ϳ0.3 mg/ml in 2 mM CHAPS buffer) was placed in one chamber of the dialyzer with increasing concentrations of ATP␥[ 35 S] (ranging from 10 to 100 M, in the same buffer) in the opposite chamber. In parallel, 125 l of buffer alone and the same increasing concentrations of ATP␥[ 35 S] were employed in separate dialyzers. The dialyzers were placed on a shaker for ϳ18 h at 4°C. After this equilibration period, 5-l aliquots of sample (in triplicate) were used to determine the amount of protein in each chamber (based on a molecular mass of 140 kDa). 30-l aliquots of each chamber sample (in triplicate) were added to 10 ml of BCS scintillant (Amersham Biosciences) for counting in a Beckman LS 6000SE scintillation counter. A standard curve was generated for ATP␥[ 35 S] to Pgp was used to calculate the binding stoichiometry. The ATPase activity of Pgp was determined before and after dialysis, and the protein was shown to retain Ͼ95% of its catalytic function after dialysis.
Fluorescence Quenching Studies-A PTI Alphascan-2 spectrofluorimeter (Photon Technology International, London, ON, Canada) was used to carry out fluorescence measurements at 22°C using a quartz microcuvette (Hellma, Germany). Fluorescence quenching experiments with nucleotides and drugs were carried out using 250 l of Pgp solution (50 g/ml in 2 mM CHAPS buffer, pH 7.5) at 22°C. The working solutions for titrations were prepared in water for nucleotides and in DMSO for drugs. For quenching of Pgp Trp fluorescence and Pgp-MIANS fluorescence, excitation/emission was at 290/320 and 320/420 nm, respectively, with 4-nm slits. Native Pgp and Pgp-MIANS samples were titrated with either nucleotides or drugs, and after each addition the samples were equilibrated for 40 s. The measured fluorescence emission intensities were corrected for dilution, scattering, and the inner filter effect as described earlier (32). Changes in fluorescence intensity were routinely ex-pressed as the percent change induced by substrate binding relative to the absolute intensity measured in the absence of substrate. Experimental data showing monophasic or biphasic quenching behavior were fitted to the equation (39) ⌬F where F o is the initial fluorescence intensity, (⌬F/F o ϫ 100) is the percent quenching (percent change in fluorescence intensity relative to the initial value), [S] is the substrate concentration, and K d1 and K d2 are the dissociation constants. Fitting to a one-site or two-site model was carried out by nonlinear regression using SigmaPlot, and values for K d and the maximum percent fluorescence quenching (⌬F max/ F o ϫ100) were extracted. Collisional Quenching Studies-Stock solutions of 5 M acrylamide, 5 M KI, and 5 M CsCl were added as 2.5-l aliquots in buffer to 250 l of 50 g/ml Pgp in 2 mM CHAPS buffer. All quencher solutions were freshly prepared, and 0.1 mM Na 2 S 2 O 3 was added to the KI stock solution to prevent I 3 Ϫ formation. Fluorescence emission was measured at 320 nm after excitation at 290 nm, and fluorescence intensities were corrected for dilution and scattering. In control titrations, KCl was added at the same concentrations as KI to correct for any ionic strength effects. Parallel experiments were carried out using N-acetyl-Ltryptophanamide to assess fluorescence quenching by the same agents when Trp is completely accessible in aqueous solution. Quenching data for a homogeneous single fluorophore system were analyzed using the Stern-Volmer equation (40), where F o and F are the fluorescence intensities in the absence and presence of quencher, respectively, [Q] is the concentration of quenching agent, and K SV is the Stern-Volmer quenching constant. For complex quenching including at least two types of fluorophores (41), the following equation was used, where f i is the fractional contribution of component i to the total fluorescence F.
Affinity of Pgp for Binding Nucleotides and Nucleotide
Analogues-Pgp from hamster and mouse contains 11 Trp residues, 8 of which are located in regions of the protein close to the membrane-water interface (42), according to the recent crystal structure (26). We previously showed that the Trp fluorescence emission maximum of Pgp is indicative of a relatively hydrophobic environment (43). The Trp residues that contribute to emission are sensitive to binding of both drugs and nucleotides, and this can be used to quantify the affinity of their interactions by fluorescence quenching titrations (43). This approach has the advantage of using native, unmodified, catalytically active Pgp. The affinity of Pgp was deter-mined for the non-hydrolyzable analogues AMP-PNP, AMP-PCP, and ATP␥S and the photoaffinity label 8-azido-ATP. ATP and ADP were also included for comparison; the binding affinity of these nucleotides was determined previously using fluorescence quenching approaches (32,43,44). Titration of Pgp with increasing concentrations of nucleotide analogues led to saturable, concentration-dependent quenching, which fit to a hyperbolic binding curve for AMP-PNP (Fig. 1A), AMP-PCP (Fig. 1B), and ATP (binding was determined at 10°C to prevent hydrolysis). Because 8-azido-ATP undergoes photolytic reaction on irradiation at 290 nm, it was not possible to estimate its binding affinity using Trp quenching. Therefore, Pgp was labeled on two Cys residues within the NBDs using the fluorophore MIANS, and the binding affinity for 8-azido-ATP was estimated by quenching of Pgp-MIANS fluorescence, as described earlier (32). Assuming an interaction with a single type of binding site, the quenching data were used to estimate the K d for binding of the nucleotide analogues (Table 1). All nucleotides displayed low affinity binding, with AMP-PNP showing comparable affinity to ATP, whereas AMP-PCP and 8-azido-ATP displayed slightly higher affinity.
The ATP␥S Trp quenching curve was characterized by more complex biphasic behavior, indicating the presence of two binding sites of differing affinity (Fig. 1C). Fitting of the data to a two-site equation revealed the existence of two binding sites for ATP␥S with remarkably different binding affinities. Indeed, one site binds ATP␥S with very high affinity (K d of 6 M; Fig. 1C, inset) and the second binds ATP␥S with much lower affinity (K d of 0.74 mM). The low binding affinity is comparable with that for unmodified ATP, and the high binding affinity exceeds that by ϳ120-fold (Table 1). Similar high binding affinity for ATP was reported in the occluded state of the catalytically arrested E552A/E1197A mutant Pgp (25,28,29), but this is the first report of two nucleotide binding sites of such widely differing affinity in wild-type catalytically active Pgp. As proposed by Senior and co-workers (25,29), the tightly bound occluded nucleotide molecule is likely committed to hydrolysis, although in this case it is not readily hydrolyzable (see below).
Pgp-mediated Hydrolysis of Nucleotides and Nucleotide Analogues-Although ATP␥S is described as a non-hydrolyzable ATP analogue, there have been reports that it can be hydrolyzed at a low rate by some transport ATPases (see for example Refs. 45 and 46). Pgp expressed in insect cells appeared to be unable to hydrolyze this analogue (30). Using a high sensitivity radiometric assay, we observed that Pgp-mediated hydrolysis of ATP␥[ 35 S] to [ 35 S]thiophosphate was linear with time over a period of 0 -4 h, with ϳ25% hydrolyzed after a 3-h incubation with 10 g of Pgp under the assay conditions. The rate of hydrolysis of ATP␥S was ϳ0.4% that determined for ATP under the same conditions. Interestingly, this is compara- ble to the rate reported for the E552A/E1197A mutant Pgp, which forms the occluded state in the presence of ATP (28). For 8-azido-ATP, the K m for hydrolysis was substantially lower than that of unmodified ATP and correlated well with the K d value for binding to Pgp. The relative V max (compared with ATP) for Pgp-mediated hydrolysis was reduced to 30% for 8-azido-ATP. Previously, a K m value of 0.4 -0.6 mM and an 8 -10-fold reduced hydrolysis rate were reported for 8-azido-ATP (27). The K m and relative V max values were also determined for hydrolysis of ⑀-ATP (Table 2); however, it was not possible to determine the binding affinity of this analogue, as its intrinsic fluorescence interferes with the quenching assays and is not environmentally sensitive. In general, the high V max values of 8-azido-ATP and ⑀-ATP limit the usefulness of these analogues, as they are rapidly hydrolyzed by Pgp.
Inhibition of Pgp-mediated ATP Hydrolysis by Nucleotides and Nucleotide Analogues-The basal ATPase activity of Pgp can be inhibited by nucleotides and nucleotide analogues that compete with ATP for interaction at the NBDs. To distinguish between competitive and noncompetitive inhibition, the rate of ATP hydrolysis was followed as a function of ATP concentration in the presence of several fixed concentrations of various nucleotide inhibitors. AMP-PNP displayed competitive inhibition ( Fig. 2A), with characteristic double reciprocal plots ( Fig. 2A, inset) that led to an estimated K i value of 0.53 mM, the same as the K m for ATP hydrolysis ( Table 2) and comparable with the estimated K d for binding (Table 1). ADP was also previously shown to inhibit ATP hydrolysis competitively (37). There has been a report that AMP-PCP did not inhibit Pgp-mediated ATP hydrolysis, even at concentrations as high as 10 mM (30). However, in our experimental system, this nucleotide analogue inhibited ATP hydrolysis, with the inhibition plot exhibiting sigmoidal character (Fig. 2B). Fitting to the Hill equation (n ϭ 1.77) gave a K i value of 1.6 mM for AMP-PCP (Table 2), which is about 10-fold higher than its K d for binding Pgp. ATP␥S inhibited ATP hydrolysis at very low concentrations (Fig. 2C), with an estimated IC 50 value of 6 M (Table 2), which agrees well with the high affinity K d value found for this nucleotide analogue ( Table 1). The IC 50 values for inhibition of ATP hydrolysis by 8-azido-ATP and ⑀-ATP could not be determined as these analogues are rapidly hydrolyzed by Pgp.
Protection of Pgp from MIANS Inactivation by Nucleotides
and Nucleotide Analogues-The fluorescent compound MIANS inactivates Pgp by a covalent reaction with Cys-413 and Cys-1074 located within the Walker A motifs of the NBDs (32,47). Although these residues are not necessary for Pgp function, they are located close to the catalytic sites, and their modifica- tion with the relatively large MIANS group leads to complete loss of ATPase activity (32). However, MIANS-labeled Pgp displays binding of both nucleotides and drugs with normal affinity. ATP can protect Pgp from MIANS inactivation in a concentration-dependent manner (32), with an IC 50 value of ϳ0.3 mM (Table 2), consistent with its K d for binding. Thus, IC 50 values for protection from MIANS inactivation by various nucleotides and nucleotide analogues were determined as an additional measure of their interaction with the NBDs of Pgp ( Table 2). The IC 50 for protection by AMP-PNP was similar to the K d for binding (Fig. 3A) and the K i for inhibition of ATPase activity; thus, this nucleotide analogue behaves as predicted. In contrast, AMP-PCP protected Pgp from MIANS inactivation with an IC 50 similar to that for inhibition of ATP hydrolysis (Fig. 3B) but ϳ12-fold higher than its K d for binding to Pgp. Thus, AMP-PCP displayed anomalous behavior. This unusual pattern suggests that AMP-PCP is not a good choice as a nonhydrolyzable nucleotide analogue for use with Pgp. ATP␥S protected Pgp from MIANS inactivation at very low concentrations (Fig. 3C, IC 50 of 5 M), consistent with its IC 50 for inhibition of ATP hydrolysis and the high affinity component of its binding.
Stoichiometry and Characterization of High Affinity ATP␥S Binding to Pgp (Occlusion)-It was previously reported that one molecule of ATP or 8-azido-ATP is tightly bound (occluded) in the catalytically arrested Walker B E556/1201Q double mutant of Pgp (28,48). We used equilibrium dialysis to measure the stoichiometry of ATP␥S binding to wild-type Pgp at low concentrations where only the high affinity binding site is significantly occupied. As shown in Fig. 4, results showed that a single molecule of ATP␥S is occluded in the NBD under these conditions. Sauna et al. (30) previously reported that ATP␥S could not be occluded at 4°C; however, our equilibrium dialysis experiments were carried out at this temperature, and occlusion clearly does take place. However, there are differences in the ATP␥S concentration dependence of the equilibrium dialysis data (Fig. 4) and the fluorescence binding assays ( Fig. 1C; carried out at 22°C), which likely arise from the different temperatures at which these experiments were conducted. Tombline et al. (28) also found that nucleotide occlusion was a highly temperature-dependent process. Sauna et al. (30) reported that ATP␥S is not occluded in the presence of a 10-fold higher concentration of ATP and attributed this to blocking of the high affinity site by ATP. In our case, this scenario seems unlikely, as the binding affinity of the occluded nucleotide is 120-fold higher than that of the more loosely bound molecule. We found that a 20-fold excess of ATP, as expected, had only a small effect on the ability of ATP␥S to bind to Pgp, as assessed by Trp quenching and the IC 50 value for inhibition of ATP hydrolysis (data not shown).
Interaction of ATP␥S with Vanadate-trapped Pgp-In the presence of vanadate, Pgp forms a stable complex in which ADP and ortho-vanadate are trapped in only one catalytic site after a single round of ATP hydrolysis (9). This trapped state is believed to be a structural mimic of the catalytic transition state and is thought to represent the conformation formed immediately "post-hydrolysis." We previously showed that the unoccupied NBD of vanadate-trapped Pgp is able to bind fluorescent ATP and ADP analogs, and vanadate-trapped Pgp-MIANS can bind ATP and ADP (35,36). When the stable vanadate-trapped Pgp complex formed in the presence of Co 2ϩ ions (35,36) was evaluated for binding of ATP␥S using Trp quenching, only a single hyperbolic curve was observed with a K d value of 13.2 M (Fig. 1D and Table 1). This binding affinity resembles that of the tightly bound occluded ATP␥S molecule found in basal-state Pgp. Effect of ATP␥S Occlusion on Drug Binding to Pgp-To determine the effect of ATP␥S occlusion on the ability of Pgp to interact with its drug substrates, we used Trp quenching to measure the binding affinity for two multidrug resistance drugs (vinblastine and daunorubicin) and two modulators (verapamil and propafenone GP12) in the absence and presence of ATP and ATP␥S (Fig. 5). The data show that for all four drugs, the dissociation constants are similar in the absence of nucleotide and with bound ATP and ATP␥S (Table 3). The K d values for ATP␥S were also estimated in the presence of vinblastine and verapamil at a concentration of 50 M, and no significant change in the nucleotide binding affinities was observed (data not shown).
Collisional Fluorescence Quenching Studies of Pgp with Occluded ATP␥S-Collisional quenching has proved to be a very useful technique to study the aqueous accessibility of fluorescent groups within Pgp and also to provide information on the polarity and charge of the region in the vicinity of the fluorophore (43). Changes in accessibility after ligand binding can be indicative of a change in protein conformation. To investigate conformational differences in more detail, Pgp in the presence of saturating concentrations of either ATP or ATP␥S was titrated with increasing concentrations of three collisional quenchers, acrylamide, I Ϫ ion, and Cs ϩ ion. In each case, a parallel quenching experiment was carried out with N-acetyl-L-tryptophanamide, a soluble Trp analogue.
As we showed earlier (43), the neutral acrylamide molecule was a poor quencher of Pgp Trp residues, with a 13-fold lower Stern-Volmer quenching constant compared with N-acetyl-L-tryptophanamide (Table 4). The completely linear Stern-Volmer plot suggests that only a single class of Trp residues within Pgp is quenched, and they all are equally accessible to acrylamide (Fig. 6A). No significant difference was observed for acrylamide quenching of Trp residues when Pgp was preincubated with saturating levels of either ATP or ATP␥S (Fig. 6A); indeed, the Stern-Volmer constants, K SV values, are very similar (Table 4). Thus, the accessibility of Trp residues to acrylamide is unchanged in the absence and presence of ATP and ATP␥S. However, a different situation was observed for collisional quenching of Pgp by I Ϫ . In the ATPbound state we observed a linear Stern-Volmer plot, again indicating the existence of a single class of Trp residues. In contrast, for the ATP␥S bound state (both high and low affinity sites occupied), the Stern-Volmer plot was curved, indicating the existence of at least two classes of fluorophore with different accessibilities to quencher (40,41). The first group of Trp residues is highly inaccessible to I Ϫ quenching as indicated by the K SV value, which is 3.5fold lower than that of nucleotide- MARCH 5, 2010 • VOLUME 285 • NUMBER 10
JOURNAL OF BIOLOGICAL CHEMISTRY 7581
free Pgp (Table 4) and represents ϳ77% of the total fluorescence intensity. The second group of Trp residues represents ϳ23% of the total fluorescence intensity and is quenched much more efficiently, with a K SV value 5.6-fold higher than that of nucleotide-free Pgp ( Table 4). The 77/23 distribution of Trp residues between these two groups cannot be readily translated into absolute numbers of residues, as not all of the 11 Trp residues found in Pgp contribute to the overall fluorescence emission (42). Overall, these results indicate that some Trp residues are more shielded from I Ϫ when ATP␥S binds, whereas others are more accessible, suggesting that the nucleotide induces heterogeneity in Trp residue accessibility that is not seen when ATP binds. One explanation is that ATP␥S binding may induce an asymmetrical protein conformation. We also conducted similar experiments with the positive collisional quencher Cs ϩ , but the level of Pgp quenching was very low (data not shown). Taken together, the results obtained with charged quenchers also suggest that the protein regions around the emitting Trp residues are positively charged, as Cs ϩ ions cannot closely approach them, whereas I Ϫ ions can.
DISCUSSION
ATP-driven dimerization of the NBDs is recognized as playing a key role in the catalytic cycle of ABC proteins; however, the details of how this is linked to ATP hydrolysis and substrate transport remain unclear. The alternating sites model for the catalytic mechanism of ATP hydrolysis by Pgp is supported by the observation of vanadate trapping in only one catalytic site and the isolation of an occluded state of the E552A/E1197A double mutant containing a single tightly bound nucleotide. More recently, it has become clear that ATP␥S also induces the occluded state in wild-type Pgp, and thus, this non-hydrolyzable analogue may be an invaluable tool to dissect the steps in the catalytic pathway. We have investigated the interactions of ATP␥S and other non-hydrolyzable ATP analogues with native, catalytically active Pgp using biochemical and fluorescence approaches. We obtained several quantitative parameters for nucleotide binding and hydrolysis, which led to a much more complete picture of the type of interactions of each species with the NBDs of Pgp, and enabled us to make some important conclusions about the mechanism of ATP hydrolysis and drug transport.
ATP␥S was hydrolyzed at a low, but measurable rate by Pgp (ϳ0.4% that observed for ATP). We cannot rule out that ATP␥S hydrolysis is carried out by an impurity in the Pgp preparation, but it seems unlikely. Other membrane-bound ATPases such as Fig. 6), expressed as values Ϯ fitting error. b NATA or Pgp samples in 2 mM CHAPS buffer in the absence or presence of ATP or ATP␥S were titrated at 22°C with increasing concentrations of acrylamide or KI solution. c Parameters for two different populations of Trp residues were determined by fitting the quenching data (see Fig. 6) to Equation 3 (see "Experimental Procedures"), expressed as values Ϯ fitting error. the Na ϩ K ϩ -and Ca 2ϩ -ATPases hydrolyze ATP␥S at very low rates compared with ATP, 0.09% (45) and 0.5% (46), respectively. It seems unlikely that a low abundance contaminant could produce the level of hydrolysis that we observe. Such an ATPase would also have to be almost completely vanadateinhibitable, as inhibition of the ATPase activity of purified Pgp by vanadate approaches 100% (36). Two molecules of ATP␥S were bound to the protein, one with low affinity and the other with 120-fold higher affinity (K d ϭ 6 M), corresponding to an occluded nucleotide. The occluded state reported by Tombline et al. (28) with a single tightly bound ATP molecule (K d ϭ 9 M) probably also contained a second loosely bound nucleotide that was lost during chromatographic separation. This asymmetric intermediate likely exists when native Pgp hydrolyzes ATP, but it is shortlived, as the tightly bound nucleotide is committed to hydrolysis and proceeds rapidly to the transition state. Previously, Sauna et al. (30) reported that Pgp had low binding affinity for ATP␥S (0.29 mM), comparable with that of ATP. However, they used an indirect method (inhibition of photoaffinity labeling) to measure binding, which failed to detect the high affinity site.
We found that the IC 50 for inhibition of Pgp-mediated ATP hydrolysis by ATP␥S was also very low, 6 M, comparable with the K d determined for its high affinity binding. This likely reflects that fact that occupation of only one NBD by ATP␥S is needed to arrest catalytic activity, as the two NBDs are tightly coupled. A low IC 50 value for inhibition of ATP hydrolysis by ATP␥S was reported in 1994 by Senior and co-workers (49), who were among the first to study the ATPase activity of Chinese hamster Pgp in detail. Interestingly, in their experiments ATP␥S stands out among all tested ATP analogues, showing at least a 10-fold higher IC 50 value compared with other nucleotides. More recently, an IC 50 value of 31 M was noted by Martin et al. (50). In contrast, Sauna et al. (30) reported an IC 50 value for ATP␥S of 0.19 mM, which represents a low affinity interaction, comparable with the K d for binding of ATP and ADP and the K m for ATP hydrolysis. The reason for this discrepancy is not clear.
We also demonstrated that ATP␥S inhibited the inactivation of Pgp by MIANS with a very low IC 50 value, whereas other ATP analogues required a Ͼ50-fold higher concentration to do so. Like MIANS, N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibit ATP hydrolysis by reacting covalently within the catalytic sites of Pgp (51,52). They also impair tight binding of ATP to the E552A/E1197A double mutant (29), suggesting that they and MIANS prevent formation of the occluded nucleotide conformation. Because reaction with MIANS "competes" with binding of ATP␥S, the IC 50 value for blocking of MIANS inactivation will reflect the high affinity binding of ATP␥S that is required to lock Pgp in the occluded state. Thus, three different indicators of ATP␥S binding affinity are in the low M concentration range.
AMP-PNP has been the analogue of choice for production of nucleotide-bound crystals of ABC proteins, but in some cases the structure resembled the ADP-bound monomeric form (53) and in others the ATP-bound dimer (15). It was previously reported that AMP-PNP inhibited Pgp-mediated ATP hydrolysis at a relatively high concentration, and it was suggested to have a 10-fold lower affinity for binding compared with ATP (30). In contrast, we show that Pgp binds this non-hydrolyzable analogue with affinity comparable with that of other nucleotides and displays competitive inhibition of ATP hydrolysis with a K i in the same range. ATP␥S is clearly different from the other non-hydrolyzable analogues in mimicking ATP and promoting formation of the correct dimer interface, likely because it has an unchanged ␥-phosphodiester bond. The potential for hydrolysis of nucleotide, even at a very low rate, may be necessary to produce the asymmetric occluded state. The failure of AMP-PNP and AMP-PCP to produce this state may arise because they have no potential for hydrolysis. ATP␥S, but not AMP-PNP, was found to mimic ATP binding to the NBD1 of MRP1 by stimulating ADP trapping at NBD2 (54), and 5 M ATP␥S competed for azido-ATP photolabeling of the NBDs of MRP1, whereas Ͼ20-fold higher concentrations of AMP-PNP were required (55).
The rate of ATP hydrolysis is known to be the rate-limiting step for a bacterial ABC transporter (16), and this is also likely to be true for mammalian ABC exporters such as Pgp. The asymmetric occluded state can likely be detected in the presence of ATP␥S because the rate of hydrolysis is very slow, thus allowing this intermediate to accumulate. In the presence of nucleotides that are hydrolyzed at a high rate (such as ATP, 8-azido-ATP, etc.), the occluded intermediate progresses very rapidly to the transition state, and it is not detectable.
When one molecule of ATP␥S is occluded while the other is bound loosely, Pgp must adopt an asymmetric conformation. We monitored conformational changes in Pgp on ATP␥S binding by collisional quenching of Trp residues. Negatively charged I Ϫ clearly showed two classes of Trp residues, indicating the induction of heterogeneity, possibly arising from an asymmetric protein conformation. The larger group of Trp residues displayed reduced quenching relative to nucleotidefree or ATP-bound Pgp, indicating that they are less accessible to I Ϫ after ATP␥S binding. Thus, ATP␥S binding and occlusion in one NBD either sterically blocks the quencher from gaining access to these Trp residues or reduces the positive charge around them. On the other hand, the second smaller group of Trp residues showed increased quenching compared with nucleotide-free or ATP-bound Pgp, implying that conformational rearrangement as a result of ATP␥S occlusion opens up access of quencher to some Trp residues or makes their local environment more positively charged. Of note, 3 of the 11 Trp residues in Pgp are located within or close to the NBDs (42), and these residues may be most affected by nucleotide occlusion.
The unoccupied NBD in vanadate-trapped Pgp was still able to bind nucleotide despite its lack of catalytic activity (35); thus, this state represents an asymmetric (but inactive) post-hydrolysis conformation. Interestingly, the vanadate-trapped state bound ATP␥S at the vacant NBD with high affinity similar to that observed in the occluded conformation. Thus, the low affinity catalytic site that did not carry out ATP hydrolysis appears to switch its conformation to high affinity immediately after catalytic turnover. We speculate that the other NBD containing trapped vanadate may be in the low affinity conforma-tion, as would be expected for a post-hydrolysis intermediate. Such simultaneous "site-switching" may play an integral role in the mechanism of ATP hydrolysis and tight coupling between the two NBDs.
A conformational switch in the drug binding site from high to low affinity is proposed to take place at some point during the transport cycle of Pgp, thus driving the dissociation/transport of substrate. The results of photoaffinity labeling studies on vanadate-trapped Pgp were interpreted as indicating that this post-hydrolysis conformation displayed very low drug binding affinity (56). However, this was contradicted by other studies showing no change in drug binding affinity in various trapped states of Pgp (57) and TAP1/2 (58,59). The ATP␥S-occluded state of Pgp was also reported to show reduced binding of two photoactive drugs (30); however, the reduction in affinity was Ͻ3-fold, which is too small to drive drug dissociation and transport across the membrane. We examined four substrates (vinblastine, daunorubicin, verapamil, and propafenone GP12) and found little change in the ability of Pgp to bind these drugs after ATP␥S occlusion. The ATP-switch model put forward for all ABC proteins by Higgins and Linton (60) proposed that ATP-induced formation of NBD dimers is the "power-stroke" for substrate transport. However, our results indicate that ATP␥S-mediated closure of the dimer interface and nucleotide occlusion do not alter drug binding affinity, implying that drug transport is not driven by ATP binding or occlusion and takes place at a later stage of the catalytic cycle. Senior proposed that drug transport is coupled to relaxation of a high chemical potential conformation of the catalytic site containing bound Mg 2ϩ ⅐ ADP⅐P i , which is generated by the process of ATP hydrolysis itself, rather than being coupled to nucleotide binding and occlusion (10,11). Our data are compatible with this proposal. The processive, clamp model put forward by Tampé and co-workers (61) for the mitochondrial ABC protein Mdl1 proposes that both ATP molecules are hydrolyzed sequentially in the same catalytic cycle, after which the two NBDs dissociate and release ADP. Such a model predicts the existence of several symmetric intermediates during catalysis, in contrast to the alternating sites proposal. In addition, both NBD dimer interfaces are open simultaneously at the end of the dual hydrolysis cycle.
We have shown in this work that progression of the conformation of Pgp containing two bound nucleotides to the asymmetric occluded state does not depend on binding of transport substrate (as proposed by the ATP switch model (60)). Indeed, this is expected, because Pgp has an uncoupled cycle and can hydrolyze ATP efficiently in the absence of drugs. In addition, a thermodynamic study of isolated NBD subunits of a bacterial ABC protein showed that NBD dimer formation is itself a facile and energetically favorable reaction in the absence of the transmembrane domains and transport substrate (62). In Fig. 7, we propose a site-switching model in which the two NBDs alternate in catalysis, and all intermediates are asymmetric to preserve memory between catalytic cycles. The symmetric state (shown outside the catalytic cycle) consists of two ATP molecules bound to Pgp with low affinity, with two open dimer interfaces. This state has been characterized in the case of catalytically arrested MIANS-Pgp or native Pgp bound to poorly hydrolyzed fluorescent and spin-labeled ATP analogues (32,35,63). When catalytic cycling starts, one of the two ATP molecules becomes tightly bound, or occluded, thus closing the dimer interface at NBD1. This ATP molecule is then commit- FIGURE 7. Proposed catalytic cycle of Pgp, including ATP-driven dimerization, ATP occlusion and hydrolysis, site-switching of nucleotide binding affinity, and drug transport across the membrane. In the symmetric state with two loosely bound ATP molecules (ATP L ), both halves of the dimer interface are open. If the pump is catalytically active, this state rapidly progresses to the asymmetric state in which one ATP molecule is tightly bound or occluded (ATP T ) in NBD1, and the dimer interface in this NBD is now closed. The tightly bound ATP molecule is committed to enter the transition state and undergoes hydrolysis, which provides the energy for movement of drug (black sphere) from the binding pocket within the transporter to the opposing face of the membrane. Binding of drug is shown after that of ATP, but these events can take place in a random order. Transport of drug is assumed to involve a conformational change in the protein transmembrane domains from an inward-facing to an outward-facing conformation. ATP hydrolysis converts the tightly bound ATP to ADP and P i , which are now loosely bound (ADP L ), resulting in opening of the dimer interface in NBD1. The other catalytic site simultaneously switches to the high affinity state, resulting in tight binding of the second ATP molecule and closure of the dimer interface in NBD2. P i dissociates from the catalytic site of NBD1 first, followed by ADP, which is replaced by another molecule of loosely bound ATP (nucleotide exchange) to achieve the asymmetric occluded state once again. A second round of ATP hydrolysis and drug transport then takes place at NBD2. Pgp exists in an asymmetric state at all points of the catalytic cycle, providing memory and mandating that the two NBDs alternate in catalysis.
ted to enter the transition state and undergo hydrolysis. Drug binding is shown as following ATP binding in Fig. 7, but it is known that these two association steps may occur in a random fashion and are not ordered (32). Hydrolysis of the tightly bound ATP has two consequences; energy is provided for drug transport across the membrane, and the nucleotide binding affinity of the two catalytic sites is simultaneously switched. The dimer interface at NBD1 opens again, allowing the now loosely bound ADP and P i products to dissociate, and the ATP molecule at NBD2 is now tightly bound/occluded at the closed dimer interface. Thus, drug movement across the membrane is driven by ATP hydrolysis, as originally proposed by Senior and co-workers (8,11), based on the large drop in free energy that accompanies dissociation of P i from the protein after catalytic turnover. ADP dissociates after P i , and is replaced by ATP; nucleotide exchange is driven by the high cellular ATP:ADP ratio, as their Pgp binding affinities are similar. At this point Pgp has again achieved the asymmetric conformation with one tightly bound and one loosely bound ATP, and the second catalytic cycle proceeds with hydrolysis at NBD2.
This model proposes ATP site affinity switching coincident with or immediately after ATP hydrolysis, so that one of the two NBD dimer interfaces is always in the tightly bound occluded state at all times. The NBD dimer, thus, never dissociates during catalytic turnover (only one-half of the interface opens after hydrolysis of an ATP molecule), and the asymmetry of the structure is maintained continuously throughout the transport cycle. Because of the high degree of structural similarity between the NBDs of many ABC transporters, the mechanism of energy utilization by these proteins is probably conserved, and this model may be applicable to many members of this family. | v3-fos-license |
2019-04-08T13:09:01.384Z | 2016-01-01T00:00:00.000 | 99407113 | {
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} | pes2o/s2orc | Geometrical parameters influence on behavior of the sandwich plates with corrugated core
The influence of geometric parameters on behavior and stiffness of sandwich plates with corrugated core is considered in this paper. The following parameters were analyzed: ratio of core sheet and facing sheet thickness and the ratio of the core's pitch to the core's depth, as well as the corrugation angle. It is shown that changes of these parameters can contribute to increase or decrease of the corrugated sandwich plate stiffness.
Introduction
The sandwich plates are the three-layer structures, which consist of the thick core and the two flat platesfaces. In general, case, the faces are made of the highly resistant materials, while the core is made of materials of the lower resistance and density. Due to high resistance of the faces and small mass of the core, one of the most important characteristics of these structures is a good ratio of the structural resistance and mass.
Advantages of the sandwich plates are the high stiffness and resistance, with respect to their mass, good heat and acoustic insulating properties, good aerodynamics, energy absorption for different types of cores, increase of the inner space and simple mounting. Due to those properties, the sandwich structures have wide application in construction industry, aircraft industry, ship building, in the field of the acoustic and heat insulation, the fire protection, etc.
The sandwich plates' core keeps the two separated faces together and stabilizes them by resisting vertical deformations. It enables the whole structure to behave like a thick plate, when the shear resistance is considered. The core also carries the part of the load in the case when the structure is subjected to bending loading. There are several types of corethe foam or the solid core, the honeycomb, prismatic, truss and corrugated core, Vinson [1].
The light and soft materials are usually used for manufacturing the core. For sandwich plates with the core made of foam or in the honeycomb form, all the planar and flexural loads are carried by the faces, while for the sandwich plates with the prismatic and corrugated core, the core also carries the part of the load.
Large number of researchers was dealing with analysis of the sandwich structures, subjected to various loadings: Allen [2]; Evans et al. [3]; Brittain et al. [4]; Wicks and Hutchinson [5][6]; Valdevit et al. [7]; Tumino et al. [8] and Cheon and Kim [9]. The most used shape of the core is the honeycomb one, since it possesses the highest stiffness and the shear strength with respect to its mass; however, it requires special attention during the manufacturing. The most studied type of the sandwich plate's failure is wrinkling, which comes to the core's resistance to deformation perpendicular to faces. It is especially prominent with plates whose cores are made of expanded materials. The cores that are made of the highly resistant materials, on the other hand, possess very good resistance to wrinkling, where especially good are the corrugated cores. They are sufficiently stiff to contribute to increase of the flexural stiffness of the whole structure. Unlike the honeycomb cores, the corrugated cores sustain well not only the vertical shear, but the bending and torsion, as well.
Influence of geometrical parameters on behavior of the sandwich plate with the corrugated core, shown in Fig. 1, is analyzed in this paper. The plate's behavior depends on its stiffness. Some geometrical parameters, like ratio of core sheet and facing sheet thickness (t c /t f ), ratio of the core's pitch and the depth (p/h c ), corrugation angle D, can contribute to increase or decrease of the stiffness (and thus the resistance) of the plate with the corrugated core.
Corrugated plate geometrical characteristics and stiffness
The sandwich plate with the corrugated core, Fig. 1, consists of two flat platesfaces of thickness t f and the corrugated core sheet thickness t c . The dimensions of the plate are as shown in Fig. 1: Dthe corrugation angle, h cthe core's depth measured from the center line at crest to center line at trough, hdistance between the middle planes of the face sheets, 2pthe face sheets and fthe length of corrugation flat segment. Subscripts c and f refer to core's and the faces' properties, respectively.
In analysis of the sandwich plates with the corrugated core, for the case of flexural loading, the following assumptions were made: the analyzed corrugated core is symmetric, the faces are made of the same material and with the same thickness, the core material is isotropic-elastic, the sandwich plate's deformations are small, the plate's thickness is significantly smaller than the core's depth, the core influences the flexural stiffness in the xdirection but not in the y-direction, the plate is infinitely long in the z-direction, the core can sustain the lateral shear stress and it also affects the flexural and axial stiffnesses, while the lateral shortening of the core is not taken into account.
The flexural stiffnesses in the x and y directions are determined as, Libove and Hubka [10]: 2 2 , Fig. 1. The geometry of the sandwich plate with the corrugated core.
The cross-section's moment of inertia of the core, parallel to the yz plane, per unit width, taken for the symmetry axis of the cross section, I c , and moment of inertia of the face, considered as the membrane, with respect to the middle plane of the sandwich plate, I f , respectively are given by: The torsional stiffness is determined as: where G f is the shear modulus of the face.
Results and discussion
In Figs This leads to conclusion that the (t c /t f ) ratio has stronger influence on the flexural stiffnesses than on the torsional one.
In Fig. 3 is shown the influence of the corrugation angle D variation on the flexural and the torsional stiffnesses, for the following geometrical parameters of the sandwich plate: the core sheet and facing sheet thickness ratio (t c /t f ) =1, the core pitch to depth's ratio (p/h c ) =1 and the core's depth to core thickness ratio (h c /t c ) =10.
Conclusion
The influence of the geometrical parameters of a sandwich plate with the corrugated core on its behavior was considered in this paper. The following parameters' influences were analyzed: ratio of the core sheet thickness to the sandwich plate face's thickness, the corrugation angle and the ratio of the core's pitch to depth.
Based on results obtained from that analysis, one can notice that all the stiffnesses of the sandwich plate (two flexural and a torsional) are decreasing with increase of the core sheet thickness to the face's thickness ratio, though the influence is more prominent for the flexural stiffnesses than for the torsional one. As that ratio increases, for the constant value of the core sheet thickness, the face's thickness decreases, what causes the decrease of the sandwich plate's flexural stiffness, since for the corrugated plates the faces are carrying the larger portion of the bending loading.
For the case of the corrugation angle increase and increase of the core's pitch to depth ratio, the main flexural stiffness increases slightly, while the one in the perpendicular direction and the torsional stiffness remain constant.
Based on analysis that was conducted within this research, certain recommendation can be given for the selection of the geometrical parameters of the sandwich plates with the corrugated core. It is shown that the lower values of the investigated parameters make the sandwich plate more resistant. It would be the best to choose the plate's parameters in such a way that face and core sheet thicknesses would be the same, (t f = t c ), that the corrugation angle value would be about 60° and that the value of the core's pitch to depth ratio (p/h c ) would be between 1 and 1.2. | v3-fos-license |
2019-07-16T14:29:51.293Z | 2019-07-15T00:00:00.000 | 196610933 | {
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} | pes2o/s2orc | Improving the Post-polymerization Modification of Bio-Based Itaconate Unsaturated Polyesters: Catalyzing Aza-Michael Additions With Reusable Iodine on Acidic Alumina
Bio-based platform molecules such as itaconic, fumaric, and muconic acid offer much promise in the formation of sustainable unsaturated polyester resins upon reaction with suitable diols and polyols. The C=C bonds present in these polyester chains allows for post-polymerization modification and such moieties are conventionally utilized in curing processes during the manufacture of coatings. The C=C modification sites can also act as points to add useful pendants which can alter the polymers final properties such as glass transition temperature, biodegradability, hardness, polarity, and strength. A commonly observed modification is the addition of secondary amines via an aza-Michael addition. Conventional procedures for the addition of amines onto itaconate polyesters require reaction times of several days as a result of undesired side reactions, in particular, the formation of the less reactive mesaconate regioisomer. The slow reversion of the mesaconate back to itaconate, followed by subsequent amine addition, is the primary reason for such extended reaction times. Herein we report our efforts toward finding a suitable catalyst for the aza-Michael addition of diethylamine onto a model substrate, dimethyl itaconate, with the aim of being able to add amine onto the itaconate units without excessive regioisomerization to the inactive mesaconate. A catalyst screen showed that iodine on acidic alumina results in an effective, heterogeneous, reusable catalyst for the investigated aza-Michael addition. Extending the study further, itaconate polyester was prepared by Candida Antartica Lipase B (CaL-B) via enzymatic polytranesterification and subsequently modified with diethylamine using the iodine on acidic alumina catalyst, dramatically reducing the required length of reaction (>70% addition after 4 h). The approach represents a multidisciplinary example whereby biocatalytic polymerization is combined with chemocatalytic modification of the resultant polyester for the formation of useful bio-based polyesters.
INTRODUCTION
An over-reliance of the chemical industry on non-renewable feedstocks has resulted in an ever growing interest in the utilization of bio-derived platform molecules to substitute petroleum-derived base chemicals as fundamental buildingblocks for the synthesis of higher value products (Werpy and Petersen, 2004;Farmer and Mascal, 2014). Due to the sheer volumes produced coupled with enormous diversity of applications, it is not surprising that the field of polymer science has shown particular interest in using platform molecules to sustainably source monomers or monomer precursors (Mathers, 2012;Gandini and Lacerda, 2015;Isikgor and Becer, 2015;Llevot et al., 2016;Zhu et al., 2016). Plastics such as poly(lactic acid) (PLA), poly(butylene succinate) (PBS), and poly(ethylene furanoate) (PEF) demonstrate how polymers with favorable properties can be partly or wholly derived from platform molecules. A more recent trend has been toward the synthesis of functionalizable polymers and in particular, the polymerization of common platform molecules, itaconic acid, muconic acid, and fumaric acid with a range of diols/polyols such as 1,2ethanediol, 1,2-propandiol, 1,3-propandiol, 1,4-butanediol, and glycerol ( Figure 1) and to produce novel, 100% bio-derived unsaturated polyesters (UPEs) (Fonseca et al., 2015Robert and Friebel, 2016;Rorrer et al., 2016;Costa et al., 2017;Kumar et al., 2017;Patil et al., 2017;Farmer et al., 2018a). Synthesis of these sustainable UPEs often employs conventional melt polymerization methods using well established metal catalysts (Ti, Al, Sn, and Zn) (Sakuma et al., 2008;Chanda and Ramakrishnan, 2015;Farmer et al., 2015;Winkler et al., 2015;Rowe et al., 2016;Schoon et al., 2017). Although valuable C=C groups present on such monomers offer enhanced functionality to the UPEs, they can also create many issues with the formation of undesired side reactions during the polymerization. In the case of itaconates, fumarates, and muconates typical side reactions include isomerization, radical cross-linking (Figure 2A) and Ordelt saturation (an oxo-Michael addition where an R-OH end-group attacks the conjugated C=C through a β-addition, Figure 2B; Farmer et al., 2015;Schoon et al., 2017).
To prevent radical crosslinking during conventional polyesterification of such monomers, scavengers such as quinol (Chanda and Ramakrishnan, 2015) and 4-methoxyphenol are used as quenchers (Schoon et al., 2017). Enzyme catalyzed polytransesterifications have proven somewhat effective in limiting isomerization but achieving high degrees of polymerization and efficient scaling-up these methods had proven elusive Pellis et al., 2015). Limiting Ordelt saturation has proven even harder to achieve, most likely due to the fact that Lewis acid catalysts promoting polytransesterification will also increases the ability of the conjugated C=C to act as a Michael acceptor to a hydroxyl end-group. Once extensive crosslinking occurs the resultant UPEs, for example when using itaconates, are typically soft and rubbery and thus are only suitable for applications which do not require inherent strength (Singh et al., 1991;Guo et al., 2011;Wei et al., 2012;Dai et al., 2015a,b,c). Undesirable isomerization of the C=C is also widely reported for UPEs of itaconate, muconate fumarate, and maleate monomers, with the latter two able to interchange between one another ( Figure 3A). In the case of itaconate containing polyesters, regioisomerization during polyesterification results in the formation of mesaconate (major) and citraconate (minor) units ( Figure 3B). Formation of these regio-isomer units lead to greater complexity in the analysis of the polyesters, whilst also effecting reproducibility of the polymers final thermal and mechanical properties.
Previous research has continually observed regioisomerization of itaconate during polyester synthesis. The extent of this undesired reaction ranges from <10% (relative to itaconate) as described by Teramoto (9%) (Teramoto et al., 2005), Farmer (8%) (Farmer et al., 2015), and Spavojevic (7%) (Panic et al., 2017), increasing up to nearly 60% in the Takasu's protocol using itaconic anhydride as the monomer (Takasu et al., 1999). In the majority of the above examples the typical reaction conditions are elevated temperatures above 160 • C, high vacuum to remove excess diol and relatively long reaction times and acidic catalysts or polymer chain ends; these conditions contribute to the promotion of mesaconate formation. The most effective method to avoid underdesired regioismerization during polycondenzation is to carry out the reaction under milder enzyme-catalyzed conditions . However, regioisomerization has also been reported to occur during addition of pendants to free unsaturated sites, with a significantly increased proportion of mesaconate being seen for high (>75%) but not complete addition (Clark et al., 2009;Farmer et al., 2016). Pendant addition to itaconate UPEs remains a very desirable pathway as significant alterations to the polymers physical properties can be achieved, whilst the pendants themselves exhibit behavior such as metal chelation. Several recent studies have demonstrated post-polymerization modification (PPM) of bio-based UPEs, allowing these polyester backbones to be altered via facile Michael additions (Fonseca et al., 2015Robert and Friebel, 2016;Rorrer et al., 2016;Costa et al., 2017;Kumar et al., 2017;Patil et al., 2017;Farmer et al., 2018a). Additions of thiols, amines and metalchelating 1,3-dicarbonyls to bio-based UPEs have been recently demonstrated, tailoring the properties of the polyesters to suit a range of applications (Lv et al., 2014;Chanda and Ramakrishnan, 2015;Farmer et al., 2016). For example Hoffmann et al. showed that amine pendant addition to itaconate polyesters can tune the hydrophilicity of resultant gels and be tailored depending on the choice of amine donor (Hoffmann et al., 2015). Amine pendant itaconate polyesters have recently been used to produced temperature switchable materials, with examples of low-temperature depolymerization promoted by primary amine addition (Guarneri et al., 2019) or secondary amine release at elevated temperatures (>190 • C) . However, in many instances long reaction times for the addition of the pendants are quoted with little or no discussion as to why this is necessary. Lv et al. reported the need for 14-20 h reaction times for the addition of thiols and amines, despite the Michael donors being used in a 15-times molar excess (Lv et al., 2014). Chanda reported 3 day long additions of both thiols and amines to poly(dodecyl itaconate) UPEs (Chanda and Ramakrishnan, 2015), while Winkler published thio-Michael additions that ran overnight using a 5-times molar excess of donor, and requiring 10 mol% hexylamine as a catalyst (Winkler et al., 2015). In a recent study it was shown that during aza-Michael addition of diethyl amine (DEA) onto dimethyl itaconate (DMI, a mimic for the itaconate unit in polyesters) regioisomerization (k 2 ) is in direct competition with desired addition (k 1 ) (Figure 4; Farmer et al., 2018b). Mesaconate (DMMes) formation was proven to be catalyzed by the amine Michael donor (DEA) but because the DMMes itself does not act as an acceptor (compounds 2 or 3 were not detected, Figure 4) the lengthy reaction times were deemed to be necessary. These long reaction times were found to be a result of the slow reformation of DMI from DMMes (k * 2 , Figure 4), with k * 2 being an order of magnitude slower than k 1 . It was deduced that a suitable catalyst might be able to selectively catalyze the desired aza-Michael addition before extensive undesired regioisomerization occurs, resulting in significantly reduced post-polymerization modification reaction times from several days to just hours. Herein we report a study into finding a suitable catalyst for the aza-Michael addition of DEA onto DMI and an extension of this method to an addition onto poly(1,8-octylene itaconate) UPE.
Chemicals and Enzymes
Dimethyl itaconate (DMI) and cerium (III) ammonium nitrate (CAN) were purchased from Alfa Aesar. Cerium (IV) ammonium nitrate (CAN) was purchased from FSA. All other chemicals and solvents were purchased from Sigma-Aldrich and used as received if not otherwise specified. For the powdered supports used as received in this study the following information is available from the supplier (Sigma-Aldrich): acidic alumina (19996-6), Brockmann I, 58 Å pore size, 150 mesh; neutral alumina (199974), Brockmann I, 58 Å pore size; basic alumina (199443), Brockmann I, 58 Å pore size; silica K60 (60738), 60 Å pore size, 220-240 mesh. Candida Antarctica lipase B (CaLB) immobilized onto methacrylic resin was purchased from Sigma-Aldrich (product code L4777, also known as Novozym 435). The enzyme was dried under
Preparation of CAN on Silica
Immobilization 1: To a 50 mL round bottomed flask, 1 gram from K60 silica gel was added with 0.2 mmols (109.7 mg) of (NH 4 )Ce(NO 3 ) 6 and 20 mL of MeOH. Equipped with a reflux condenser, the solution was stirred slowly using a magnetic stirrer bar, at room temperature for 2 h. The solvent was then slowly evaporated over an hour under increasing reduced pressure until 10 mbar vacuum was achieved. The resultant bright orange material was then place under high vacuum (<1 mbar) for 2 h and then stored under an inert purge until required. Actual quantities used resulted in a loading of 0.156 mmol g −1 CAN on silica.
Preparation of Iodine on Alumina or Silica
For the generation of the various forms of iodine (I 2 ) on alumina or silica, the standard loading of I 2 was 0.1 mmol (as I 2 ≡ 0.2 mmol elemental I) per gram of alumina/silica (Deka and Sarma, 2001). To create this, 0.3804 g of I 2 was dissolved in 30 mL dichloromethane, before 15 g of the either alumina or silica was added to the reaction mixture. This suspension was then stirred for 30 min, before the excess solvent was removed under reduced pressure at 40 • C. The orange/red powder obtained was then left to dry fully overnight before use. For higher loadings the iodine amount was multiplied (2x, 3x, and 4x) as required while all other reagent amounts and procedures were followed as above.
Enzymatic Polycondenzation for Formation of Poly(1,8-Octylene Itaconate) (POI) The solventless synthesis procedure was taken from previous literature (Pellis et al., 2018. Briefly: 6 mmols of dimethyl itaconate and 6 mmols of 1,8-octanediol were accurately weighed into a 25 mL round bottom flask. The mixture was then stirred at 85 • C until a homogeneous melt was obtained. Ten percent w w −1 calculated on the total amount of the monomers of Novozym 435 was then added to the reaction mixture. The reactions were run for 6 h at 1,013 mbar. A vacuum of 20 mbar was subsequently applied for an additional 18 h maintaining the initial reaction temperature (total reaction time: 24 h). The reaction product was recovered by adding THF to dissolve the POI, the supported CaLB catalyst was removed via vaccumassisted Buchner filtration (Fisherbrand filter paper QL100, 70 mm diameter), and the solvent from the filtrate evaporated under vacuum to yield the POI polymer product. The polymer was characterized ( 1 H-NMR spectroscopy in CDCl 3 , Figure S10) without any additional purification steps prior to use.
Aza-Michael Addition of Diethylamine (DEA) Onto Dimethyl Itaconate (DMI) Using Cerium Catalysts 2.5 mmol of dimethyl itaconate and the selected catalyst (2 mol% Ce relative to DMI) were accurately weighed into an 8 mL flat bottomed sample vial. Twenty millimoles of diethylamine was added to the reaction and stirred at room temperature for 2 or 6 h when kinetic studies were made (taking aliquots at 2 hourly intervals for analysis by 1 H-NMR spectroscopy, CDCl 3 solvent). For the recovery of the catalyst the reaction mixture was filtered via vacuum-assisted Buchner filtration (Fisherbrand filter paper QL100, 70 mm diameter), with no solvent washing, the collected catalyst was left to air-dry over night between reuses.
Aza-Michael Addition of Diethylamine (DEA) Onto Dimethyl Itaconate (DMI) Using Molecular Iodine Catalysts 2.5 mmol of dimethyl itaconate and the selected amount of catalyst (no catalyst, 1.5, 5, 12.5 mol% of I 2 relative to DMI) were accurately weighed into an 8 mL flat bottomed sample vial. Twenty millimoles of diethylamine was added to the reaction and stirred at room temperature for 2 h. An aliquot of the reaction mixture was dissolved in CDCl 3 for analysis by 1 H-NMR spectroscopy.
Aza-Michael Addition of Diethylamine (DEA) Onto Dimethyl Itaconate (DMI) Supported Iodine Catalysts 2.5 mmol of dimethyl itaconate and the selected catalyst (5%mol w.r.t. I 2 per mole of DMI = 1.28 g of standard catalyst 1x loading catalyst) were accurately weighed into an 8 mL flat bottomed sample vial. Twenty millimoles of diethylamine was added to the reaction and stirred at room temperature for 2 h. The catalyst was removed by filtration and an aliquot of the filtrate was dissolved in CDCl 3 for analysis by 1 H-NMR spectroscopy. For the recovery of the catalyst the reaction mixture was filtered via vacuum-assisted Buchner filtration (Fisherbrand filter paper QL100, 70 mm diameter), with no solvent washing, the collected catalyst was left to air-dry over night between reuses. A dry freeflowing powder was obtained from each air drying. For the iodine FIGURE 4 | Competitive reaction between either the addition of diethyl amine (DEA) to dimethyl itaconate (DMI) or regioisomerization of DMI to form dimethyl mesaconate (DMMes, dominant isomer) or dimethyl citraconate (DMCit, minor isomer). on alumina loading study the relative quantity of I 2 (relative to DMI) was increased to 12.5%mol.
Extended Recovery and Reuse (10 Cycles) of Iodine on Acidic Alumina for the Aza-Michael Addition of Diethylamine (DEA) Onto Dimethyl Itaconate (DMI) 2.5 mmol of dimethyl itaconate and 1.28 g of 0.1 mmol g −1 I 2 on Al 2 O 3 catalyst (5%mol w.r.t. I 2 per mole of DMI) were accurately weighed into an 8 mL flat bottomed sample vial. 20 mmol of diethylamine was added to the reaction and stirred at room temperature for 2 h. The catalyst was removed via vacuumassisted Buchner filtration (Fisherbrand filter paper QL100, 70 mm diameter), with no solvent washing, the collected catalyst was left to air-dry over night between reuses. For each used an aliquot of the filtrate was dissolved in CDCl 3 for analysis by 1 H-NMR spectroscopy.
Aza-Michael Addition of Diethylamine (DEA) Onto
Poly(1,8-Octyleneitaconate) (POI) 2.5 mmol of POI (0.6 g, based on constitutional repeat unit of 240.29 g mol −1 ) and 0.64 g of 0.2 mmol g −1 I 2 on Al 2 O 3 catalyst were accurately weighed into an 8 mL flat bottomed sample vial. Twenty millimoles of diethylamine was added to the vial and stirred for 24 h with aliquots taken at various intervals for analysis by 1 H-NMR spectroscopy (CDCl 3 solvent). After 24 h the reaction mixture was filtered to remove the spent catalyst (where used), excess DEA was removed in vacuo and the resultant viscous polymer analyzed by 1 H-NMR (CDCl 3 solvent) to compare against known previous literature characterization for this material . For the non-catalyzed reaction 0.52 g and 0.54 g (duplicate) of polymer was recovered, for the catalyzed reaction 0.33 g and 0.22 g (duplicate) of polymer was recovered.
Nuclear Magnetic Resonance (NMR) Spectroscopy
Gel Permeation Chromatography (GPC) GPC was carried out using a PSS SDV High set composed of 3 analytical columns (300 × 8 mm, particle diameter 5 µm) of 1,000, 1,000 × 10 5 and 10 6 Å pore sizes, plus guard column (Polymer Standards Service GmbH, PSS) installed in a PSS SECcurity SEC system. Elution was with THF at 1 mL min −1 with a column temperature of 30 • C and detection by refractive index. Twenty microliter of a ∼2 mg mL −1 sample in THF, adding a drop of toluene as reference standard, was injected for each measurement and eluted for 50 min. Calibration was carried out in the molecular weight range 370-25,20,000 Da using the ReadyCal polystyrene standards supplied by Sigma Aldrich and referenced to the toluene peak.
Nitrogen Porosimetry
Nitrogen adsorption measurements were carried out at 77 K using a Micromeritics Tristar Porosimeter. Prior to analysis, the catalyst samples were outgassed at 180 • C for 8 h under a flow of nitrogen gas. The specific surface areas were evaluated using the Brunauer-Emmett-Teller (BET) method in the P/P 0 range 0.05-0.3 (linear range). Pore size distribution curves were calculated using the adsorption branch of the isotherms and the Barrett-Joyner-Halenda (BJH) method, and pore sizes were obtained from the peak positions of the distribution curves.
Thermogravitmetric Analysis (TGA)
TGA was performed on a PL Thermal Sciences STA 625 thermal analyzer. 10 mg of sample was weighed into an aluminum cup, placed in the furnace with a N 2 flow of 100 mL min −1 and heated from 30 to 625 • C at a heating rate of 10 • C min −1 .
RESULTS AND DISCUSSION
Aza-Michael addition onto unsaturated polyesters are typically performed without catalyst, however as previously mentioned this requires extensive reaction times (Blaha et al., 2018;Pellis et al., 2019). Contrastingly, catalysts are often used for aza-Michael additions when synthesizing drug molecules, allowing lower activation energies and thus increased rates of reaction. Example catalysts include boric acid , lipases (Dhake et al., 2010), sulfonated zirconia (Reddy et al., 2008), copper(II) acetylacetonate , and indium trichloride (Yang et al., 2007) but more commonly observed is the use of lanthanide metal-centered catalysts (e.g., SmI 2 Reboule et al., 2005 and Yb(OTf) 2 Jenner, 1995). Such catalysts have been shown to be particularly efficient for promoting aza-Michael additions, their activity assumed to be a result of Lewis acid behavior drawing electron density away from the C=C and making the β-position FIGURE 5 | Example 1 H NMR spectrum of the crude product from aza-Michael reaction between DMI and DEA with 3.8-3.5 ppm expanded region of the spectrum shown. Spectra of the crude reaction mixture was recorded in CDCl 3 after 2 h of reaction. more susceptible to attack from the nucleophilic amine. Of the lanthanide series, cerium (Ce) is one of the most intriguing as it can readily switch between the (IV) and (III) oxidation states whereas other lanthanides generally tend to only be stable in the (III) oxidation state. Cerium's (IV) state has high oxidizing power (Kilbourn, 1986) and high redox potential meaning that cerium has been widely used as a single electron oxidant, though the salts of cerium as Lewis acids have received somewhat less attention (Sridharan et al., 2007).
Investigations Into Cerium Ammonium Nitrate as a Catalyst for Aza-Michael Additions
Cerium ammonium nitrate (CAN) is one of the most documented amongst the cerium-based catalysts (Sridharan and Menendez, 2010;So and Leung, 2017). CAN is able to act as a catalyst for a multitude of reactions, including carbon-nitrogen bond forming reactions such as aza-Michael additions (Nair and Deepthi, 2007) and has the benefit of being widely available and relatively cheap. Furthermore, it has also been shown to be an efficient catalyst toward both aliphatic and aromatic amine additions to Michael acceptors, thus offering a broad substrate scope (Duan et al., 2006).
Despite its high activity, recycling of cerium complexes as catalysts has not been well-documented in the literature and whilst a recent review (Molnar and Papp, 2017) on catalyst recycling mentioned two cerium containing complexes, both catalysts had cerium as a minor component. As such, we additionally sought to find an appropriate means of catalyst recovery and reuse in this work.
In alignment with our previous study into the un-catalyzed addition of amines on polyesters (Farmer et al., 2018b;Pellis et al., 2019), we first elected to study the room temperature addition of DEA using DMI as a model compound (Figure 4) representing the UPRs constitutional repeat unit (CRU). Use of a small model compound also aided analysis and avoided issues of changing reaction viscosity and consequently, effects of mass transfer. 1 H NMR analysis of the DMI and DEA reaction mixture showed peaks A and I, confirming the presence of regioisomerization product DMMes (Figure 5). Peak H shows CH 2 of the excess unreacted DEA, whilst there is significant overlap between the amine protons and CH 3 peaks J, making these unquantifiable for some samples. Peaks F is a complex multiplet as the surrounding H atoms are inequivalent, with a similar observation seen for signal G. To calculate the relative amounts of DMI and DMMes, peaks labeled D (methyl ester groups, CO 2 CH 3 ) were integrated as 6 protons. These peaks are expanded in Figure 5 to demonstrate the detectable difference. Although 6 different environments are possible for the methyl ester groups only five signals are observed; D2 is a combination of the unsaturated ester of DMI and one of the ester groups of DMMes. Using these signals we were able to quantify the relative molar ratios of DMI (D2 + D3), DMMes (D1 + D2) and desired adduct 1 (D4 + D5) during the course of reaction. The following As shown in Figure 6, non-immobilized CAN(IV) (Figure 6, R0, white boxes) noticeably increases the formation of aza-adduct over the studied timescale (2-6 h) relative to the conventional un-catalyzed system (Figure 6, white circles). Interestingly, the CAN(IV) catalyst remained insoluble in the reaction media for the duration of the reaction and was therefore investigated as a potentially recoverable and reusable heterogeneous catalyst (Figure 6, R1-R3, shades of gray boxes). However, CAN's activity was found to steadily decrease upon re-use, particularly upon the third reuse (R3, Figure 6). As such, experiments were conducted to further understand the mechanism of the catalyst deactivation in the DMI + DEA model system, in the hope that it would highlight approaches to both improved adduct yield and enhanced catalyst recycling. The reduction of Ce(IV) to Ce(III), with regeneration of Ce(IV) by an external oxidant was considered as one possible mechanism of catalysis. If this was the case, it was thought that the addition of a constant supply of oxygen to act as an oxidizing agent for Ce(III), would improve the recyclability allowing Ce(IV) to be constantly regenerated. However, as shown in Figure S1, this was not successful, where the two Ce oxidation states might exhibit similar activity and the addition reaction could be independent of oxidation state. Decrease in catalytic activity of the recycled catalyst was possibly due to the CAN itself deactivating overtime. CAN(IV) has 2 ammonium and 6 nitrate groups, and CAN(III) has 2 ammonium and 5 nitrate groups. In order to test whether the CAN(IV) was losing its nitrate groups, and consequently catalytic ability, the molar equivalent of sodium nitrate was added to the reaction on CAN's 4th recycle in an attempt to regenerate the CAN(IV). However, there appeared to be little benefit in addition of the sodium nitrate to prevent deactivation of either CAN(III) or CAN(IV), though the latter did show a slightly higher activity after 4 reuses with nitrate addition (Figure S2).
Activity of CAN (IV) and CAN(III) was shown to exhibit slight differences, where CAN(IV) was found to be more efficient at maintaining recyclability but the CAN(III) more efficient for the initial run. The increasing ionic charge might explain this, where increased Lewis acid capability allows CAN(IV) to act as a better catalyst. As for explaining the cases where CAN(III) is a better catalyst, further experiments were conducted. Michael additions with CAN were attempted with various amines to see how steric bulk affected the Michael addition and further probe differences between the two oxidation states; Figure S3 shows the dipropylamine (DPA, Figure S3A) and dibutylamine (DBA, Figure S3B). Interestingly, the difference between CAN(IV) and CAN(III) appears to increase with increasing steric bulk of the amine. Dicyclohexane (DCHA) and diisopropylamine (DIPA) were also tested however no reaction appeared to take place, most likely due to the much more bulky sterics of diisopropyl and cyclohexyl groups. Ce(III) is a bigger ion due to reduced orbital contraction and the increased relative size may have the effect of allowing increasing co-ordination with substrates. This together with one less nitrate group and the increased nucleophilicity of dibutylamine (and relatively small increase in sterics in comparison with the propyl group) might explain the increase rates of reaction seen with the large amine group and Ce(III).
Another consideration was that the Michael adduct, 1, might be hindering the catalytic ability of CAN (both III and IV). Therefore, prior to the Michael addition reaction, CAN(IV) and CAN(III) were stirred for 2 h with a small amount of purified product 1, this was then removed before performing the reaction as normal. The results are shown in Figure S4 (Figure S4B, shows a comparison without this extra pre-reaction step). Initial observations suggest that the addition of product 1 actually improves the reaction; however this is very unlikely to be the case. Incomplete removal of the addition product from the CAN increases the amount of addition product in the reaction, which invalidates the experiment. However, considering the increases in adduct yield as time passes, it is reasonable to conclude that the addition product had no effect, positive or negative, on the catalytic ability of CAN.
Interestingly, a consistent observation was that the CAN(IV) catalyst starts as an orange powder, but after the first reuse the color changes dramatically to give a brown sticky residue that adheres to the side of the reaction vessel. It was considered that this change in physical state might reduce available reactive surface area as well as the catalyst structure itself. In order to avoid agglomeration of the catalyst to a residue, CAN (IV) was physisorbed on to silica, with the aim of retaining small particle sizes with good accessibility. Two different loadings of CAN(IV) on silica were prepared to investigate the effect of loading on reuse and catalytic activity. Figure S5 shows a comparison of catalytic activity between the free and two different immobilized CAN(IV) catalysts and surprisingly, the same loss of activity after reuse is observed and appears to be independent of catalyst loading. Although catalytic activity appears not to be a result of surface area loss, the immobilized CAN(IV) was more easily recovered a reused and therefore this approach still offers some merit, despite not overcoming the catalyst deactivation.
Alternative Catalysts to CAN for Aza-Michael Additions
CANs deactivation issues led us to search for alternative catalysts for the reaction between DMI and DEA. Complexes containing trifluoromethanesulfonate (OTf) ligands are known to be useful in metal-centered catalysis due to their ability to make the central metal ion highly electrophilic, and further to this, there have been many literature reports of complexes containing OTf being recyclable as well as efficient for Michael additions (Kobayashi et al., 1992). Both Ce(IV) and Ce(III) OTf were tested and initially showed much more promising yields to Michael adduct 1, being considerably higher than those achieved with the CAN catalyst ( Figure 7A). Unfortunately, Figure 7B shows that the catalytic ability of CeOTfs reduces significantly upon reuse, achieving similar conversions to the non-catalyzed background syntheses, suggesting almost total deactivation. A comparison between the two oxidation states for the OTf salts showed that the 4+ state is a better catalyst until it is consumed or made ineffective, thereafter the 3+ state dominants, contrary to the trend observed for the CAN catalysts. It is possible that in CeOTf, Lewis acidity is the primary mechanism of catalysis (more so than the CAN) in which case a more electrophilic Ce(IV) is a better Lewis acid.
This data led us to seek an alternative to lanthanide catalysts. A further screen of the literature suggested molecular iodine (I 2 ) as a promising candidate since it is reported to catalyze the addition of various aliphatic Michael acceptors and donors at high yields (>80%) (Borah et al., 2010). The amount of catalyst (based on relative %mol of I 2 to Michael acceptor) was screened at 1.5, 5, and 12.5%mol (entries 2-4, Table 1). Molecular iodine gave impressive yields (76-90%) for all three loadings, giving considerably higher yields than using no catalyst (entry 1, 26-28%), and producing limited amounts of the unwanted DMMes isomer. However, the molecular I 2 dissolved entirely in the excess of DEA, making it unrecoverable. Saikia et al. previously showed that I 2 can be supported onto alumina and used for other aza-Michael additions. This approach was therefore considered and further investigated (Saikia et al., 2009). Acidic alumina (i.e., without I 2 ), used as received, was found to have no catalytic ability (entry 5, Table 1), giving similar results to the no catalyst system (entry 1) with the exception of a slight increase in the isomerization of DMI to DMMes. The low catalytic ability of acidic alumina differs from observations of Bosica and Abdilla who found it to be suitable catalyst for aza-Michael additions of aromatic amines (Bosica and Abdilla, 2016). However, acidic alumina was found to be a suitable support for supporting I 2 .
Using the preparation method given by Deka and Sarma a freeflowing heterogeneous catalyst was prepared and found to give reasonable yields of adduct even upon recovery (via vacuumassisted Buchner filtration and air drying) an subsequent reuse (entry 6, Table 1; Deka and Sarma, 2001). Nitrogen porosimetry (Figure S6) showed the BET surface area of the acidic alumina (120 m 2 g −1 ) decreased slightly upon supporting 0.1 mmol g −1 fresh I 2 (110 m 2 g −1 ), and decreased further for the recovered catalyst (86 m 2 g −1 ). Pore size distribution would suggest this reduction in surface area was a result of partial blocking of the mesopores (Figure S7), though despite this reduction in surface area the 0.1 mmol g −1 I 2 on acidic alumina maintained its catalytic ability upon reuse (entry 6, Table 1). Thermogravimetric analysis (TGA) corresponds to the porosimetry trends, showing the mass loss up to 625 • C for the acidic alumina support (∼5% loss, Figure S8A) increases slightly to 7-8% loss following 0.1 mmol g −1 loading of I 2 (Figure S8B). Of note is that the loss of I 2 does not appear to give a specific narrow temperature range for desorption, but instead seems to occur over a broad temperature range. The recovered catalyst's TGA trace ( Figure S8C) shows an additional mass loss over the 30-220 • C range, this attributed both to some residual amine (30-80 • C, diethylamine b.p. is 55.5 • C) and trapped itaconate, mesaconate, and adduct (100-220 • C). The residual organics observed by TGA likely also caused the surface area and pore size reduction seen from porosimetry.
Equivalent supported forms of 0.1 mmol g −1 I 2 on neutral (entry 7, Table 1) and basic (entry 8, Table 1) alumina were also prepared and trialed for the addition of DEA to DMI, but the original system on acid alumina was found to remain superior. 0.1 mmol g −1 I 2 on K60 silica (I 2 -SiO 2 ) was also assessed as a potential heterogeneous form of iodine (entry 10, Table 1), though this proved less efficient than the alumina supported systems. The I 2 -SiO 2 seemingly promoted undesirable isomerization to a greater extent (>30%) compared to the other catalysts, an observation similarly observed for K60 silica without iodine (entry 9, Table 1). Amberlyst-15, an acidic heterogeneous resin, has also been reported to catalyze aza-Michael additions though for our substrate was found to have limited activity (entry 11, Table 1; Das and Chowdhury, 2007).
Extended Re-use Study for I 2 on Acidic Alumina
An extended recyclability study to 10 full cycles was carried out using the standard loading 0.1 mmol g −1 I 2 on acidic alumina catalyst (Figure 8) for the addition of DEA onto DMI. The first use and subsequent first four recycles retain good catalytic activity, whilst a slight reduction in activity was observed after the 5th recycle. Despite yields to adduct 1 dropping to ∼48% for the last three uses, this remained considerably higher than with no catalyst at all (26-28% entry 1, Table 1, and Figure 9) or FIGURE 8 | Extended re-use study of I 2 on acidic alumina catalyst for addition of diethyl amine onto dimethyl itaconate. 2.5 mmol DMI, 20 mmol DEA, 1.28 g of 0.1 mmol g −1 I 2 on Al 2 O 3 (standard loading) catalyst, stirred for 2 h at room temperature, reaction mixture filtered then filtrate analyzed by NMR spectroscopy (CDCl 3 solvent) while catalyst was recovered and reused with fresh reactants. Following filtration the catalyst was air-dried overnight prior to reuse between each run. All experiments were performed in duplicates and are shown in the figure as the average of two independent experiments ± the standard deviation.
The catalyzed experiment started with 1.28 g of catalyst but after the 10 full cycles 1.06 and 0.97 g (duplicate) of final catalyst was recovered. As such the final runs had catalyst loadings reduced to 76-83% of the original and this mass loss may have contributed to some of the reduced adduct yield observed over the extended reuse study. TGA analysis of the fresh (B, Figure S9) and recovered catalyst after 10 uses (C, Figure S9) suggest the latter contains 10-11% additional mass contributed by residual organics, this likely blocking some active sites and further reducing catalytic activity as observed in Figure 8. Nevertheless, I 2 -Al 2 O 3 was proven to be a more appropriate heterogeneous and recyclable catalyst for the aza-Michael addition compared to the lanthanide catalysts trialed above.
Effects of I 2 Loading on I 2 on Acidic Alumina Catalyst
Increasing I 2 loading on acidic alumina was investigated by multiplying the amounts of I 2 deposited onto the same amounts of catalyst (2x, 3x, and 4x). For their use in the aza-Michael addition the mass of each catalyst was concurrently reduced so as to ensure the same amount of I 2 (12.5%mol of I 2 with respect to DMI) was present in each run despite less acidic alumina support as loading increased. Results in Table 2 show that the standard loading (1x = 0.1 mmol g −1 I 2 -Al 2 O 3 ) and double loading (2x = 0.2 mmol g −1 I 2 -Al 2 O 3 ) gave very similar results for 1st use and reuse, suggesting these suffer little from I 2 leaching. Increasing to 3x and 4x loadings both gave marginally higher yields of adduct for the first use, and the 4x loading suffered from a noticeable drop in yield upon reuse suggesting some leaching of I 2 occurred during the first run. Free, homogenous I 2 is extremely active as a catalyst for this reaction (see entries 2-4, Table 1, entry 4 being the direct comparison with 12.5%mol I 2 relative to DMI) and therefore leaching for the 4x, and possibly 3x, is likely responsible for the 1st use giving a greater yield. Based on this data it was concluded that the 2x loading (0.2 mmol g −1 I 2 -Al 2 O 3 ) was the optimum catalyst and this was used for a study into the catalyzed addition of DEA onto an itaconate polyester.
Use of I 2 on Acid Alumina for Catalyzing the Aza-Michael Addition Onto Itaconate Polyesters
DEA addition onto poly(1,8-octylene itaconate) (POI, Figure 9) was selected as an example reaction to study whether iodine on acidic alumina remained catalytic for reactions involving polymer substrates where viscosity is significantly increased and mass transfer thus reduced. The 2x loaded catalyst (0.2 mmol g −1 I 2 on Al 2 O 3 ) was selected from the screen above. Maximizing the loading of I 2 would reduce the mass transfer limitations by lowering the overall quantity of required solid. A comparison against the uncatalysed system was made, and in both instances an 8:1 molar ratio of DEA to itaconate units was used, thus the excess DEA also acting as a solvent. The POI polyester was prepared using a previously reported enzymatic catalyzed polymerization method, this to minimize isomerization caused during polymerization. The method used for determining the extent of addition and isomerization via 1 H-NMR spectroscopy was modified from the model system ( Figure 5) as a result of some peak overlap and loss of resolution ( Figure S10 for POI, Figure S11 following addition of DEA to the polymer). The reference peak changed from the methyl ester group to the combined signals around 4.2 ppm set to an integral of 4 (CH 2 s of the 1,8-octanediol nearest to the esters). The amount of addition was determined based on loss of alkene proton signals (5.5-6.8 ppm), whilst the amount of isomerization to mesaconate was determined by comparing signal integrals of the alkene protons of mesaconate (6.8 ppm, 1H) and itaconate (5.7 and 6.3 ppm, 1H each). A dramatic increase in rate of formation of aza-Michael adduct pendanted polyester was seen when using the I 2 on acidic alumina catalyst, monitored over a 24 h period (Figure 10). The catalyzed reaction had a 92% conversion rate to the Michael adduct polyester after 24 h, with only 60% seen for the conventional uncatalysed system. The catalyst also attained adduct yields of >70% after just 4 h, and this with a concurrent significant reduction in extent of isomerization to mesaconate. The rapid reduction in the amount of itaconate units, with nearly complete conversion after 6 h, further demonstrates this catalyst to be very effective in promoting aza-Michael additions onto itaconate whilst not promoting undesired regio-isomerization. It was noted that the catalyzed reaction resulted in a polymer of darker orange coloration compared to that of no catalyst. Thermogravimetric analysis (Figure S12) of the fresh and recovered catalyst was thus used to ascertain if an appreciable quantity of I 2 was lost from the surface of the catalyst during the course of the reaction. The fresh 0.1 mmol g −1 I 2 on Al 2 O 3 catalyst showed a prolonged gradual mass loss over the full TGA range (30-625 • C) representing ∼7-8%. Complete loss of all I 2 from this sample should have resulted in a mass loss of just 2.5%, therefore it was hypothesized that water adsorbed onto the Al 2 O 3 was also contributing to the observed mass loss. This was confirmed by comparison to the acidic alumina as received (Sigma-Aldrich), where a gradual mass loss of ∼5% is indeed also observed ( Figure S8A). The recovered spent catalyst following the addition of DEA to DMI also shows this prolonged mass lost but has an additional mass loss at 190-270 • C. This new mass loss event matches the previously reported temperature for retro-aza-Michael addition of the DEA unit from the polymer, suggesting residual polymer is contained on or within the catalyst . The gradual mass loss associated to I 2 and H 2 O release matches roughly with that for the fresh catalyst suggesting leaching of I 2 was only a minimal cause of the observed coloration, and further supports the sound reusability of this catalyst.
CONCLUSION
Although a versatile and useful reaction for the derivatization of α,β-unsaturation carbonyls, aza-Michael additions typically require long reaction times of several days. This is particularly problematic for the addition of amine pendants onto bio-based itaconate polyesters as undesired regioisomerization results in formation of mesaconate units which significantly reduce the rate of aza-Michael addition. A screen of various heterogeneous catalysts using the model reaction of addition of diethylamine onto dimethylitaconate found that I 2 supported onto acidic alumina produced an effective, recoverable and reusable catalyst for this aza-Michael addition. Although initially promising it was eventually concluded that various catalysts based on a cerium metal center were ineffective due to rapid deactivation after their first use. Despite extensive studies we were unable to fully ascertain the cause of this deactivation and hence sought an alternative. I 2 supported on acidic alumina demonstrated far better reusability, with an extended reuse study still showing significant catalytic activity remained. The best supported iodine catalyst gave yields of aza-Michael adduct of >70% after 2 h, even after reuse, while the equivalent non-catalyzed reaction had yields of adduct of 26-28%. A screen of I 2 loading found that catalysts of ≤0.2 mmol g −1 I 2 on alumina maintained their original efficiency upon reuse, while higher loadings of I 2 saw a drop that was likely caused by leaching of iodine into the reaction media. The optimum catalyst was subsequently used for the addition of diethylamine onto poly(1,8-octylene itaconate), this unsaturated polyester prepared via enzymatic polycondenzation. Use of the catalyst for post-polymerization modification showed that >90% amine pendant addition was possible after 24 h, this was far greater than the non-catalyzed equivalent with just 60% adduct and extensive undesired mesaconate units remaining.
More impressively the catalytic system showed >70% addition after just 4 h, the non-catalyzed equivalent reached than half this over the same period. This study thus concludes that I 2 supported on acidic alumina is a very effective catalyst for aza-Michael additions on bio-based itaconate polyesters, and holds much promise in considerably reducing the lengthy times typically used for these reactions.
DATA AVAILABILITY
The raw data generated for the iodine on acidic alumina study and used to confirm the results can be found via doi: 10.15124/0bf5104e-70aa-4bb8-958b-a7501dcd2b48. Raw | v3-fos-license |
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} | pes2o/s2orc | Inactivation of Fam20C in Cells Expressing Type I Collagen Causes Periodontal Disease in Mice
Background FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues. Methods By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20Cfl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20Cfl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches. Results The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice. Conclusion Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice.
FAM20C is expressed at significant levels in the mineralized tissues and a number of soft tissues including dentin, enamel, bone, cementum, periodontal ligament (PDL), cerebrum cortex, basal ganglia, skeletal cartilage, heart, liver and kidney [1,8,9]. Previously, our group showed that global inactivation of Fam20C in mice led to hypophosphatemic rickets, along with a downregulation of certain osteoblast differentiation markers, an elevation of fibroblast growth factor 23 in the serum, and a reduction of serum phosphorus [10]. These Fam20C-deficient mice also showed remarkable enamel and dentin defects [11].
In this study, by crossbreeding the Fam20C floxed/floxed (Fam20C fl/fl ) mice [10] with transgenic mice expressing Cre-recombinase driven by the 2.3 kb Col 1a1 promoter, we generated 2.3 kb Col 1a1-Cre;Fam20C fl/fl (cKO) mice, in which Fam20C was inactivated in the cells expressing Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology and scanning electron microscopy approaches. We performed immunostaining for BSP, OPN, DMP1, dentin sialoprotein (DSP, the NH 2 -terminal fragment of DSPP), periostin and fibrillin-1 to examine if the levels and distribution of these potential substrates of FAM20C were altered in the Fam20C-deficient periodontium. We observed that the Fam20C-deficient mice developed periodontal diseases, along with reduced levels of the above secretory proteins in the periodontium.
Ethics statement
The use of animals in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&M University Baylor College of Dentistry (approved protocol numbers: 2011-09-BCD and 2012-03-BCD) and was in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. mice, which we refer to as ''conditional knockout'' (cKO) mice in this report. The Fam20C fl/+ or Fam20C fl/fl mice from the same litters created during the crossbreeding regime were used as normal controls. Previous studies in our group [10,11] have shown that Fam20C fl/fl mice or mice losing one allele of Fam20C (i.e., heterozygous Fam20C knockout mice) are normal. In this investigation, we also observed that 2.3 kb Col 1a1-Cre;Fam20C fl/+ mice were not different from the wild type mice. Using the Fam20C fl/+ or Fam20C fl/fl littermates of 2.3 kb Col 1a1-Cre;Fam20C fl/fl (cKO) mice as normal controls not only reduced the number of mice needed but also prevented potential variances that may result from comparing mice from different litters. DNA samples isolated from mouse tails were analyzed by polymerase chain reaction (PCR) genotyping with primers specific for the Cre transgene and Fam20C floxed allele, as we previously described [10,11].
Generation
We observed that the periodontal ligament of the 4-week-old cKO mice did not have significant inflammation and the junctional epithelium in their molars was at normal position. Thus, we selected the 4-week-old mice as the starting point of observation, and chose the 12-and 24-week-old mice to evaluate the progression of periodontal defects in the cKO mice. Samples from the normal mice at the same ages were used as controls in this study. Four to seven mice were analyzed for each age group of the cKO or normal mice. The study was performed in accordance with the Guidelines laid down by the National Institutes of Health in the USA regarding the care and use of animals for experimental procedures. The animal protocol was approved by the Animal Welfare Committee of Texas A&M University Baylor College of Dentistry.
Plain X-ray radiography and micro-computed tomography (mCT) The mandibles dissected from the normal and cKO mice at the ages of 4, 12 and 24 weeks were analyzed using plain X-ray radiography (Faxitron MX-20DC12 system; Faxitron Bioptics, Tucson, Arizona, USA). While we also used X-ray radiography to assess the long bone of the cKO mice at the above ages, this report focuses on the loss-of-function effects of Fam20C on the periodontal tissues in the mandible. The mandibles dissected from these mice were examined by mCT radiography (Scanco mCT35 imaging system; Scanco Medical, Brüttisellen, Switzerland) using a low-resolution scan (12-mm slice increment) for morphological observations, as previously reported [10,11]. The data acquired from the high-resolution scans (6-mm slice increment) of the samples from 4 mice (n54) at 12 and 24 weeks were used for quantitative analyses. The quantitative data were reported as mean ¡ SD and analyzed by Student's t test. P,0.05 was considered statistically significant in the quantitative analyses.
Resin-casted scanning electron microscopy (SEM)
For the SEM analyses, the mandibles from 4-week-old mice were dissected and fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer solution (pH 7.4) at 4˚C for 24 hours. The tissue specimens were dehydrated in ascending concentrations of ethanol and then embedded in methyl methacrylate (MMA, Buehler, Lake Bluff, Illinois, USA). After adjusting a suitable comparable position of the samples, sandpaper was used to grind the acrylic block in an increasing order of grit fineness. These samples were then polished using a micro cloth with Metadi Supreme Polycrystalline diamond suspension of 0.1, 0.25 and 0.05 microns in size (Buehler). These samples were then washed ultrasonically and placed in the vacuum system for 2 days. To assess the osteocyte and lacunocanalicular structures, the surface of the MMA-embedded mandible was polished, acid-etched with 12% phosphoric acid for 7 seconds, washed with 5% sodium hypochlorite for 35 minutes, coated with gold and palladium, and then examined using a FEI/Philips XL30 Field emission environmental SEM system (JSM-6010LA, JEOL, Tokyo, Japan).
Preparation of decalcified sections and haematoxylin and eosin (H&E) staining
The mandibles from 4-, 12-and 24-week-old mice were fixed overnight at 4˚C with 4% paraformaldehyde in phosphate buffered saline (PBS) solution and then decalcified in 15% ethylenediaminetetraacetate (EDTA) solution (pH 7.4) at 4˚C for 5,14 days, depending on the ages of the animals. The samples were processed for paraffin embedding, and 5-mm serial sections were prepared for H&E staining, picro-sirius red staining, and immunohistochemistry analyses.
Picro-sirius red staining
For picro-sirius red staining, the sections were immersed in haematoxylin solution for 8 minutes to stain the nuclei and washed for 10 minutes in water. The sections were then stained in picro-sirius red for one hour, washed in two changes of acidified water, dehydrated in three changes of 100% ethanol, cleared in xylene and mounted. We analyzed the structure and organization of collagen fibers in the periodontal ligament under bright-field and polarized light microscopy.
Immunohistochemistry (IHC) Staining
The IHC experiments on paraffin-embedded sections were carried out using ABC kit and DAB kit (Vector Laboratories, Burlingame, California, USA) according to the manufacturer's instructions. The polyclonal anti-FAM20C antibody [9] was diluted at 1:400 and used to analyze the presence or absence of FAM20C in the mandible sections from the normal or Fam20C-cKO mice. We employed polyclonal antibodies against BSP (LF87, a gift from Dr. Larry Fisher of the National Institute of Dental and Craniofacial Research), OPN [32], DMP1 [33], and a monoclonal antibody against DSP [34] to detect the individual members of the SIBLING family as we previously reported [34][35][36]. An affinity-purified polyclonal antibody against periostin at a concentration of 1 mg/ml (Innovative Research, Atlanta, Georgia, USA) and an affinity-purified polyclonal antibody at a concentration of 20 mg/ml against fibrillin-1 (Sigma-Aldrich, St. Louis, Missouri, USA) were employed to detect these two ECM molecules in the periodontal tissues according to the manufacturers' instructions. In the IHC analyses for each type of antibodies, the specimens from the normal and cKO mice from the same litters were stained in the same batch of experiments to ensure that exactly the same conditions were applied to the normal and cKO groups. The same concentrations of normal rabbit serum or rabbit IgG were used to replace the polyclonal antibodies serving as negative controls for the IHC experiments detecting BSP, OPN, DMP1, periostin and fibrillin-1. The same concentration of mouse IgG was used to replace the anti-DSP antibody, functioning as a negative control for this monoclonal antibody. The IHC sections were counterstained with methyl green.
X-ray radiography
X-ray radiography revealed that the cKO mice have defects in the periodontium, dentin abnormalities in the teeth and a rachitic appearance in the skeleton. The cKO mice also had a smaller stature and a lower level of serum phosphorus compared to the normal mice (data not shown); the serum phosphorus level in the 12-week-old cKO mice was reduced by approximately 50%, similar to that in the mice in which Fam20C was globally inactivated (10). This report focuses on the periodontal defects associated with the inactivation of Fam20C.
At 4 weeks after birth, plain X-ray examinations showed radiolucency in the furcation region between the first and second mandibular molars of the cKO mice, while the height of the interdental alveolar bone appeared similar in the normal (control) and cKO mice (Figure 1a). At 12 weeks, the interdental region between the first and second mandibular molars in the cKO mice had remarkable bone loss compared with the normal mice (Figure 1b). At 24 weeks, very little alveolar bone remained in the interdental region, and the remaining alveolar bone in the cKO mice had a much lower radiopacity than the normal mice ( Figure 1c).
Histology
Histological evaluation of the periodontium showed bone defects, disorganization of the collagen fibers in the periodontal ligament (PDL) and detachment of the junctional epithelium, along with the formation of periodontal pockets in the 12and 24-week-old cKO mice (Figures 3 and 4). The amounts of cellular cementum in the 12-and 24-week-old cKO mice appeared to be reduced compared to the normal mice of the same ages. The histological findings were consistent with results from the X-ray analyses, further confirming that these Fam20C-deficient mice developed periodontal disease.
At 4 weeks after birth, H&E staining showed that the PDL of the cKO mice did not have significant inflammation, and the junctional epithelium was at a position close to the cemento-enamel junction, similar to that observed in the normal mice Figure 1. Plain X-ray radiography analyses of 4-, 12-and 24-week-old mice. The mandibles dissected from the 4-, 12-and 24-week-old normal mice (images in the upper portion) and cKO mice (lower portion) were examined by X-ray radiography. At 4 weeks, the furcation region between the mesial and distal roots of the first mandibular molars in the cKO mice had apparent radiolucency compared with the same area of the normal mice (a, arrows). At 12 weeks, the interdental region between the first and second mandibular molars in the cKO mice revealed remarkable bone loss compared with the normal mice (b). At 24 weeks, alveolar bone in the furcation and interdental regions of the cKO mice showed dramatically lower radiopacity than the normal mice (c). Figure 3a (4-week-old normal mice), respectively. b1 and b2 were the higher magnification views of black and blue box area in b (4-week-old cKO mice). c1 and c2 were the higher magnification views of black and blue box area in c (12-week-old normal mice). d1 and d2 were the higher magnification views of black and blue box area in d (12-week-old cKO mice). e1 and e2 were the higher magnification views of black and blue box area in e (24-week-old normal mice). f1 and f2 were the higher magnification views of black and blue box area in f (24-week-old cKO mice). Black arrows indicate the cemento-enamel junctions. Blue arrows indicate the alveolar crests. Black arrowheads indicate the severe inflammation regions. Blue arrowheads indicate the abscesses. At 4 weeks (a, b), the height and area of alveolar bone in the interdental and interradicular regions of the cKO mice were similar to those of the normal mice, and PDL had no significant inflammation. At 12 weeks (c, d) and 24 weeks (e, f), the cKO mice showed typical features of periodontitis, which include PDL inflammation, alveolar bone (Figures 3a2, 3b2, black arrows). The height and area of the alveolar bone in the interdental and interradicular regions of the cKO mice (Figures 3b, 3b1, 3b2) were similar to those of the normal mice (Figures 3a, 3a1, 3a2). However, picro-sirius red staining showed that the collagen fibers in the PDL of the 4-week-old cKO mice were remarkably thinner and more disorganized than in the normal mice ( Figure 4). Some collagen fibers in the Fam20C-deficient PDL appeared broken or detached from the alveolar bone or root surface (Figures 4b1, 4b2).
In the interdental area of the 12-week-old cKO mice, significant inflammation was observed in the PDL, the majority of the alveolar bone was lost, the junctional epithelium had migrated to the apical region, and deep periodontal pockets had formed (Figures 3c2, 3d2). The picro-sirius red staining revealed that the majority of the collagen fibers in the interdental area were lost (data not shown). The furcation region (Figure 3d1) in the cKO mice also showed bone loss and inflammation although the defects in this area were not as severe as those in the interdental region (Figure 3d2). Figure 4a (normal mice), respectively. b1 and b2 were the higher magnification views of left and right box area in b (cKO mice). Ab, alveolar bone; D, dentin; arrows indicate collagen fibers in the PDL. In the normal PDL (a, a1, a2), the thick collagen fibers were evenly distributed. In the Fam20C-deficient PDL (b, b1, b2), the collagen fibers were remarkably thinner and unevenly distributed, with some collagen fibers detached from the alveolar bone or root surface. Bar in a or b: 200 mm; bar in a1, a2, b1 or b2: 20 mm. The periodontal defects in the 24-week-old cKO mice (Figures 3e, 3f) were worse than in the 12-week-old mice. At 24 weeks, nearly all of the interdental alveolar bone was lost and certain areas of the PDL were necrotized, accompanied by the formation of abscesses. Due to significant bone absorption, the alveolar bone in the furcation regions of the cKO mice became island-like (bone spicules), giving rise to a network appearance; inflammatory cells and fibroblasts were present within these networks of spicules. The picro-sirius red staining revealed that nearly all of the collagen fibers in the interdental region were broken down (data not shown).
Backscattered and acid-etched scanning electron microscopy (SEM)
Using backscattered SEM, we observed that in the 4-week-old normal mice, minerals were evenly distributed around the osteocyte lacunae in the interradicular alveolar bone of the first molar (Figures 5a, 5a1), while the mineral level was lower in the same region surrounding the osteocytes in the cKO mice ( Figures 5b, 5b1). The normal mice had a considerable amount of cementum in the apical region (Figure 5a2), while the cKO mice had significantly less cementum, which also appeared to have a lower level of mineralization ( Figure 5b2).
The resin-infiltrated sections were acid-etched to reveal three-dimensional images of the osteocytes and their processes contained in the lacuno-canalicular systems of the alveolar bone ( Figure 6). The lacunae of the normal osteocytes in the interdental region or furcation region were highly organized and regularly spaced with numerous canaliculi appearing to radiate out orderly from the osteocyte lacunae (Figures 6a1, 6a2). In comparison, the lacunae of the osteocytes in the alveolar bone of the cKO mice appeared to be larger and irregularly distributed with fewer disorganized canaliculi, giving the impression of being ''collapsed'' (Figures 6b1, 6b2). These observations indicated that the osteocytes and their processes in the alveolar bone were abnormal.
Immunohistochemistry (IHC) Staining
IHC was performed to assess the presence or absence of FAM20C and to analyze the expression and distribution of BSP, OPN, DMP1, DSP, periostin and fibrillin-1 in the normal and cKO mice at 4, 12 and 24 weeks after birth. In this report, the representative images from the IHC analyses of 4-week-old mice are presented.
Anti-FAM20C immunostaining analyses showed that in the normal mice, FAM20C was present in the odontoblasts, osteoblasts and PDL fibroblasts, while the signal for this protein was not seen in the corresponding components of the cKO mice (Figure 7). These observations indicated that in the 2.3 kb Col 1a1-Cre;Fam20C fl/fl mice, FAM20C was effectively nullified in the Type I collagenexpressing cells.
In the 4-week-old normal mice, BSP was mainly detected in the alveolar bone and cementum, and the immunoreactivity was stronger along the reversal lines in the alveolar bone (Figures 8a, 8a1). The signal for BSP was weaker in the cementum (arrows) and alveolar bone (arrowheads) of the cKO mice (Figures 8b, 8b1) compared to the two tissues of the normal mice (Figures 8a, 8a1). Additionally, BSP in the Fam20C-deficient alveolar bone showed a diffused distribution pattern (Figure 8b1), in contrast to the protein of the normal mice that was concentrated along the reversal lines in the alveolar bone (Figure 8a1).
In the normal mice, OPN was detected in the alveolar bone, cementum and PDL (Figures 8c, 8c1). In the cementum and alveolar bone of the cKO mice (Figures 8d, 8d1), the level of OPN was remarkably reduced in comparison to Figure 5. Backscattered SEM analyses of periodontal tissues in 4-week-old mice. a1 and a2 were the higher magnification views of the blue box area (furcation region) and yellow box area (apical region) in Figure 5a (normal mice), respectively. b1 and b2 were the higher magnification views of the blue box area (furcation region) and yellow box area (apical region) in b (cKO mice). In a2 and b2, the cementum was outlined by the yellow-dotted lines. In the images of backscattered SEM, the black areas represent unmineralized or hypomineralized areas, and a greater degree of whiteness represents the presence of a higher level of mineral. The network appearance in the furcation region of the cKO mice was primarily due to the presence of the unmineralized osteoid within the osseous masses; the alveolar bone images in a1 and b1 were from the upper portion of the furcation bone, which contained little or no central spongiosa. These images revealed that the alveolar bone in the furcation region of the cKO mice (b1) had a lower level of mineralization compared to the same region of the normal mice (a1). Note that the cKO mice (b2) had much less cementum than in the normal mice (a2). Bar in a or b: 500 mm; bar in a1, a2, b1 or b2: 100 mm.
doi:10.1371/journal.pone.0114396.g005 their normal littermates. The level of OPN in the PDL of cKO did not seem to be significantly different from that of the normal mice.
In the periodontium of the normal mice, DMP1 was observed in the alveolar bone and cementum (Figures 8e, 8e1). The level of DMP1 was remarkably lower in the Fam20C-deficient alveolar bone and cementum (Figures 8f, f1) than in the normal tissues.
In the periodontium of normal mice, DSP was mainly detected in the alveolar bone, in particular, the alveolar bone of the furcation region (Figures 8g, 8g1). DSP was undetectable in the alveolar bone of the cKO mice (Figures 8h, 8h1).
In the normal mice, strong signals for periostin were observed across the PDL, with an accentuated accumulation along the thick collagen fibers (Figures 9a, 9a1). The level of periostin in the PDL of the cKO mice was dramatically reduced ( Figures 9b, 9b1).
Strong signals for fibrillin-1 were detected in certain areas of the PDL of the normal mice (Figures 9c, 9c1). The fibrillin-1 signals were weaker in the PDL of the cKO mice (Figures 9d, d1) than in the normal mice.
Discussion
FAM20C has been studied only to a limited extent. Previously, we analyzed the spatiotemporal expression of FAM20C in mouse tissues and found that this protein is expressed at significant levels in osteoblasts, cementoblasts and PDL fibroblasts [9]. In this study, we analyzed the periodontal tissues of the 2.3 kb Col 1a1-Cre;Fam20C fl/fl (cKO) mice, in which Fam20C was inactivated in the cells expressing Type I collagen. Since osteoblasts, cementoblasts and PDL fibroblasts Figure 6. Acid-etched SEM analyses of the alveolar bone in the 4-week-old mice. a1 (normal) and b1 (cKO) were SEM images taken from the alveolar bone in the furcation region (from the blue box areas). Images of a2 (normal) and b2 (cKO) were taken from the alveolar bone in the interdental region (yellow box). The lacunae of Fam20C-deficient osteocytes appeared ''collapsed'' (b1, b2). The lacuno-canalicular networks in the cKO mice were disorganized with fewer canaliculi that appeared thicker and more randomly distributed compared to the normal mice. Bar in a or b: 500 mm; bar in a1, a2, b1 or b2: 10 mm. express Type I collagen, the alveolar bone, cementum and PDL in the cKO mice were Fam20C-deficient, allowing us to analyze the effects of Fam20C inactivation on the health of periodontium.
At 4 weeks after birth, histology analyses using H&E staining revealed that there was no obvious inflammation in the PDL and no significant migration of the junctional epithelium in the Fam20C-deficient mice. However, picro-sirius red staining showed that the collagen fibers in the Fam20C-deficient PDL were very thin, sparsely distributed and disorganized. The backscattered SEM analyses showed that the mineralization level of the alveolar bone and cementum in the cKO mice was lower than in the normal mice, and the cKO mice also had less cementum. The acid-etched SEM analyses demonstrated that the lacunae of osteocytes in the Fam20C-deficient alveolar bone appeared to have ''collapsed'', and the process-encompassing canaliculi were disorganized. There was a sharp reduction of the SIBLING proteins: BSP, OPN, DMP1 and DSP in the Fam20Cdeficient alveolar bone and/or cementum. Previous studies have shown that lossof-function of BSP [18], DMP1 [16], or DSPP [19] leads to periodontal defects in mice. The reduction of these SIBLING molecules in the periodontium of the cKO mice could be a contributing factor to the development of periodontal disease in these mice at later stages. Periostin is an adhesion molecule produced by the fibroblasts and secreted into the PDL [21]. Studies have shown that inactivation of periostin leads to periodontal disease in mice [24,25,28]. In the present investigation, we showed a remarkable reduction of periostin in the PDL of the cKO mice. Another ECM molecule, fibrillin-1, whose inactivating mutations are associated with severe periodontal diseases [26,27], was also reduced in the PDL of the cKO mice. These structural and molecular changes in the cKO mice indicate the Fam20C-deficient periodontium had intrinsic (inherent) defects. Collectively, these intrinsic defects may lead to the severe periodontal disease observed in the 12-and 24-week-old cKO mice. Figure 8a (normal mice, anti-BSP immunostaining). b1 was the higher magnification view of the box area in b (cKO mice). c1 was the higher magnification view of the box area in c (normal mice, anti-OPN). d1 was the higher magnification view of the box area in d (cKO mice). e1 was the higher magnification view of the box area in e (normal mice, anti-DMP1). f1 was the higher magnification view of the box area in f (cKO mice). g1 was the higher magnification view of the box area in g (normal mice, anti-DSP). h1 was the higher magnification view of the box area in h (cKO mice). Arrows indicate cementum, and arrow heads indicate alveolar bone. Note that the signals (brown) for BSP, OPN and DMP1 in the cementum and alveolar bone of the cKO mice were weaker compared to the same tissues of the normal mice. DSP signals were clearly observed in the alveolar bone of normal mice, but were undetectable in the same tissue of the cKO mice. These data indicate that the levels of these SIBLING family members were reduced in the periodontal tissues of the cKO mice. In the IHC analyses for each type of the antibodies, the specimens from the normal and cKO mice from the same litters were stained in the same batch of experiments. Bar in a, b, c, d, e, f, g or h: 500 mm; bar in a1, b1, c1 d1, e1, f1, g1 or h1: 50 mm. doi:10.1371/journal.pone.0114396.g008 At 12 or 24 weeks after birth, the cKO mice revealed a significant reduction of alveolar bone and cementum, remarkable inflammation in the PDL, formation of deep periodontal pockets, and disorganization of PDL fibers. These findings demonstrated clearly that the Fam20C-deficient mice developed periodontal disease. It should be noted that mice younger than 12 months do not naturally develop periodontal diseases [37], and thus, the periodontal disease in the cKO mice must be attributed to the inactivation of Fam20C. The cKO mice also had inflammation in the dental pulp at 12 or 24 weeks, which might spread to the PDL via the apical foremen. Therefore, the inflammation in the PDL at these stages may be attributed to two factors: 1) direct infiltration of bacteria from the periodontal pockets that were formed in association with the intrinsic defects of Fam20C-deficient periodontium (primary), and 2) spreading of inflammation from the infected pulp (secondary). We believe that the inherent defects caused the lack of proper formation of alveolar bone, cementum and PDL, which subsequently leads to the apical migration of the epithelial attachment, inflammation in the PDL and formation of periodontal pockets, while the secondary effects (spreading of inflammation from the infected pulp) might further aggravate the periodontal disease in the cKO mice. These observations indicate that FAM20C plays a fundamental role in maintaining the structural integrity of the periodontal structures. While the 4-week-old cKO mice had intrinsic defects in their periodontium, they did not form periodontal pockets. The 12-week-old cKO mice formed deep periodontal pockets and the defects Figure 9. IHC analyses of periostin and fibrillin-1 in the periodontal tissues of 4-week-old mice. a1 was the higher magnification view of the box area in Figure 9a (normal mice, anti-periostin). b1 was the higher magnification view of the box area in b (cKO mice). c1 was the higher magnification view of the box area in c (normal mice, anti-fibrillin-1). d1 was the higher magnification view of the box area in d (cKO mice). Strong signals for periostin were seen in the PDL, in particular, along the collagen fibers in the normal mice (a, a1). The level of periostin in the PDL of the cKO mice was reduced (b, b1). Fibrillin-1 signals were strong in certain areas of the PDL and its signals were weaker in the PDL of cKO mice (d, d1) compared to the normal mice (c, c1). Bar in a, b, c or d: 500 mm; bar in a1, b1, c1 or d1: 100 mm. became much worse at 24 weeks after birth. These observations indicate that the periodontal deterioration progressed rapidly in the absence of FAM20C.
In vitro studies have revealed that FAM20C is a Golgi kinase that phosphorylates serine residues in the S-X-E motifs of members in the secretory calcium binding phosphoprotein family [12,13], which includes the SIBLING molecules and certain enamel proteins [13,38]. Mouse periostin has three S-X-E motifs in its amino acid sequence [30,31], and mouse fibrillin-1 has seven S-X-E motifs [29]; thus, these two ECM molecules are potential substrates of FAM20C. In this investigation, we observed a significant reduction of the SIBLING proteins (BSP, OPN, DMP1, DSP), periostin and fibrillin-1. At this point, we do not have a clear answer to the question of why the inactivation of Fam20C leads to the reduction of these secretory proteins. We hypothesize that a partial or complete failure of the phosphorylation of these ECM proteins may send feedback signals to the corresponding cells in the cKO mice and instruct the cells to reduce the synthesis of these proteins in order to avoid ''wasting'' their products. It is also possible that the ECM proteins with a partial or complete failure in phosphorylation may be degraded faster than their natural forms, leading to the reduction of these molecules in the ECM of the periodontal tissues in the mutant mice. Clearly, future studies are warranted to examine the phosphorylation status of these ECM proteins in the Fam20C-deficient tissues. | v3-fos-license |
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} | pes2o/s2orc | Protein Tyrosine Nitration of the Flavin Subunit Is Associated with Oxidative Modification of Mitochondrial Complex II in the Post-ischemic Myocardium*
Increased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} and NO production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. A crucial segment of the mitochondrial electron transport chain is succinate ubiquinone reductase (SQR or Complex II). In SQR, oxidative impairment and deglutathionylation of the 70-kDa flavin protein occurs in the post-ischemic heart ( Chen, Y. R., Chen, C. L., Pfeiffer, D. R., and Zweier, J. L. (2007) J. Biol. Chem. 282, 32640-32654 ). To gain insights into the oxidative modification of the 70-kDa protein in the post-ischemic myocardium, we used the identified S-glutathionylated peptide (77AAFGLSEAGFNTACVTK93) of the 70-kDa protein as a chimeric epitope incorporating a “promiscuous” T cell epitope to generate a high titer polyclonal antibody, AbGSC90. Purified AbGSC90 showed a high binding affinity to isolated SQR. Antibodies of AbGSC90 moderately inhibited the electron transfer and superoxide generation activities of SQR. To test for protein nitration, rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion. Tissue homogenates were immunoprecipitated with AbGSC90 and probed with antibodies against 3-nitrotyrosine. Enhancement of protein tyrosine nitration was detected in the post-ischemic myocardium. Isolated SQR was subjected to in vitro protein nitration with peroxynitrite, leading to site-specific nitration at the 70-kDa polypeptide and impairment of SQR electron transfer activity. Protein nitration of SQR further impaired its protein-protein interaction with Complex III. Liquid chromatography/tandem mass spectrometry analysis indicated that Tyr-56 and Tyr-142 were involved in protein tyrosine nitration. When the isolated SQR was subjected to in vitro S-glutathionylation, oxidative modification and impairment mediated by peroxynitrite were significantly decreased, thus confirming the protective effect of S-glutathionylation from the oxidative damage of nitration.
Mitochondrial dysfunction in ischemia-reperfusion injury is caused by oxidative stress (1)(2)(3)(4)(5)(6)(7). In the ischemic myocardium oxygen delivery to the myocyte is not sufficient to meet the need for mitochondrial oxidation during the physiological conditions of hypoxia, leaving the mitochondrial electron transport chain in a highly reductive state. This results in increased electron leakage from the electron transport chain that in turn reacts with residual molecular oxygen to give superoxide (O 2 . ) (8,9). Because of the lack of ADP, re-introduction of oxygen with reperfusion will greatly increase electron leakage along with a decrease in scavenging capacity, leading to O 2 . and O 2 . -derived oxidants being overproduced in mitochondria.
Specifically, an increased hyperoxygenation induced by reperfusion in the post-ischemic heart has been detected by in vivo EPR oximetry (10 -12). The overproduction of reactive oxygen species (ROS) 2 also initiates oxidative impairment of Complex II (or succinate-ubiquinone reductase (SQR)) as reported previously (7). Myocardial ischemia provides a stimulus to alter NO metabolism (13)(14)(15)(16)(17). Increased NO production and subsequent peroxynitrite (OONO Ϫ ) formation have been detected in the postischemic heart (10,11,14,15). Alterations in the generation of NO occurring in hearts subjected to ischemia/reperfusion have been linked to NO synthase (NOS)-dependent (10,11,14,15) and NOS-independent (18,19) pathways, including involvement of endothelial NOS (eNOS), increased nitrite disproportionation, and increased expression of inducible NO synthase (iNOS) in chronic reperfusion injury (11). Therefore, myocardial ischemia/reperfusion indirectly changes the balance between NO and ROS (especially O 2 . ) in mitochondria. It has been documented that OONO Ϫ -mediated nitration of mitochondrial proteins can be detected in the endothelial cells after ischemia/reperfusion under flowing conditions (20).
Mitochondrial Complex II (EC 1.3.5.1. succinate:ubiquinone oxidoreductase) is a key membrane complex in the tricarboxylic acid cycle that catalyzes the oxidation of succinate to fumarate in the mitochondrial matrix (21). Succinate oxidation is coupled to reduction of ubiquinone at the mitochondrial inner membrane as one part of the respiratory electron transport chain. SQR mediates electron transfer from succinate to ubiquinone through the prosthetic groups of flavin adenine nucleotide (FAD), [2Fe-2S] (S1), [4Fe-4S] (S2), and [3Fe-4S] (S3) and heme b. The enzyme is composed of two parts, a soluble succinate dehydrogenase and a membrane-anchoring protein fraction. Succinate dehydrogenase contains two protein subunits, a 70-kDa protein with a covalently bound FAD, and a 27-kDa iron-sulfur protein hosting S1, S2, and S3 iron-sulfur clusters (21). The membrane-anchoring protein fraction contains cytochrome b hosting two hydrophobic polypeptides (CybL/14 kDa and CybS/9 kDa) with heme b binding (22).
In previous studies we have demonstrated that oxidative impairment (ϳ42% reduction of thenoyltrifluoroacetone-sensitive electron transfer activity) of mitochondrial Complex II was detected in the post-ischemic heart from in vivo regional ischemia-reperfusion models (7). A decrease in the redox modification of protein S-glutathionylation was marked at the 70-Da FAD binding subunit. In vitro studies indicated that removal of S-glutathionylated (GS) binding from the specific cysteine residue(s) of Complex II moderately increased enzyme-mediated O 2 . production and decreased electron transfer efficiency. Therefore, oxidative post-translational modification of the SQR 70-kDa subunit is logically hypothesized to follow deglutathionylation of the SQR 70-kDa subunit. The molecular events regarding oxidative post-translational modification of the flavin protein in Complex II after myocardial ischemia/reperfusion are not clear and remain to be defined. Furthermore, in vitro studies have also identified a tryptic peptide, AAFGLSEAGFNTACVTK (aa 77-93), which is involved in S-glutathionylation (or GS binding) (7). Whether the identified GS binding domain is essential for the enzymatic function of SQR remains to be determined. This study was undertaken to address the fundamental questions regarding the redox biochemistry of Complex II and its molecular mechanism of oxidative post-translational modification implicated in the post-ischemic myocardium. We have employed immunochemistry to define the functional role of the GS binding domain in SQR. Furthermore, we detected an increase in protein tyrosine nitration associated with the FAD binding subunit of Complex II in the post-ischemic heart in vivo. In in vitro studies using isolated SQR from the myocardial tissue, we have characterized the specific tyrosyl residues involved in detected protein nitration mediated by OONO Ϫ in the 70-kDa subunit in nitrated SQR.
Peptide Synthesis and Purification
Peptide synthesis was performed on a Milligen/Biosearch 9600 solid-phase peptide synthesizer (Bedford, MA) using Fmoc/t-butyl chemistry. Preloaded Fmoc-amino acids on CLEAR ACID resin (0.36 meq/g) (Peptides International, Louisville, KY) were used for the peptide synthesis with the PyBop/ HoBt coupling method. The B-cell epitope was assembled by choosing regioselective side chain protection on Cys residues as Cys (Trt) or cys (Acm) essentially as described by Kaumaya et al. (23,24). The B-cell epitope was synthesized co-linearly with a promiscuous T-helper epitope (MVF) derived from the measles virus fusion protein (amino acids 288 -305). Also, an MVF T-helper epitope with a four-residue linker (GPSL) was incorporated for independent folding and was assembled on the B-cell epitope. All peptides were cleaved from the resin using global deprotection reagent B (trifluoroacetic acid:phenol: water:triisopropylsilane, 90:4:4:2). The protecting group from Cys (Trt) comes off in the global cleavage reaction. Crude peptides were purified on preparative reverse phase HPLC using a C-4 Vydac column in a water (0.1% trifluoroacetic acid), acetonitrile (0.1% trifluoroacetic acid) gradient system. Pure fractions were analyzed using analytical HPLC, pooled together, and lyophilized in a 10% acetic acid solution. The purified peptide was hydrolyzed dry and kept at Ϫ20°C to prevent oxidation of free sulfhydryl groups of Cys residues.
Peptide Immunization and Antibody Purification
Two New Zealand White rabbits (6 -8 weeks old, female outbreed) were purchased from Harlan (Indianapolis, IN) and immunized with an MVFGSC90 chimeric peptide (1 mg) dissolved in H 2 O (500 l) with 100 mg of a muramyl dipeptide adjuvant, nor-MDP (N-acetylglucosamine-3-yl-acetyl-L-alanyl-D-isoglutamine). Peptides were emulsified (50:50) in a Montanide ISA 720 vehicle (Seppic). Two ml of blood were drawn for pre-immunization sera. All rabbits were immunized subcutaneously at four spots on the back. After the first immunization the same dose was administered three more times as booster injections 3, 6, and 9 weeks later. Sera were collected by bleeding from the ear of the rabbit after each immunization for determination of antibody titers. Antibody titers were determined by enzyme-linked immunosorbent assay.
High titer sera were purified on a protein A/G-agarose column (Pierce). Eluted antibodies were concentrated and exchanged in phosphate-buffered saline using 100-kDa cut-off centrifuge filter units (Millipore, Bedford, MA).
In Vivo Myocardial Regional Ischemia-Reperfusion Model
The procedure for the in vivo ischemia-reperfusion rat model was performed by the technique reported in the literature (10,25). Sprague-Dawley rats (ϳ300 -350 g) were anesthetized with Nembutal administered intraperitoneally (80 -100 mg/kg). After the rats were fully anesthetized, they were intubated and then ventilated with room air (1.0 ml, rate of 100 breaths/min) using a mechanical ventilator Model 683 (Harvard Apparatus, Holliston, MA). The rats then underwent a left lateral thoracotomy, the pericardium was opened, and a pericardial cradle formed to allow adequate exposure of the heart surface. The left anterior descending coronary artery (LAD) was then occluded by placing a suture (6.0 nylon) around the origin of the LAD.
After 30 min of ischemia, the suture around the coronary artery was untied, allowing reperfusion to occur. Following the reperfusion, all wounds were closed and infiltrated with 0.5% xylocaine (Ͻ0.3 ml). The muscular layers and skin incisions were closed with 4.0 nylon sutures. A chest tube (2.5 cm PE 50 tubing) was inserted at the wound site and maintained in position, whereas the animal was taken off respiratory support.
Upon spontaneous breathing, the chest tube was removed, and a surgical clip was applied over the withdrawal site. The animal was allowed to recover, and a physiological assessment was performed. During the recovery period the animals received supportive post-operative care as needed. Body temperature was maintained at 37°C by a thermal heating pad. By 6 h post-operation the animals had recovered sufficiently to eat and drink independently.
At 24 h post-infarction the rats were placed under deep anesthesia with Nembutal (200 mg/ml). The left anterior descending coronary artery was reoccluded, and Evans blue (4%) was injected from the inferior vena cava to delineate the non-ischemic myocardial tissue. Rats were then sacrificed, and the hearts were excised and placed in phosphate-buffered saline buffer. The infarct area was identified by 2,3,5-triphenyltetrazolium chloride staining. The risk region of myocardial tissue without 2,3,5-triphenyltetrazolium chloride staining was excised and subjected to biochemical analysis.
Cell Culture and Confocal Fluorescence Microscopy
Rat cardiac myoblasts (H9c2 cell line from ATCC, Manassas, VA) were grown and maintained in Dulbecco's modified Eagle's medium (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin antibiotic in 35-mm polystyrene tissue culture dishes at 37°C in the presence of 4.5% CO 2 . Confluent cells with Ͼ90% viability were used to conduct immunoblotting.
Cardiac myoblasts were placed on sterile coverslips (Harvard Apparatus, 22 mm 2 ) in 35-mm sterile dishes at a density of 10 4 cells/dish and incubated at 37°C in a humidified 5% CO 2 , 95% air mix before hypoxia/reoxygenation treatment. Cells were plated in a Modular Incubator Chamber (Billups-Rothenberg, Inc., Del Mar, CA). Hypoxic treatment was accomplished by flushing nitrogen gas on the surface of the glucose-free medium and incubating for 1 h, after which reoxygenation was carried out by incubating in medium with glucose for 1 h. At the end of the experiment, cells attached to coverslips were washed with phosphate-buffered saline and fixed with 3.7% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 containing 0.01% Tween 20 in Tris-buffered saline (TTBS) for 5 min, then blocked for 30 min with 1% bovine serum albumin (BSA) in 0.01% TTBS and incubated with AbGCS90 (1:2000) for visualization of the SQR 70-kDa subunit and the anti-3nitrotyrosine monoclonal antibody (1:1000, Upstate Biotechnology, Inc., Lake Placid, NY) in 0.01% TTBS containing 1% BSA for 1 h at room temperature. After treatment of cells with the chosen primary antibodies, they were incubated with secondary anti-rabbit AlexaFluor 488-conjugated and secondary anti-mouse AlexaFluor 568-conjugated antibodies (1:1000) for 1 h at room temperature. The coverslips with cells were then mounted on a glass slide with the antifade mounting medium, Fluoromount-G, viewed with confocal fluorescence microscopy (LSM 510; Zeiss, Thornwood, NY) with a 60ϫ objective, and overlaid with LSM Image Brower software, generating a merged image for each co-stained specimen.
Preparations of Mitochondrial Succinate-Cytochrome c Reductase (SCR) and SQR
Bovine heart mitochondrial SCR was prepared and assayed according to the published method developed by Yu and Yu (26). The purified SCR contained ϳ4 -4.2 nmol of heme b per mg of protein and exhibited an activity of ϳ8.5 mol of cytochrome c reduced/min/mg of protein. Purified SCR was stored in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA.
SQR and QCR were isolated from SCR by calcium phosphate-cellulose chromatography under non-reducing conditions according to the published method developed by Yu et al. (26,27). SQR-containing fractions obtained from the second calcium phosphate-cellulose column were concentrated by 43% ammonium sulfate saturation and centrifuged at 48,000 ϫ g for 20 min (27). The precipitate obtained was dissolved in 50 mM sodium/potassium phosphate, pH 7.8, containing 0.2% sodium cholate and 10% glycerol. The specific activity of the purified SQR was ϳ15.2 mol of succinate oxidized (or dichlorophenol indophenol (DCPIP) reduced/min/mg of protein).
Analytical Methods
Optical spectra were measured on a Shimadzu 2401 UV-visible recording spectrophotometer. The protein concentration of rat heart tissue homogenates was determined by the Lowry method using bovine serum albumin as a standard. The heme b concentration of SQR was calculated from the differential spectrum between dithionite reduction and ferricyanide oxidation using an extinction coefficient of 28.5 mM Ϫ1 cm Ϫ1 for the absorbance difference of A 560 nm -A 576 nm . The concentration of Q 2 was determined by absorbance spectra from NaBH 4 reduction using a millimolar extinction coefficient ⑀ (275 nm-290 nm) of 12.25 mM Ϫ1 cm Ϫ1 (28). The enzyme activity of SQR was assayed by measuring Q 2 -stimulated DCPIP reduction by succinate as described in the literature (27).
To measure the electron transfer activity of SQR, an appropriate amount of SQR was added to an assay mixture (1 ml) containing 50 mM phosphate buffer, pH 7.4, 0.1 mM EDTA, 75 M DCPIP, 50 M Q 2 , and 20 mM succinate as developed by Hatefi (29). The SQR activity was determined by measuring the decrease in absorbance at 600 nm. The specific activity of SQR (nmol of DCPIP reduced (or succinate oxidized)/min/mg of SQR) was calculated using a molar extinction coefficient ⑀ 600 nm of 21 mM Ϫ1 cm Ϫ1 . SQR-mediated O 2 . generation was assayed by an acetylated cytochrome c reduction. The reaction mixture contained acetylated cytochrome c (50 M), succinate (2 mM), and diethylenetriaminepentaacetic acid (1 mM) in 50 mM sodium/potassium phosphate buffer, pH 7.4. The kinetics of acetylated cytochrome c reduction was initiated by SQR (5 pmol, based on heme b), and the absorption increase at 550 nm was monitored at room temperature. O 2 . -mediated acetylated cytochrome c reduction was measured by pre-addition of Cu,Zn-SOD (0.67 unit/l) in the same assay mixture.
Immunoblotting Analysis
The reaction mixture was mixed with the Laemmli sample buffer at a ratio of 4:1 (v/v), incubated at 70°C for 10 min, and then immediately loaded onto a 4 -20% Tris-glycine polyacrylamide gradient gel. Samples were run at room temperature for 2 h at 100 V. Protein bands were electrophoretically transferred to nitrocellulose membranes in 25 mM Tris, 192 mM glycine, and 10% methanol. Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TTBS) and 5% dry milk (Bio-Rad). The blots were then incubated overnight with anti-3-nitrotyrosine polyclonal antibody (1:2000, Upstate) or AbGSC90 (1:50,000, 0.22 g/ml) at 4°C. Blots were then washed 3 times in TTBS and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG in TTBS at room temperature The blots were again washed twice in TTBS and twice in TBS and then visualized using ECL Western blotting detection reagents (Amersham Biosciences).
Electron Paramagnetic Resonance Measurements
EPR measurements were performed on a Bruker EMX spectrometer operating at 9.86 GHz with 100 kHz modulation frequency at room temperature. The reaction mixture was transferred to a 50-l capillary, which was then positioned in the HS cavity (Bruker Instrument, Billerica, MA). The sample was scanned using the following parameters: center field, 3510 G; sweep width, 140 G; power, 20 milliwatts; receiver gain, 2 ϫ 10 5 ; modulation amplitude, 1 G; time of conversion, 163.84 ms; time constant, 163.84 ms. The spectral simulations were performed using the WinSim program developed at NIEHS by Duling (30). The hyperfine coupling constants used to simulate the spin adduct of DEPMPO/ ⅐ OOH were isomer 1: a N ϭ 13.14 G, a H  ϭ 11.04 gauss, a H ␥ ϭ 0.96 G, a p ϭ 49.96 G; isomer 2: a N ϭ 13.18
Mass Spectrometry
The sample of nitrated SQR was prepared by incubating isolated SQR (1 M based on heme b) with OONO Ϫ (100 M) at room temperature for 1 h. The obtained nitrated SQR (50 pmol) was subjected to SDS-PAGE in the presence of -mercaptoethanol using 4 -12% gradient Bis-tris polyacrylamide gel. Protein bands on the gel were stained with Coomassie Blue for 1 h, and the staining background was rapidly removed by methanol/acetic acid/water (40:10:50). The gel was then equilibrated with distilled water at 4°C overnight before in-gel digestion and MS measurement.
In-gel Digestion-Gels were digested with sequencing grade trypsin (Promega, Madison WI) and chymotrypsin (Roche Diagnostics) using the Montage In-Gel Digestion kit from Millipore following the manufacturer's recommended protocols with minor changes for optimization of peptide extraction. Briefly, the bands of interest were trimmed as closely as possible to minimize background polyacrylamide material. After being washed twice in 50% methanol, 5% acetic acid for several hours, the gel bands were dehydrated with acetonitrile and washed again with cycles of acetonitrile and 100 mM ammonium bicarbonate buffer. The gels were then dried using a speed vacuum. A 50-l aliquot of trypsin (20 ng/l) or chymotrypsin (25 ng/l) in 50 mM ammonium bicarbonate buffer was added to the dehydrated gel. The gel was set on ice for 10 min for rehydration before the addition of another 20 l of 50 mM ammonium bicarbonate buffer. The mixture was then incubated at room temperature overnight. The peptides were extracted from the gel using 50% acetonitrile with 5% formic acid several times and pooled together. The extracted pools were concentrated in a speed vacuum to ϳ25 l.
Nano-LC MS/MS (LC/MS/MS)-Capillary-liquid
chromatography-nanospray tandem mass spectrometry (nano-LC/ MS/MS) was performed on a Thermo Finnigan LTQ mass spectrometer equipped with a nanospray source operated in positive ion mode. The LC system was an UltiMate TM Plus system from LC-Packings (Sunnyvale, CA) with a Famos autosampler and Switchos column switcher. Five microliters of each sample were first injected into the trapping column (LC-Packings) and washed with 50 mM acetic acid. The injector port was switched to inject, and the peptides were eluted off the trap onto a 5-cm, 75-m (inner diameter) ProteoPep II C18 reversephase column (New Objective, Inc., Woburn, MA) packed directly in the nanospray tip. Solvent A was 50 mM acetic acid in water, and solvent B was acetonitrile. Peptides were eluted directly off the column into the LTQ system with a gradient of 2-80% solvent B at a flow rate of 300 nl/min. The total run time was 65 min. The scan sequence of the mass spectrometer was programmed for MS/MS scans of the 10 most abundant peaks in the spectrum. Dynamic exclusion was used to exclude multiple MS/MS of the same peptide after detecting it three times. Sequence information from the MS/MS data were processed using the Mascot 2.0 active Perl script with standard data processing parameters. Data base searching was performed against the NCBInr data base using MASCOT 2.0 (Matrix Science, Boston, MA). The mass accuracy of the precursor ions was set to 1.5 Da to accommodate accidental selection of the 13 C ion, and the fragment mass accuracy was set to 0.5 Da. The number of missed cleavages permitted in the search was 2 for both tryptic and chymotryptic digestions. The considered modifications (variable) were cysteine oxidation and nitration on tyrosine and tryptophan.
RESULTS AND DISCUSSION
Generation of Antibody against the Epitope of Glutathione Binding Domain of the SQR-In the previous study we identified a specific tryptic peptide, 77 AAFGLSEAGFNTACVTK 93 , that is involved in the GS binding domain of the flavoprotein of SQR (7). To gain insight into the functional role of the identified GS binding domain, we generated a polyclonal antibody against this domain.
Based on the published three-dimensional structure of SQR (32), a 17-residue peptide sequence (aa 77-93) exhibited an ␣-helix--turn-sheet conformation (aa 72-93) as indicated in supplemental Fig. 1. To stabilize the ␣-helical conformation, the peptide p-GSC90 sequence 72 GAGLRAAFGLSEAGFN-TACVTK 93 was designed with additional residues at the N terminus. This B cell epitope 22-residue sequence contains an ␣-helix--turn-sheet structure as indicated in supplemental Fig. 1. We have previously demonstrated that the design, synthesis, and immunological and structural characterization of such motifs can be achieved successfully (33). The peptide of p-MVFGSC90 was synthesized as chimeric constructs incorporating an 18-residue promiscuous T-helper measles virus (MVF sequence, 288 KLLSLIKGVIVHRLEGVE 305 ) T cell epitope linked via a 4-residue linker (GPSL) and p-GSC90 B cell epitope on a Milligen/Biosearch 9600 solid-phase peptide synthesizer as described under "Experimental Procedures." The crude peptide was purified to homogeneity by reverse phase HPLC and fully characterized by matrix-assisted laser desorption ionization mass spectroscopy, which gave the exact mass unit ((MϩH) ϩ 4478.96] as calculated [(MϩH) ϩ 4478.48).
Two New Zealand White rabbits (6 -8 weeks old) were immunized with the immunogen p-MVFGSC90. Sera were collected, and antibodies were purified as described under "Experimental Procedures." The generated antibody is termed AbGSC90.
Immunological Specificity of Antibodies-The immunological cross-reactivity of purified antibodies was analyzed by enzyme-linked immunosorbent assay (supplemental Fig. 2) using mitochondrial electron transfer complex including SQR (complex II), SCR (supercomplex containing SQR and QCR), NADH-ubiquinone reductase (or complex I), and ubiquinolcytochrome c reductase (QCR or complex III) as antigens. When each antigen, at a fixed protein concentration (see supplemental Fig. 2), was titrated with various amounts of antibody preparation, the AbGSC90 antibodies reacted at a high titer, with antigens containing a 70-kDa FAD binding subunit in the form of SQR or SCR. There was a very low binding detected between AbGSC90 and NADH-ubiquinone reductase or QCR. The protein concentrations of SCR used for enzyme-linked immunosorbent assay were 3 times higher than those of SQR because the amount of SQR present in SCR is about 33%.
When the immunological specificity of AbGSC90 was characterized by a Western blot (Fig. 1), they were seen to bind specifically to the 70-kDa subunit of SQR and SCR (Fig. 1A). As expected, no binding was observed with NADH-ubiquinone reductase and QCR (data not shown). Antibodies were further used to test the myocardial tissue homogenates obtained from normal and post-ischemic hearts with glyceraldehyde-3-phosphate dehydrogenase as individual protein loading controls. As indicated in Fig. 1B, they bind specifically to the 70-kDa subunit of SQR in the myocardial tissue homogenates. This result also illustrates that there was no significant change in the level of protein expression of the 70-kDa subunit during ischemia/reperfusion injury (7).
The antibodies were further used to probe the 70-kDa subunit of SQR using the rat cardiac myoblast cell line H9c2. Cells were subjected to hypoxia (1 h)/reoxygenation (1 h) and then lysed by sonication. Cell lysate was subjected to SDS-PAGE and then probed with AbGSC90. -Actin was used as an individual protein loading control. As shown in Fig. 1C, antibodies bind specifically to the SQR 70-kDa polypeptide in the cell lysate of H9c2 cells. There is no significant change in the protein expression of the 70-kDa subunit during hypoxia/reoxygenation. These results together demonstrate that the rationale-based designed antibodies against B cell epitope, p-GSC90, are highly specific with high sensitivity and are, thus, suitable for in vivo or in vitro studies.
Immunoinhibition of Succinate-ubiquinone Reductase by AbGSC90-The effect of AbGSC90 binding on the electron transfer and O 2 . generation activities of SQR was measured (supplemental Fig. 3). When the isolated SQR was incubated with various amounts of antibodies, the electron transfer activity (ETA) of SQR decreased as the amount of antibodies increased. A maximum inhibition of 25% was observed with 400 g of antibody per nmol (based on heme b) of SQR, indicating that the binding of AbGSC90 with the epitope that is involved in GS binding (aa 77-93) moderately decreased the electron transfer efficiency catalyzed by SQR.
In the previous studies (7) a modest increase of ETA resulted from in vitro S-glutathionylation of SQR. However, the binding of antibodies against p-GSC90 led to a moderate reduction of ETA in the SQR. These results support that the peptide of the GS binding domain may play a regulatory role in the redox function of SQR. This result also indicates that the peptide of the GS binding domain is surface-exposed, which is confirmed Oxidative Post-translational Modification of SQR OCTOBER 10, 2008 • VOLUME 283 • NUMBER 41 by the x-ray structure of mammalian SQR (32). The property of surface exposure renders it accessible to AbGSC90.
The binding of antibodies also moderately decreased the O 2 . generation mediated by SQR by 26% as measured by the acetylated cytochrome c reduction assay (supplemental Fig. 3A) or EPR spin trapping with DEPMPO (supplemental Fig. 3B). The decreased O 2 . generation can be explained by the ETA decrease resulting from antibody binding since the electron transfer activity of SQR is required for the electron leakage for O 2 . production (34).
Protein Tyrosine Nitration of SQR 70-kDa FAD Binding Subunit in the Post-ischemic Myocardium-A previous study established that deglutathionylation of the SQR 70-kDa flavin protein is an early event during myocardial ischemia/reperfusion injury (7). Protein tyrosine nitration in mitochondria has been detected using the myocyte model subjected to hypoxia/ reoxygenation. In a separate study Han et al. (20) reported a similar observation in the mitochondria of endothelial cells under the conditions of ischemia/reperfusion. Therefore, oxidative modification with protein nitration at the SQR 70-kDa subunit is expected to follow after the highly reductive conditions of myocardial ischemia. To test this hypothesis, the AbGSC90 was immobilized onto the commonly used Affi-Gel 10 (Bio-Rad) on the basis of the protein's isoelectric point as outlined in the product literature (bulletin no. 1085). The immobilized AbGSC90 was subsequently used to immunoprecipitate the 70-kDa FAD binding subunit from the tissue homogenates of post-ischemic myocardium followed by immunoblotting with a polyclonal antibody against 3-nitrotyrosine (3-NT, Upstate). As indicated in Fig. 2, we have detected a very weak signal of nitrotyrosine on the 70-kDa subunit of SQR from tissue homogenates of the normal heart (panel A, lane 2). The signal intensity of protein nitration on the 70-kDa subunit of SQR was greatly enhanced in tissue homogenates of the post-ischemic myocardium (panel A, lane 1). The detected Western blot signal was abolished by pretreatment of sample with dithionite due to reduction of 3-nitrotyrosine to 3-aminoty- rosine (panel A, lane 3). The tissue homogenates were further immunoblotted with AbGSC90 to measure the protein loading of SDS-PAGE (Fig. 2, panel B) and confirm the protein nitration occurring at the 70-kDa. The ratio of signal intensity obtained from anti-3nitrotyrosine antibody that of AbGSC90 is ϳ6 for the post-ischemic myocardium and ϳ2 for normal myocardium.
To confirm that deglutathionylation of 70-kDa subunit occurred during myocardial ischemia-reperfusion, the same sample was subjected to SDS-PAGE under the non-reducing conditions and immunoblotted with anti-GSH monoclonal antibody (Viro-Gen, Watertown, MA). As indicated by Western blots (supplemental Fig. 4A), the intrinsic protein S-glutathionylation on the 70-kDa subunit of SQR of non-ischemic tissue was subsequently decreased (by 57.8 Ϯ 11.9%) in the post-ischemic myocardium. This result was consistent with the previous observation (7).
To further demonstrate that protein tyrosine nitration at the 70-kDa subunit was enhanced under the physiological conditions of hypoxia/reoxygenation (H/RO), rat cardiac myoblasts (H9c2) were subjected to the conditions of control or H/RO at 37°C before immunofluorescence staining using AbGSC90. Similar fluorescence intensities of AbGSC90 staining were detected by confocal microscopy under the control and H/RO conditions (Figs. 2, C and D, left pictures). To determine the effects of H/RO-mediated protein tyrosine nitration, 3-NT in myoblasts was probed with monoclonal antibody against 3-NT and then examined by immunofluorescence confocal microscopy. A very low level of 3-NT staining was observed in myoblasts under the control conditions (Fig. 2C, middle). Exposing myoblasts to hypoxia (1 h) followed by reoxygeneration (1 h) resulted in substantially more intense 3-NT staining that showed modest but significant localization of 3-NT in the 70-kDa subunit of SQR (Fig. 2D, middle and right), further demonstrating that reactive nitrogen species-mediated protein nitrotyrosine formation enhanced in the SQR 70-kDa subunit under the physiological conditions of ischemia/reperfusion injury.
Enhancement of protein tyrosine nitration in mitochondrial protein (the SQR 70-kDa subunit in this study) during myocardial ischemia/reperfusion is likely OONO Ϫ -mediated and caused by the alternations of NO metabolism. Production of excess NO is likely mediated by eNOS and NOS- DTN). Membrane was probed with anti-3-nitrotyrosine antibody. B, same as A except that membrane was probed with AbGSC90. C, H9c2 cardiac myoblasts were fixed and stained for 3-nitrotyrosine and the SQR 70-kDa subunit. Fluorescence images were acquired with confocal microscopy and merged to determine whether the increase in 3-nitrotyrosine signal was located in the SQR 70-kDa subunit. D, same as C except that cells were subjected to hypoxia (1 h) and reoxygenation (1 h). independent nitrite disproportionation (reduction of nitrite to NO) in the early phases of reperfusion after ischemia. This mechanism has been demonstrated in the mouse model of eNOS Ϫ/Ϫ , in which immunostaining of 3-NT is significantly decreased in the post-ischemic myocardium of eNOS Ϫ/Ϫ (10,11,14). Furthermore, post-ischemic myocardial oxygen consumption mediated by eNOS-derived NO has been linked to oxidative inactivation of the mitochondrial electron transport chain (10).
NO generated from a NOS-independent pathway has been reported in the ischemic heart by direct reduction of nitrite to NO (18,19). Specifically, mitochondria have been suggested to be major players in catalyzing nitrite disproportionation in heart tissue (19,35). Presumably, this pathway mediated by the mitochondria of ischemic tissue can be greatly facilitated under highly acidic and reductive conditions. Stimulation of iNOS up-regulation has been marked in the post-ischemic myocardium (11). NO generated by iNOS overexpression has been suggested to be the major pathway during chronic reperfusion after ischemia (11). We have analyzed the mRNA and protein expression of iNOS from the post-ischemic myocardium by real-time quantitative PCR and immunoblotting with anti-iNOS polyclonal antibody (BD Transduction Laboratories) in this study. Our real-time quantitative PCR analysis indicated that the mRNA expression of iNOS was increased 3.0 -3.7-fold in the 24-h postischemic rat myocardium compared with that of the nonischemic rat myocardium (supplemental Fig. 4B). Significant enhancement of iNOS expression was also detected in the post-ischemic myocardium (supplemental Fig. 4B). Because this result was consistent with previous reports using the mouse model (10,11,14), we suggest that OONO Ϫ formation is indeed because of overproduction of NO (34)). Therefore, overproduction of NO would be expected to block mitochondrial respiration, increase electron leakage, and subsequently stimulate OONO Ϫ formation in the post-ischemic myocardium. Presumably, hypoxia-induced deglutathionylation of SQR increases the electron leakage for O 2 . generation from the FAD binding subunit (7), thus facilitating local OONO Ϫ formation and subsequently enhancing protein tyrosine nitration at the SQR 70-kDa subunit. Furthermore, our recent research progress of in vitro EPR studies shows that excess NO stimulates SOD-dependent oxygen-free radical generation through the formation of a dinitroso-iron intermediate at the S1 center (2Fe-2S) of SQR. 3 Therefore, it is likely that binding of NO to the S1 center blocks electron flow and stimulates O 2 . production on the flavin protein subunit, consequently leading to nitration of the 70-kDaderived subunit protein tyrosine.
In Vitro Protein Tyrosine Nitration of SQR-To gain a deeper insight into the molecular mechanism of SQR-de-rived tyrosine nitration, the isolated SQR was reduced with dithiothreitol (1 mM) and then passed through a Sephadex G-25 column. SQR (1 M) was subjected to in vitro protein nitration with OONO Ϫ (100 M) treatment. The resulting OONO Ϫ -treated SQR was subjected to SDS-PAGE in the presence of -mercaptoethanol followed by immunoblotting with anti-3-nitrotyrosine antibody. It was observed that the 70-kDa subunit of SQR was involved in site-specific protein nitration as indicated in Fig. 3. The Western blot signal increased in proportion to the dosage of OONO Ϫ used (data not shown). Both native and nitrated SQRs were further blotted with AbGSC90. There was no significant difference (less than 5%) observed in the Western signal of 70-kDa, indicating that protein nitration of 70-kDa did not affect AbGSC90 binding at a significant level (Fig. 3).
The protein band of the 70-kDa subunit of OONO Ϫtreated SQR (lane 2 in Fig. 3 Single nitration of native protein will increase the molecular weight by 45 Da. Therefore, the mass spectra from the proteolytic digest of nitrated SQR 70-kDa polypeptides were examined for the addition of 45 ϫ n Da (n is the number of tyrosine residues in the peptide). In the tryptic digest MS/MS results, a mass difference of 45 Da was observed in two peptide fragments, 130 -163 ( 130 GSDWLGDQDAIHYMTEQAPASVVEL-ENYGMPFSR 163 , NOY142) and 48 -76 ( 48 VSDAISAQYPVV-DHEFDAVVVGAGGAGLR 76 , NOY56), indicating that these two peptides were nitrated. Further analysis suggested that nitration occurred on the specific tyrosine residues, Tyr-142 and Tyr-56 . As shown in Fig. 4 and Table 1, in the MS/MS spectrum of (NOY142) 3ϩ at m/z 1282.76 3ϩ , some of the structurally informative fragment ions including y23-y27, y29-y30, b20, b22, b24-b30, and b32-b33 were observed with a mass shift of 45 Da compared with native fragment ions, thus allowing unequivocal assignment of the nitrated adduct to the tyrosine-142 residue of the tryptic peptide 130 GSDWLGDQDAIHYMT-EQAPASVVELENYGMPFSR 163 . Other sequence informative ions, y4-y17, y19, y21, b5, and b7-b10, provided the evidence to ensure that the sequence of (NOY142) 3ϩ was matched to the sequence of aa residues 130 -163 of the 70-kDa subunit of the SQR. These data allow unequivocal assignment of tyrosine-142 as the site of nitration.
Likewise, in the MS/MS spectrum of (NOY56) 3ϩ at m/z 982.90 3ϩ (supplemental Fig. 6), some of the structurally informative ions including y21, y24, b23-b24, and b27-b28 were detected with a mass shift of 45 Da. Another series of detected sequence informative ions, y3 and y5-y17, match the sequence of aa residues 48 -76. Together these data show that tyrosine-56 is involved in the nitrated adduct of the tryptic peptide NOY56.
Substantial evidence from our previous investigation using mouse/rat models strongly support that increasing protein nitration marked in the post-ischemic myocardium is caused by OONO Ϫ formation in vivo (10,11,14,15). Therefore, in vitro OONO Ϫ -mediated protein tyrosine nitration provided an appropriate way to enrich protein 3-NT in the SQR 70-kDa, thus facilitating the detection of MS/MS. The value of in vitro results should be recognized as a model since this system yielded a protein tyrosine nitration in a highly site-specific manner and resulted in impairing enzymatic function, which meets the criteria of in vivo situ- ation and supports OONO Ϫ to be the source of nitrating agent in vivo.
S-Glutathionylation of SQR 70-kDa Subunit Preserves Enzyme Inactivation by Peroxynitrite in Vitro-In a previous study we demonstrated that S-glutathionylation of SQR dimin-ishes the SQR-derived protein radical formation induced by oxygen free radical(s), which supports the protective role of protein S-glutathionylation (7). To learn whether protein S-glutathionylation exerts a similar protective effect on SQR function related to OONO Ϫ -mediated injury, we S-glutathionylated isolated SQR in vitro with GSSG according to a published procedure (7). The resulting GS-SQR was incubated with OONO Ϫ (100 M) for 1 h at room temperature. Excess degraded product NO 3 Ϫ was removed by passing through a Sephadex G-25 column. The sample was subjected to probing with a monoclonal antibody against 3-NT. As indicated in Fig. 5A, the Western signal of SQR-derived protein nitration was significantly diminished (by 77.2%, n ϭ 3) in the S-glutathionylated SQR, thus supporting the protective role of S-glutathionylation. In the control experiment, the same membrane was probed with AbGSC90. As indicated in the Fig. 5A (lower panels), the signal intensity obtained was slightly decreased by 13% of control (n ϭ 3) for the GS-SQR, confirming roughly equal amounts of protein loading. Furthermore, the signal intensity probed with anti-GSH monoclonal antibody was shown to be significantly higher for GS-SQR (Fig. 5A), indicating satisfactory efficiency of protein S-glutathionylation induced by GSSG.
OONO Ϫ -treated SQR and GS-SQR were further subjected to analysis of electron transfer activity of Q 2 -mediated DCPIP reduction. Both SQR and GS-SQR were incubated with different concentrations of OONO Ϫ (from 0 -400 M) at room temperature for 20 min before the activity assay. As indicated in Fig. 5B, significant protection of the electron transfer activity from OONO Ϫ inactivation was observed in GS-SQR. Specifically, we have observed that the protective effect of S-glutathionylation is observed at a lower concentration of OONO Ϫ (e.g. 96 versus 67% ETA remaining at 100 M OONO Ϫ in Fig. 5B).
The identified nitrated peptide ( 130 GSDWLGDQDAIHYMTE-QAPASVVELENYGMPFSR 163 ) containing Tyr-142 (Tyr-99 in the mature protein) of bovine SQR is highly conserved in proteins from humans ( 129 GSDWLGDQDAIHYMTE-QAPAAVVEENYGMPFSR 162 ), mice, rats, yeast ( 120 GSDWLG-DQDSIHYMTREAPKSIIELEHYGVPFSR 153 ), and Escherichia coli ( 75 GSDYIGDQDAIEYMCKTGPEAILELEHMGLPFSR 108 ). From this information together with the results of the current in vitro study, we suggest that the Tyr-142 of the flavoprotein subunit of SQR is the critical tyrosine susceptible to oxidative damage induced by OONO Ϫ , and S-glutathionylation of Cys-90 should play a role in antioxidant defense to combat oxidative attack from OONO Ϫ .
Based on the x-ray crystal structure of mammalian SQR (PDB ID 1ZOY), the flavin subunit has a Rossman-type fold with four major domains (32) (Fig. 6A). Tyr-142 is located in the major helix (residues 136 -158 in the precursor; see the cyan helix in Figs. 6, A and B) of a floating subdomain (residues 105-196 in the precursor) which is a part of the large FAD binding domain (residues 53-316 and residues 404 -488 in the precursor). Specifically, Tyr-142 is highly surface-exposed (Fig. 6A) and situated in the hydrophilic environment, suggesting that this specific tyrosine is susceptible to nitration by OONO Ϫ . The x-ray structure reveals that Tyr-142 is ϳ20 Å away from the isoalloxazine ring of FAD. Cys-90 (Cys-47 in the mature protein) is located within the part of the N-terminal  barrel subdomain (residues 53-104 in the precursor, yellow ribbon of Mitochondrial Functions in the Post-ischemic Myocardium-To verify the effect of protein nitration occurring in the post-ischemic myocardium on the mitochondrial function, mitochondria were isolated from the tissue of risk region and subjected to measurement of respiratory control ratio by the polarographic method using an oxygen electrode. As indicated in Figs. 7, A and B, ADP-stimulated respiration (state 3) was decreased from 370 Ϯ 5.1 (Sham control) to 130.6 Ϯ 3.4 nmol of O 2 /min/mg of protein after myocardial infarction. The respiratory control ratio was decreased from 3.3 Ϯ 0.1 (sham control) to 1.3 Ϯ 0.1, suggesting protein tyrosine nitration caused by ischemia/reperfusion injury may contribute to defects in the mitochondrial integrity due to marked reduction of ADP-stimulated respiration.
Impairment of Protein-Protein Interaction between Complex II and Complex III by Protein Nitration of SQR-Direct exposure of intact mitochondria (0.5 mg/ml) to OONO Ϫ (100 M) impaired ADP-stimulated respiration by 45% and decreased respiratory control ratio by 42%. However, the treatment failed to induce protein nitration of SQR as OONO Ϫ is nearly membrane-impermeable. To better define the role of SQR-derived tyrosine nitration in the impairment of electron transport chain, the systems of SCR and reconstituted SCR were employed. SCR is the supercomplex hosting SQR and QCR (or complex III). The enzymatic activity of SCR in catalyzing electron transfer from succinate to cytochrome c is derived from protein-protein interaction between SQR and QCR (26,37). Pretreatment of intact SCR (3 M, based on heme b) with OONO Ϫ (100 M) resulted in the protein tyrosine nitration at the multiple polypeptides including the 70-kDa subunit of SQR. As indicated in Fig. 7C, the ETA of SQR in the SCR was decreased by 34%, and the ETA of SCR was decreased by 67%. However, the ETA of QCR did not suffer significant loss by OONO Ϫ , indicating the impairment of protein-protein interaction between SQR and QCR. It should be noted that similar inhibitory profile was also observed when intact SCR was pretreated with a different dosage of OONO Ϫ (supplemental Fig. 7).
To further demonstrate the ability of SQR-derived tyrosine nitration to impair protein-protein interaction, isolated SQR was subjected to in vitro protein nitration with OONO Ϫ (Fig. 3). Nitrated SQR was reconstituted with native QCR in vitro before measuring the ETA of SCR. The reconstituted SCR suffered a dramatic loss of ETA by 63% compared with that of the control (in vitro reconstitution of native SQR and native QCR) as indicated in Fig. 7C. Replacement of nitrated SQR with GS-SQR pretreated with OONO Ϫ in the system restored the reconstituted SCR activity (Fig. 7C), thus further supporting the protective role of S-glutathionylation. It is worth noting that the ETA of native QCR was not significantly affected by OONO Ϫ treatment. O 2 . generation by reconstituted SCR was induced by succinate and measured with EPR spin-trapping with DEPMPO. As shown in Fig. 7D (EPR spectra provided in supplemental Fig. 8), O 2 . production (based on spin quantitation of DEPMPO/ ⅐ OOH) by the SCR reconstituted from nitrated SQR with QCR was enhanced by 33% compared with that of control, presumably due to the impairment of protein-protein interaction by protein nitration of SQR.
The evidence of protein-protein interaction between SQR and QCR has been demonstrated with EPR spin labeling and differential scanning calorimetry (37). It is likely that SQR impairment detected in the post-ischemic myocardium can decrease its interaction with QCR and subsequently diminish the electron transfer activity from succinate to cytochrome c in vivo, leading to weakening mitochondrial function. FIGURE 7. A and B, State 3 and state 4 respiratory rates of mitochondrial (Mito.) preparations from the risk region of post-ischemic myocardium. Post-ischemic myocardium was obtained as described under "Experimental Procedures." The tissue of risk region was excised and subjected to mitochondrial preparations according to published method (48). The oxygen consumption by mitochondria (0.5 mg/ml) was induced by succinate (Suc., 5 mM) and measured by oxygen polarographic method at 30°C. State 3 oxygen consumption was stimulated by the addition of ADP (200 M), and state 4 oxygen consumption was determined after addition of oligomycin (2 M) followed by ADP addition. C, effect of OONO Ϫ -mediated protein nitration on the ETA of intact SCR and reconstituted SCR. Intact SCR (3 M, based on heme b) and SQR (1 M) were incubated with OONO Ϫ (100 M) at room temperature for 30 min. Nitrated SQR was then reconstituted with the native QCR (2 M) at 0°C for 1 h. Reconstituted SCR and intact SCR were subjected to ETA measurement. SCR and QCR activities were assayed as reported previously (7). D, effect of OONO Ϫ -mediated protein nitration of SQR on the O 2 . generation catalyzed by reconstituted SCR. An aliquot of enzyme solution (1 M of heme b) was withdrawn and added to a mixture containing succinate (180 M), DEPMPO (20 mM), and diethylenetriaminepentaacetic acid (1 mM) before EPR measurement. The DEPMPO/ ⅐ OOH in each spectrum was quantitated by double integration of simulated spectrum (supplemental Fig. 8).
Oxidative Post-translational Modification of SQR
It is known succinate-induced O 2 . generation by SCR is mainly controlled by QCR and operated through Q-cycle mechanism. Impairment of protein-protein interaction between SQR and QCR presumably increased unstable semiquinone radical formation and subsequently enhanced O 2 . production. Therefore, results of current studies implicate that protein nitration of SQR plays a significant role in triggering mitochondrial dysfunction during myocardial ischemia/reperfusion, perhaps through diminishing protein-protein interaction and augmenting oxidative stress. Peroxynitrite-mediated Cysteine S-Sulfonation of SQR 70-kDa Subunit-The MS/MS spectra obtained from nitrated SQR were further examined for cysteine oxidation involved in S-sulfonation (conversion of -SH to -SO 3 H), identified by the addition of 48 Da. It was observed that Cys-267, Cys-476, and Cys-537 were S-sulfonated. In the MS/MS spectrum of doubly protonated ion (m/z 813.35 2ϩ and supplemental Fig. 9) of the tryptic peptide hosting Cys-476, a mass shift of 48 Da was observed in the fragment ions of y6, y8-y11, and b11-b14. The fragment ions of y4-y5, b3-b6, and b9 provided additional evidence of sequence information matched to amino acid residues 461-481. Likewise, a mass shift of 48 Da was detected in the MS/MS spectra of doubly protonated ions of tryptic peptides containing Cys-267 (aa residues 263-283 m/z 1121.56 2ϩ ) and Cys-537 (aa residues 529 -548 m/z 1101.67 2ϩ ), (data not shown).
Cys-267, Cys-476, and Cys-537 are involved in OONO Ϫ -mediated S-sulfonation. Cys-267 (Cys-224 in the mature protein) is located in the hydrophilic pocket of the -barrel subdomain of the large FAD binding domain. Cys-476 (Cys-433 in the mature protein) is situated at the terminus of an ␣-helix in the FAD binding domain. Cys-537 (Cys-494 in the mature protein) is located on the surface of a three-helix bundle from the helical domain. All of the cysteines are surface-exposed, thus logically susceptible to OONO Ϫmediated oxidation in vitro.
S-Sulfonation of Cys-537 was verified in this study. Cys-537 has been reported to be a secondary site (Cys-90 is the primary site) involved in S-glutathionylation induced by GSSG in vitro (7), implicating the possibility of S-sulfonation after deglutathionylation at the same cysteine residue of Cys-537 in the SQR 70-kDa subunit.
The milieu of the mitochondrial matrix is almost anoxic in the presence of the GSH/GSSG pool under normal physiological conditions (38). Analysis of redox compartmentation indicates that the relative redox states from most reductive to most oxidative are as follows: mitochondria Ͼ nuclei Ͼ endoplasmic reticulum Ͼ extracellular space (38). Thus, it is expected that very low oxygen tension in the mitochondrial environment of ischemic tissue should facilitate the free thiol state for most cysteines and that mitochondrial thiols are the targets of oxidants such as OONO Ϫ . They are vulnerable to oxidation such as S-sulfonation. Presumably, cysteine S-sulfonation is a mechanism of oxidative modification by mitochondrial proteins in response to oxidative stress.
We have attempted to map the specific sites of protein sulfonation/nitration by immunoprecipitating the 70-kDa polypeptide from tissue homogenates of the infarct area using AbGSC90. However, MS/MS analysis of tryptic peptides revealed ambiguous results with low sequence coverage, and we were not able to identify 3-NT/cysteine sulfonic acid peptides. Several possible reasons may contribute to our failure in obtaining satisfactory MS/MS data for 3-NT/ cysteine sulfonic acid-containing peptides from tissue. They are as follows. (i) Low abundance of 3-NT in 70-kDa was caused by generally low levels of protein nitration in vivo. Specifically, accumulation of 3-NT in protein may undergo dynamic progress involved in protein tyrosine denitration (39 -41) or increased turnover of nitrated proteins (42) in which even we have employed the pathological tissue. (ii) Other factors are low expression of SQR in tissue, poor recovery of 3-NT/cysteine sulfonic acid-containing peptide from gel, and insufficient amounts of sample for MS/MS sequencing. These limitations may be overcome by a more direct approach, but it is not likely to isolate large amounts of the target protein from very limited amounts of infarct tissue. The physiological conditions of hypoxia and reoxygenation during ischemia and reperfusion trigger overproduction of ROS from the mitochondrial electron transport chain, including complex I and complex III. Oxidative stress induces alterations in the redox state including deglutathionylation of SQR, which moderately increases SQR-derived superoxide generation activity (7). Up-regulation of the iNOS expression and nitrite disproportionation during ischemia/ reperfusion trigger NO overproduction (10,11,18,19), which reacts with superoxide produced by the electron transport chain to form peroxynitrite. Peroxynitrite causes oxidative modifications of the SQR 70-kDa subunit with protein tyrosine nitration (Y-NO 2 ). Fum, fumarate.
Conclusion-The diagram of Fig. 8 illustrates the relationship between deglutathionylation, overproduction of ROS and NO, and protein tyrosine nitration of SQR in mitochondria during myocardial ischemia/reperfusion. Physiological conditions of hypoxia and reoxygenation during ischemia and reperfusion trigger overproduction of NO through up-regulation of three major pathways, including eNOS, iNOS, and nitrite reduction (10,11,18,19). These conditions also stimulate O 2 .
generation from the mitochondrial electron transport chain, including Complex I and Complex III (8,34,(43)(44)(45)(46)(47). Based on the results of in vitro studies, deglutathionylation of SQR increases SQR-mediated O 2 . production (7). The enhanced ability of SQR to produce O 2 . should augment the overall magnitude of OONO Ϫ formation and consequent protein nitration in the post-ischemic heart. Specifically, we have detected the enhancement of SQR-derived protein tyrosine nitration in the post-ischemic myocardium. It is important to recognize the physiological significance of this study was to elucidate the redox pathway in which protein nitration occurs after deglutathiolation of SQR in the post-ischemic myocardium. This redox pathway contributes to marked mitochondrial dysfunction and offers a marker of oxidative stress in mitochondria during ischemia/reperfusion. Although the detected redox modifications seem to have modest effects on the electron transfer activity of SQR, the impact on protein-protein interaction and the consequent mitochondrial respiration is significant. Defining this molecular mechanism is important for understanding how oxidants modulate post-ischemic injury caused by mitochondrial dysfunction. | v3-fos-license |
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} | pes2o/s2orc | Fischer–Tropsch Synthesis: Computational Sensitivity Modeling for Series of Cobalt Catalysts
: Nearly a century ago, Fischer and Tropsch discovered a means of synthesizing organic compounds ranging from C 1 to C 70 by reacting carbon monoxide and hydrogen on a catalyst. Fischer–Tropsch synthesis (FTS) is now known as a pseudo-polymerization process taking a mixture of CO as H 2 (also known as syngas) to produce a vast array of hydrocarbons, along with various small amounts of oxygenated materials. Despite the decades spent studying this process, it is still considered a black-box reaction with a mechanism that is still under debate. This investigation sought to improve our understanding by taking data from a series of experimental Fischer–Tropsch synthesis runs to build a computational model. The experimental runs were completed in an isothermal continuous stirred-tank reactor, allowing for comparison across a series of completed catalyst tests. Similar catalytic recipes were chosen so that conditional comparisons of pressure, temperature, SV, and CO / H 2 could be made. Further, results from the output of the reactor that included the deviations in product selectivity, especially that of methane and CO 2 , were considered. Cobalt was chosen for these exams for its industrial relevance and respectfully clean process as it does not intrinsically undergo the water–gas shift (WGS). The primary focus of this manuscript was to compare runs using cobalt-based catalysts that varied in two oxide catalyst supports. The results were obtained by creating two di ff erential equations, one for H 2 and one for CO, in terms of products or groups of products. These were analyzed using sensitivity analysis (SA) to determine the products or groups that impact the model the most. The results revealed a significant di ff erence in sensitivity between the two catalyst–support combinations. When the model equations for H 2 and CO were split, the results indicated that the CO equation was significantly more sensitive to CO 2 production than the H 2 equation.
Introduction
Fischer-Tropsch synthesis (FTS) is a process used to produce a vast range of organic compounds. Discovered nearly a century ago, FTS reacts H 2 and CO over a metal catalyst to produce C 1 -C 70 -chain compounds, including gasoline and alternate materials [1,2]. Thus, it is often used to generate fuels, monomers for common polymers, rubber, etc. Despite the long history of research and industrial use of FTS, it is still unknown how this process works on a microscopic level [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. There are at least three proposed mechanisms, namely, the carbide originally proposed by Fischer and Tropsch, CO insertion, and formate mechanisms. Yet, none of these can fully describe the entire FTS process, mainly because of the diversity of materials generated by different active metals [1].
To further comprehend FTS, many have attempted to use mathematical modeling as a useful means of describing the process [23][24][25][26][27][28]. Density functional theory (DFT) calculations, paired with experimental procedures, have provided further insight into the FTS mechanism [18][19][20][21]. These models generally fall into one of two categories-probabilistic models and kinetic models. Probabilistic models attempt to describe the propagation of the carbon chain to generate the products observed [23][24][25]. Kinetic models focus more specifically on the rate of the reaction, determining a mechanism to describe the reaction as a whole [26][27][28]. While these models have significant application for understanding FTS, they do not exist without limitation. Kinetic models depend on the mechanism of the reaction, which, as stated earlier, is not fully understood due to the diverse array of products. Furthermore, they become quite mathematically complex. Probabilistic models focus on the likelihood of a specific carbon chain propagating or terminating. This type of modeling is limited by the monomer used for propagation, which is not fully understood.
This research sought to create a model that describes the rate of change of hydrogen and carbon monoxide as the reaction progresses. Using a series of industrially relevant cobalt-catalyst runs with varying supports, a system of differential equations was created to model the unreacted syngas in terms of the products generated. Each chemical product output term (such as moles of methane, water, carbon dioxide, etc.) was scaled by an impact parameter, a value in units of hr-1. The model was then tested using sensitivity analysis (SA) to determine which impact parameter and, consequently, which associated chemical product term altered the solution of the model the most. From the results of this analysis, conclusions were drawn about the chemical implications, which may then be used to further examine catalytic runs.
Computational Modeling
The computational model was established using chemical engineering process principles from the stirred-tank slurry reactor and basic chemical kinetics. The rate of H 2 and CO coming out of the reactor (unreacted syngas) was modeled using ordinary first-order differential equations (ODEs) [29]. More specifically, the rate of change of H 2 in the reaction can be described by the hydrogen flow into the reactor minus everything coming out of the reactor (H 2 , water, methane, hydrocarbon products). All the products (excluding unreacted hydrogen) were scaled by an "impact parameter". This model was applied to CO as well to produce two ODEs as follows: where Q H2 and Q CO are input rates of H 2 and CO in moles per hour, respectively; q H2 and q CO are the output rates for H 2 and CO in units of hr-1, respectively; MH 2 (t), MCO(t), MH 2 O(t), MCO 2 (t), MCH 4 (t), MC 2 C 4 (t), and Mliq(t) are the time-dependent functions for the moles of H 2 , CO, CO 2 , H 2 O, CH 4 , C 2 -C 4 products, and C 5 + (or liquid) products, respectively; and Z1-10 are the impact parameters in units of hr-1. It is important to note that MH 2 (t) and MCO(t) are the unknown functions for this system. All other mole functions were interpolated from the experimental data (see Supplementary Materials for data), but these two are the functions for which the system is solved. The MH 2 (t) and MCO(t) solutions of the ODE system were compared to the experimentally observed values of these functions (see Supplementary Materials) and the resulting sum of squared error was the "response" for the sensitivity analysis. The * symbol at the end of the hydrogen equation is to note that the bracketed term was added later, thus explaining the variance in sequence of impact parameters. Originally, the carbon dioxide term was not included in the hydrogen equation but was later added to explore the possibility of a water-gas shift (WGS). Due to the innovative nature of the differential equations modeling the reactor, the solver used to evaluate the solutions to this model was the Livermore Solver for Ordinary Differential Equations (LSODA). LSODA performs a test for numerical stiffness as the time steps evolve. A stiff differential equation is one which exhibits solutions with different time scales. For example, stiffness may occur in chemical modeling when simulating reactions with very different reaction rate constants. To solve stiff equations numerically, one must use an implicit solver that requires time-consuming nonlinear equation solutions (e.g., Newton's method for nonlinear systems) for each time step. The LSODA solver primarily uses a non-stiff solver for speed but dynamically checks the solution to determine if a stiff method is required and applies an implicit solver for those time steps. Since the values of the impact parameters were unknown, this approach provided the most flexibility in the numerical solutions. Initial testing of the solution showed good qualitative agreement with the data, indicating that the LSODA solver worked properly with this model. Once the solution was established, sensitivity analysis (SA) was performed. SA is a branch of computational methods that provide insight into how much the total change in the output of a model can be divided up and allocated to the "input" variables/parameters of the model [30]. In other words, SA measures the effect on the output of the model that results from changing the input factors. While there are two types of SA, local and global, local was not used in this work since it involves only measures the change in output at a specified point. Global SA measures the output across a specified range for the input parameters, which was more practical in analyzing this model [30].
While there are many methods for global SA, the Sobol method and Morris method [31][32][33] were of primary interest for application to the model. These are commonly used for such systems and they are both well validated in multiple applications [32,34,35]. The Sobol method is a variance-based global SA method that generates sensitivity indices from ratios of variance terms [33]. The limitation of Sobol is that calculating the variances is very complex mathematically, and the method requires a large number of samples (often more than 10,000) to counteract any error from negative indices. Furthermore, the primary function of the Sobol method is to analyze the interactions by looking at the second-and higher-order indices, but since the primary focus of this work was the impact of a factor on the model output, the Morris method was used as the SA tool for this preliminary study.
The Morris method is known as a one-at-a-time (OAT) global SA method [31]. This method measures the impact of each input factor on the model by changing only one input factor at a time and observing the change in the model output. Specifically, Morris generates a set of elementary effects (EEs) for each input factor across the given range [31]. An EE is generated by starting with the model evaluated at a specific set of input factors. Then, the model is evaluated again with only one factor increased or decreased by a specified amount, D. The unchanged model output is subtracted from the altered model output, the absolute value is taken for the difference, and this absolute value is divided by D, as follows: In the equation above, Y represents the model, X 1 . . . i is the set of parameters, and i = {1, 2, . . . , n}, n being the number of parameters in the model. A set of these EEs using multiple values for D is generated for each input parameter across the given range. Since each set, known as the distribution of EEs, can be quite large, they are sampled. The original Morris sampling technique was a type of random sampling that did not use a high number of samples (10-50), but Campolongo et al. discovered a better sampling technique [31]. In this new technique, 500-1000 original samples are taken from the distribution of EEs. These are then analyzed to determine the optimal 10-50 samples (those that most closely describe the set as a whole). The optimal samples, or optimal trajectories, create a new set that is much smaller and easier to analyze statistically. The average, µ*, and the standard deviation, σ, are taken for each optimal sample set. µ* represents the impact of the specific parameter on the model, and σ represents the interaction between a parameter and the other input factors [31]. The greater the magnitude for these values, the greater the impact (µ*) and interaction (σ) for the corresponding input parameter. Since Morris is a relatively simple method that focuses more on the impact and less on the interactions between parameters, it was chosen as the global SA method for this work. It is important to note that, when performing SA on this model, the impact parameters were the input parameters. While each impact parameter has a corresponding output product (e.g., k 1 corresponds to the hydrogen term for H 2 O output), there is not a necessary relationship between the degree of impact and the product itself. However, this work hypothesizes that there is at least a small direct correlation between the magnitude of impact for an impact parameter and the amount of corresponding product produced.
Results and Discussion
To ensure the effectiveness of this modeling, the mass closure for these runs had to be accounted for. Since this work was to understand, specifically, how the reactants affected each product through the sensitivity analysis, our entire process would collapse and be non-functional if mass closure was not accounted for. To do this, the mass flow controllers were calibrated beforehand, the flow in and out was measured regularly, and all liquids pulled from the system were weighed and accounted for. The difference between output and input displayed almost 96% closure for mass balance for all our work.
Using the data collected from the experimental runs, the mole functions were interpolated, the differential equations were solved, and the solution was then tested using Morris method SA. Initially, the DEs were loosely coupled, meaning that the solutions for MH 2 (t) and MCO(t) could be somewhat dependent on one another and the input parameters (Z1-Z9) could potentially interact. Each set of experimental data was analyzed nine times with Morris, on varying ranges from 0.1-50 to 0.1-500. The number of samples generated varied between 500 and 1000, and the optimal trajectories varied between 0 and 50, as described by Campolongo et al. [31]. The µ* and σ values were collected from the Morris output and the average for all nine analyses was taken. The µ* and σ averages for each data set were compared to the other data sets with the same catalyst-support combination and then compared to the values for the other category of data to discern any variance between the two types of supports.
Next, the two DEs were uncoupled. The CO 2 term for the hydrogen equation, along with k 10 , was added to explore the possibility of WGS occurring in the 0.5 wt% Pt, 25 wt% Co/Al 2 O 3 catalyst. Both equations were analyzed using Morris, following the same procedure as described above. The µ* and σ values were recorded for both the H 2 and the CO equations. These were then compared, primarily the µ* values for k 5 and k 10 , the CO 2 impact parameters for the CO and H 2 , respectively. In theory, should µ* for k 10 consistently be greater than that of k5, it could be indicative of hydrogen having a more substantial role in producing CO 2 . This could only happen through WGS, and thus if Z10 > Z5, it could indicate the possibility of WGS. Otherwise, if Z5 > Z10, this would most likely perpetuate the prevailing theory, primarily for cobalt catalyst FTS, that the Boudouard reaction is producing the CO 2 [36,37].
Listed below are the data collected from the outlined modeling and sensitivity analysis procedure. As a key, Table 1 is the list of the five product categories and the corresponding impact parameters for the two ODEs in the model. For example, Z 4 is the impact parameter for water in the carbon monoxide equation.
Recall that the µ* value for each parameter indicates how much that parameter influences the model. In other words, changing the impact parameter with the highest µ* value will alter the output of the model more than any of the other parameters. The µ* value does not indicate any level of interaction among the parameters, only how significantly that parameter affects the model. To examine the interactions between the parameters effectively, Sobol method SA should be used. Tables 2 and 3 show the data collected from Morris method SA performed on the coupled DEs.
The * is to note that Z 10 was added during the second set of sensitivity analysis runs with the split equations. It does not appear in the first set of data. The last column is an average of µ* across all three runs for each impact parameter. The last column is an average of µ* across all three runs for each impact parameter.
From these data, the order of impact for the nine impact parameters can be observed for both supports. For 20 wt% Co/SiO 2 , the order of impact is Z 7 > Z 3 > Z 4 > Z 1 > Z 6 > Z 2 > Z 8 > Z 9 > Z 5 . In contrast, the order for 0.5 wt% Pt, 25 wt% Co/Al 2 O 3 is Z 7 > Z 3 > Z 4 > Z 1 > Z 6 > Z 2 > Z 8 > Z 5 > Z 9. Although a subtle difference, the order of k 9 and k 5 is inverted, indicating that k 5 had a greater impact on the model with the cobalt-alumina catalyst than the cobalt-silica. These orders of impact held constant across all three sets of data for each type of support, as can be seen in Tables 2 and 3. Table 4 lists the averages for each category side-by-side in order to compare the two supports more closely. From the hypothesis, this variation in the order of the parameter impact indicates that CO 2 , the corresponding product for Z 5 , is more impactful on the alumina support than the silica. Furthermore, based on the proposed correlation between the products and corresponding parameters, alumina produces a greater amount of carbon dioxide than does silica. To verify this hypothesis, the original data from the data sets were examined, and the theory was confirmed. As Table 5 shows, the cobalt-alumina catalysts did in fact produce consistently more carbon dioxide than the cobalt-silica. Due to this CO 2 production difference, the two DEs were separated and examined individually using the Morris method. Only the Category 2 data were explored since the CO 2 parameter was more impactful for the cobalt-alumina support. The additional carbon dioxide must come from an additional source. According to the hypothesis, that source could be either water-gas shift (WGS) or the Boudouard reaction. In order to account for any CO 2 produced as a result of hydrogen interaction, thus allowing for the possibility of WGS, the parameter Z 10 was added to the hydrogen equation along with the CO 2 mole function. Tables 6 and 7 show the data collected from Morris method SA for the separated equations. Hydrogen equation only. The last column is an average of µ* across all three runs for each parameter. The row in bold is the parameter of interest (i.e., the parameter for CO 2 ).
The parameters Z 5 and Z 10 were of primary interest. As is evident from the data, the impact of k 5 was invariably greater in magnitude for the CO equation than the impact of Z 10 for the H 2 equation across all three data sets in Category 2. Since Z 5 > Z 10 , the likelihood of carbon dioxide being produced from WGS is very minimal. The results indicate that the extra CO 2 is a product of the Boudouard reaction. Carbon monoxide equation only. The last column is an average of µ* across all three runs for each parameter. The row in bold is the parameter of interest (i.e., the parameter for CO 2 ).
Catalyst Preparation
The cobalt and platinum starting materials were cobalt and platinum nitrate purchased from Alfa Aesar (Haverhill, MA, USA). A 25% cobalt alumina catalyst was prepared through a slurry impregnation procedure using Catalox 150 (high purity γ-alumina, ≈150 m 2 /g, Sasol, Hamburg, Germany) as a support. Next, the calcination of the catalysts was performed under air at 623 K for 4 h using a tube furnace (Lindberg/MPH, Riverside Road Riverside, MI 49084). The alumina was calcined for 10 h before impregnation and then cooled under an inert gas to room temperature. Co(NO 3 ) 2 ·6H 2 O (99.9% purity) was used as the precursor for Co. In this method, which follows a Sasol patent [38], the ratio of the volume of solution used to the weight of alumina was 1:1, such that approximately 2.5 times the pore volume of solution was used to prepare the loading solution. A two-step impregnation method was used allowing a loading of two portions of 12.5% Co by weight. After the impregnation, the catalyst was then dried under vacuum by means of a rotary evaporator (Buchi R-100, Flawil, Switzerland) at 353 K, then slowly increased to 368 K. After the second impregnation/drying step, the catalyst was calcined under air flow at 623 K for 4 h.
The 20% Co/SiO 2 catalyst was also prepared using the aqueous slurry phase impregnation method, with cobalt nitrate as the cobalt precursor. The support was PQ-SiO 2 CS-2133, surface area about 352 m 2 /g. The catalyst was calcined in flowing air or flowing~5% nitric oxide [39] in nitrogen at a rate of 1 L/min for 4 h at 623 K.
BET Surface Area and Porosity Measurements
Surface area, pore volume, and average pore radius of the catalyst calcined at 623 K was evaluated through Brunauer-Emmett-Teller (BET) by using a Micromeritics Tri-Star 3000 gas adsorption analyzer system (Norcross, GA, USA) ( Table 8). About 0.35 g of the catalyst was first weighed out, then added to a 3/8 in. o.d. sample tube. The adsorption gas was N 2 ; sample analysis was performed at the boiling temperature of liquid nitrogen. Prior to the measurement, the chamber temperature was gradually increased to 433 K, then evacuated for several hours to approximately 6.7 Pa.
Hydrogen Chemisorption
Hydrogen chemisorption was conducted using temperature-programmed desorption (TPD), Table 8, with a Zeton-Altamira AMI-200 instrument (Pittsburgh, PA, USA). The Co/Al 2 O 3 (≈0.22 g) was activated in a flow of 10 cm 3 /min of H 2 mixed with 20 cm 3 /min of argon at 553 K for 10 h and then cooled under flowing H 2 to 373 K. The sample was held at 373 K under flowing argon to remove and/or prevent adsorption of weakly bound species prior to increasing the temperature slowly to 623 K, the temperature at which oxidation of the catalyst was carried out.
The TPD spectrum was integrated and the number of moles of desorbed hydrogen determined by comparing its area to the areas of calibrated hydrogen pulses. The loop volume was first determined by establishing a calibration curve with syringe injections of hydrogen into a helium flow. Dispersion calculations were based on the assumption of a 1:1 H:CO stoichiometric ratio and a spherical cobalt cluster morphology. After TPD of hydrogen, the sample was reoxidized at 623 K using pulses of oxygen. The percentage of reduction was calculated by assuming that the metal reoxidized to Co 3 O 4 . Results of chemisorption are reported in Table 9. Table 9. H 2 chemisorption (temperature-programmed desorption (TPD)) and pulse reoxidation of metallic phases of cobalt-supported catalysts (cat.).
Catalyst Activity Testing
Reaction experiments were conducted using a 1 L stirred-tank slurry reactor (STSR; Fort Worth, TX, USA) equipped with a magnetically driven stirrer with turbine impeller, a gas inlet line, and a vapor outlet line with a stainless steel (SS) fritted filter (2 mm) placed external to the reactor. A tube fitted with a SS fritted filter (0.5 mm opening) extending below the liquid level of the reactor was used to withdraw reactor wax (i.e., wax that is solid at room temperature), thereby maintaining a relatively constant liquid level in the reactor. Separate Brooks Instrument mass flow controllers (Hatfield, PA, USA) were used to control the flow rates of hydrogen and carbon monoxide. Carbon monoxide, prior to use, was passed through a vessel containing lead oxide on alumina to remove traces of iron carbonyls. The gases were premixed in an equalization vessel and fed to the STSR below the stirrer, which was operated at 750 rpm. The reactor temperature was maintained constant (≈274 K) using a temperature controller.
A 12 g amount of the oxide cobalt catalyst (sieved 63-106 µm) was loaded into a fixed-bed reactor for 12 h of ex situ reduction at 623 K and atmospheric pressure using a gas mixture of H 2 /He (60 L/h) with a molar ratio of 1:3. The reduced catalyst was transferred to an already capped 1 L STSR containing 310 g of melted Polywax 3000 (Baker Hughes, Houston, TX) under the protection of inert nitrogen gas. The reactor used for the reduction was weighed three separate times, before and after reduction, and after catalyst transfer, in order to obtain an accurate amount of reduced catalyst that was added. The transferred catalyst was again reduced in situ at 503 K at atmospheric pressure using pure hydrogen (20 L/h) for another 24 h before starting the FT reaction. Each run was held at 493 K and 18.7 atm (275 psig). The CO:H 2 ratio was 1:2 for each experiment. The weight hourly space velocity (WHSV) varied from 3.0 to 6.0 slph/g catalyst, depending on the purpose of the experiment.
Gas, water, oil, light wax, and heavy wax samples were collected daily and analyzed. Heavy wax samples were collected in a 473 K hot trap connected to the filter-containing dip tube. The vapor phase in the region above the reactor slurry passed continuously to the warm (373 K) and then the cold (273 K) traps located external to the reactor. The light wax and water mixture were collected daily from the warm trap and an oil plus water sample from the cold trap. Tail gas from the cold trap was analyzed with an online HP Quad Series Micro gas chromatograph (GC) (Palo Alto, CA, USA), providing molar compositions of C 1 -C 5 olefins and paraffins, as well as H 2 , CO and CO 2 . The analysis of the aqueous phase used an SRI 8610C GC (20720 Earl St. Torrance, CA 90503, USA) with a thermal conductivity detector (TCD).
Products were analyzed with an Agilent 6890 GC (Santa Clara, CA, USA) with a flame ionization detector (FID) and a 60 m DB-5 column. Hydrogen and carbon monoxide conversions were calculated based on GC analysis of the gas products, the gas feed rate, and the gas flow that was measured at the outlet of the reactor.
Conclusions
In conclusion, the mathematical model created in this work accurately described the FTS process in terms of rate of change of syngas. When the model was analyzed using Morris method SA, a significant difference was observed between cobalt-silica and cobalt-platinum-alumina catalysts, namely a difference in the effect of the impact parameters k 5 and k 9 . Based on the initial hypothesis, this difference indicated that the cobalt-platinum-alumina generated more carbon dioxide than did the cobalt-silica. This was verified by the raw data as well. These conclusions are highly specific to the two catalyst-support types in question, and thus more general conclusions must come from further research. Thus, the equations were separated, and the cobalt-platinum-alumina catalyst data were reexamined, now with an additional CO 2 term on the hydrogen equation to test for the possibility of WGS. The observed results showed that the carbon monoxide contribution to CO 2 was consistently higher than the hydrogen contribution. Since WGS is the only pathway in which hydrogen produces CO 2 during FTS, WGS was excluded as a source of CO 2 production. Hence, the Boudouard reaction was determined to be the more probable source of additional carbon dioxide. Though the Boudouard reaction yields a high amount of carbon on the surface, to the best of our knowledge, there is no direct evidence that this reaction leads directly and solely to inert graphitic carbon (coke). Moreover, one of the main mechanistic routes proposed for FT is the carbide mechanism, where CO completely dissociates before reacting with hydrogen. In order to understand this rate of formation for C, evidence of more than one type of carbon on the catalyst surface, an active phase and an unreactive phase, has been observed on multiple catalysts such as cobalt [40] and Fe [41].
Further indications using sensitivity analysis show that both the H 2 and CO equations displaying Z 3 and Z 7 obtained the highest value, indicating the liquids are most sensitive to any deviations brought on by H 2 and CO. Though expected, these details could further indicate the importance for a specific balance in the FTS system between the two reactants, suggesting the difficulty in tuning a specific catalyst for C 5 + products as a whole. Additionally, the C 5 + products are more sensitive in the CO reaction than the H 2 , which implies that the balance for the FTS system lies more heavily in the CO than for H 2 , primarily describing the importance of CO, possibly due to the metal's ability to back donation [42] in the FTS process. While this research is preliminary, the model and analysis produced some excellent descriptive results for FTS. Although this work only examined cobalt catalyst data, future work could include the incorporation of multiple catalysts and supports. Impact parameters could potentially be optimized in future work to find the ideal values for a given catalyst. The model can be adapted to include terms for olefins and paraffins, and the liquid (C 5+ ) term could be broken down into individual terms for each number of carbons per chain (e.g., C 5 , C 6 , C 7 , etc.). The equations could be changed to predict specific product amounts based on initial conditions such as catalyst, support, temperature, pressure, flowrate, etc. Ultimately, this type of FTS modeling not only describes the process in various and versatile ways but can also be adapted and altered to potentially produce a predictive model. Funding: This research received no external funding.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | Discrimination of Melanoma Using Laser-Induced Breakdown Spectroscopy Conducted on Human Tissue Samples
Discrimination and identification of melanoma (a kind of skin cancer) by using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics methods are reported. -e human melanoma and normal tissues are used in the form of formalinfixed paraffin-embedded (FFPE) blocks as samples. -e results demonstrated higher LIBS signal intensities of phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), and calcium (Ca) in melanoma FFPE samples while lower signal intensities in normal FFPE tissue samples. Chemometric methods, artificial neural network (ANN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and partial least square discriminant analysis (PLS-DA) are used to build the classification models. Different preprocessing methods, standard normal variate (SNV), mean-centering, normalization by total area, and autoscaling, were compared. A good performance of the model (sensitivity, specificity, and accuracy) for melanoma and normal FFPE tissues has been achieved by the ANN and PLS-DA models (all were 100%). -e results revealed that LIBS combined with chemometric methods for detection and discrimination of human malignancies is a reliable, accurate, and precise technique.
Introduction
Cancer is an increasing concern worldwide and has become a major threat to human health. e leading causes of death are still due to various types of cancers. Skin cancer is one of the most frequently diagnosed types of cancer worldwide. For the identification and diagnosis of melanoma skin cancer analytical methods such as synchrotron radiation microanalysis (SXRF) [1][2][3], or transmission electron microscopy combined with energy dispersive X-ray analysis (TEM-EDX) [4,5], laser ablation inductively coupled plasma spectrometry, mass spectrometry (LA-ICP-MS) [5][6][7][8], and Raman Spectroscopy are already used [9]. In terms of LIBS sensitivity and spatial resolution with other techniques such as LA-ICP-MS, TEM, Nano-SIM (nanosecondary ion mass spectrometry), EPMA (electron probe micro-analysis), TEM, and SEM (scanning electron microscope), as indicated in [1], which illustrated that LIBS achieves micrometer scale resolution with part-per-million range sensitivities. e LIBS detection range is much more than other techniques. In brief, comparison of LIBS feature that LIBS is better than XRF is as follows: for example, elemental detection range, isotope detection, spectral line interference, lateral spatial resolution, spatial resolution of depth, surface contamination sensitivity, rapidity of analysis, and safety during use [3]. Micro-SXRF is ideal for elemental mapping and chemical speciation with a spatial resolution down to less than 100 nm and a detection limit in the order of less than parts per million because of its high brightness and linear polarized existence [5]. e sensitivity and resolution given by SXFR or microXRF is very high and is primarily used for Cu, Fe, and Zn imaging. Quantification with SXRF, however, remains difficult and appropriate calibration requirements should be achieved in an appropriate matrix [6,7]. LIBS has been the only all-optical technique that is entirely consistent with optical microscopy, providing part-per-million-range sensitivity (accessible for most metals) and micrometer-scale resolution [8]. e acquisition rate can be very high in LIBS since it is constrained only by the speed of the detector and the frequency rate of the laser. A pace of acquisition up to kilohertz has already been seen [9]. Although these analytical approaches showed high performance in terms of spatial resolution and sensitivity, their relatively slow analysis and the complexity of the required equipment make their use rather restrictive and difficult for routine medical diagnosis. erefore, it is important to develop a precise and accurate method to identify and differentiate the melanoma samples from the normal.
In recent years, laser-induced breakdown spectroscopy (LIBS) has become a well-recognized and valuable analytical spectroscopic technique for the identification and analyses of different tissues with the ability to detect all the elements on the periodic table, including low-and high-Z elements [10][11][12]. Some additional advantages of LIBS are acknowledged such as its ability to work at room temperature and atmospheric pressure, ease of use, simplicity in apparatus, full compatibility with optical microscopy, and the prospect of standoff and on-site operations, and there is no sample size or shape restrictions. In LIBS, the laser pulse is focused on the sample surface. Plasma can be formed if the irradiance at the focal spot overreach a specific threshold valued. Excitation and deexcitation of ions and atoms in the plasma of the ablated sample will give out fingerprint emission of the different elements. e chemical components of the samples can be firmed by analysing atomic and ionic emissions, and the sample can then be discriminated and classified [13].
In the literature, LIBS has been widely used to investigate various types of malignancies discrimination. Kumar et al. used LIBS spectra intensities difference ratios of various elements of the histological sections of dog liver, to distinguish normal liver and hemangiosarcoma [14]. Han et al. used LIBS spectra in the presence of argon flow rate � 15 L/ min of mice sample (in the form of tissue and pellets) for a distinction of surrounding dermis and melanoma via LDA model for quantitative analysis difference of magnesium and calcium [13]. Gaudiuso et al. used mice blood and tissues as a sample, for the early diagnosis of melanoma; they obtained an accuracy of up to 96% [15]. To the best of our knowledge, here we, for the first time, evaluated the use of LIBS to obtain elemental analyses, the classification and discrimination of paraffin-embedded human biopsies of healthy and malignant skin tissues combined with chemometric methods.
In the present work, LIBS combined with chemometrics approaches has been used for investigations and discriminations of human melanoma FFPE tissue samples instead of animal samples. We performed our experiment in an open environment while using no inert gasses during sample ablation in experiments. We also investigate the performance of the model for different types of input data (10 lines intensities, 27 lines intensities, and all lines intensities). For precise and quantified results, data preprocessing plays a vital role. e analyst uses different procedures for data analysis. e main difference in the results can come from the use of different algorithms to perform deionizing and/or baseline subtraction [16,17], to extract the emission signals for the selection of spectral line analysis. We also proposed different preprocessing methods like standard normal variate (SNV), autoscaling (auto), mean-centering (mean), and normalization by area (norm). We investigated which preprocessing method performed better and which data set line intensity is suitable in ANN, QDA, LDA, and PLS-DA models by comparing their impacts on the final classification accuracies, respectively. e performances of the models are determined in terms of accuracy, sensitivity, and specificity along with suitable preprocessing methods, and also reported P (phosphorus) line of the FFPE tissue samples, while the above-reported results about melanoma references [13,15] did not report any P lines in their spectra because of mice samples (tissue, pellets, and blood) and early melanoma diagnosis. Based on these results, we analysed distinguishable elements from melanoma FFPE tissues, reflecting the clinical situation. Figure 1, a flashpumped Q-switched Nd: YAG laser at 1064 nm with pulse duration 5 ns, repetition frequency 1 Hz, energy 64 mJ/pulse, and beam diameter ∅6 mm was used as the excitation source. e laser pulse was focused on the sample surface by three mirrors and a convex lens with a 100 mm focal length. e fiber with a diameter ∅ 600 μm was used to collect plasma emission by another convex lens with a 36 mm focal length. Two-channel spectrometer (AvaSpec 2048-2-USB2 and Avantes) was connected to the outlet of optical fiber. e samples were placed on a 3D translation stage in order to ablate the fresh tissue samples spots. e coverage range of the spectrometer was 190 nm-1100 nm with 0.2∼0.3 nm resolution. A digital delayer (SRS-DG535, Stanford Research System) and a photodetector were also used in the system. When the laser signal is detected by a photodetector, the spectrometer was triggered by DG535 after a preset delay time, 1.28 μs in our case, to reduce the bremsstrahlung radiation. e time of integration of CCD detector was set to 2 ms. e experiment was performed in an open environment without any buffer gasses.
Samples.
e melanoma and normal FFPE tissues were used as the samples in the study; some images of samples are shown in Figure 2 before and after laser ablation. e Based on the anatomic level of invasion of the layers of skin and vertical thickness of the lesion in millimetres, two classification schemes have been developed for skin cancer. For accurate prediction of future tumour behaviour, the most exclusive classification, the Breslow classification scheme is used recently. e stage of thin (T1) melanoma was classified by Clark level from the last decades [18]. In our research, the normal FFPE samples were collected from stage I melanoma patients and the melanoma FFPE samples were from stage III C patients. e number of the normal and the melanoma spectra was 111, respectively.
Data Preprocessing.
Usually, due to interference of surrounding air, fluctuation of laser energy, and inhomogeneity of sample surface, spectral fluctuations are produced between the measurements of each pulse. To reduce data fluctuations, suitable preprocessing methods are needed [19]. For obtaining a reliable data matrix, proper preprocessing of the raw data is very important, to perform the actual statistical analysis. In addition, proper data preprocessing appreciably upgrade and clarify the data analysis [20][21][22]. Four types of preprocessing methods are used in the present study, that is, autoscaling, normalization by total area, mean-centering, and SNV. e scaling process gives equal importance to all data sets and should not be suitable for noisy (i.e., poor signal-to-noise ratio) data [22].
Classification Models.
In the present study, four types of classification models (ANN, PLS-DA, LDA, and QDA) are used on the four types of different preprocessing methods (autoscaling, mean-centering, normalization by total area, and SNV). For ANN networks, feedforward networks are used because of their excellent ability of self-adapting and self-learning. e two common and principal network types of feedforward networks are radial basis function (RBF) and multilayer perceptron (MLP). For data analysis when ANN is used, it is very essential to determine between the ANN model (the network's arrangement) and ANN algorithms (computation that eventually produces the network output). e network that has been constructed for an application is ready to be trained. ere are two types of training approaches, which are supervised and unsupervised. A fully connected ANN is used most frequently, with a backpropagation learning rule supervised network. ANN model of this type is better for discrimination purposes [23].
PLS-DA is one of the supervised methods and needs a learning step prior to its application to unknown samples. e interclass difference increases while the intraclass difference decreases in the PLS-DA model, which revealed the model recognition ability. PLS-DA model is very suitable for binary classification, while complexity will increase in the case of multiclassification PLS-DA model [24]. PLS-DA has also been applied to the classification of LIBS data and discriminates the tissue samples [25].
For classification purposes, both LDA and QDA are also used and achieved good accuracy results [26,27]. LDA and QDA models are derived from simple probabilistic models, which model the class conditional distribution of the data P (X|y � k) for each k class. Bayes rules are used for predicting the class as shown in where X and y are events, P (y � k|X) is a conditional probability, and k is the class which maximizes the conditional probability. Sensitivity is the ability of a test which decides the disease cases correctly, while specificity is defined as the ability of a test which decides and determines the healthy cases. Similarly, accuracy is defined as the ability of a test to demarcate the disease and healthy cases precisely. Sensitivity, specificity, and accuracy are calculated by the following equations, respectively: where (TP) is true-positive, the number of cases precisely recognized as a disease, (FP) is false-positive, the number of cases incorrectly identified as a disease, (TN) is true-negative, the number of cases precisely recognized as healthy, and (FN) is false-negative, the number of cases incorrectly identified as healthy.
LIBS Spectra Measurement
Results. e typical LIBS spectra of human melanoma and normal FFPE tissue samples are illustrated in Figure 3(a), which is the average of 111 spectra in the spectral range of 200-900 nm of each sample. e LIBS spectra are usually normalized for discrimination purposes to reduce the spectral fluctuations that are produced due to the matrix effects and experimental variations. e normalization is operated comparatively to a specific line.
e Carbon C 247.482 nm line is used for normalization purposes because the C emission is a specific line [13]. Carbon C 247.482 nm is used for normalization purposes because the C emission is firmed among all the measurement of LIBS spectra. e normalized LIBS spectra of the samples in the spectral region of 200 to 900 nm are also illustrated in Figure 3 For further classification analysis, these 27 element lines are selected as features variables of the LIBS spectra. e main spectral lines can be seen, which is located at the ionic and atomic lines from Ca, Fe, H, K, Mg, Na, O, and P. ese atomic and ionic emission lines of melanoma are more vigorous than those of the normal FFPE tissue samples. Other emission lines like Fe and H are frail and weaker. erefore, these emission lines are listed in Table 1 for the classification of FFPE melanoma and normal tissue sample.
We detected significant changes in the LIBS spectra between melanoma and normal samples. In melanoma FFPE tissue samples, the peak intensities of Ca, K, Mg, and P increase relative to the normal FFPE tissue peak intensity. Magnesium plays an important role in regulating cell division as well as Ca.
e melanin-containing pigment granules have an enormous amount in melanoma cells, which are wealthy in Ca [28]. e high levels of Ca are also reported in previous literature in different cancer tissues, like breast cancer, colorectal cancer [13], uterine cancer [29], and canine hemangiosarcoma [14]. Magnesium has also an important role in cell proliferation and the biosynthesis of proteins [30]. In the LIBS imaging technique, the elemental images of the tumour reported a gradient concentration for Ca, Mg, and P in metastatic melanoma cases, and also reported the wide dynamic ranges of concentration for elements, such as Fe and Na from low to high concentration [31]. A bioelemental study of melanoma lymph nodes also showed a clear distinction of P from the normal tissue [32]. e normalized intensities of 27 emission lines of the melanoma and normal FFPE tissue samples are shown in the bar graph in Figure 4. ese 27 emission lines are selected as features emission lines of normal and melanoma spectra. en, the area average was taken for these 27 elemental lines and selected only 10 lines on the basis of their normalized spectral intensities to draw a threshold line, which is also shown in Figure 4. For discrimination purposes, three types of input variables are used as input data for all the classification models (10 lines, 27 lines, and all lines intensities, resp.).
Classification Results.
Classification accuracy is defined as the division of the number of correct predictions by predictions number, multiplied by 100% to gain the percentage accuracy. We performed all the models with the training set including 83 spectra of melanoma and normal sample (166 spectra in all) and a test set including 27 spectra of melanoma and normal sample (54 spectra in all).
For the ANN model, the number of hidden layers of neurons l, which needs to be optimal, is calculated according to the formula e accuracy of the ANN model for different input variable data sets (10 lines, 27 lines, and all lines) is illustrated in Figure 5. In 10-line input variable data set, the highest accuracy is 100% for an autoscaling method in all hidden layers of a neuron, while the mean-centering, normalization by total area, and SNV preprocessed method showed 98% and 97%, respectively, which is shown in Figure 5(a). Similarly, for 27-line intensity, input variables data set mean-centering showed the highest accuracy 100%, while the autoscaling and SNV showed 98% accuracy for different hidden neurons' layers as shown in Figure 5(b). Furthermore, for the whole input spectra variable data set, the ANN accuracy is 98% in autoscaling and 99% for normalization in all hidden layers, while the mean-centering and SNV showed very low accuracy for all the hidden layers as shown in Figure 5(c). Overall, the normalization data set revealed the best sensitivity, specificity, and accuracy results, for the ANN model. So, for tissue analysis, the normalization by area is the best and suitable compared to other preprocessing methods in the ANN model; the accuracy % result of the ANN model is illustrated in Figure 6(a). PLS-DA is also used to discriminate against the tissue samples. e 10-line intensity data set showed the highest accuracy result for PLS-DA model classification. Both meancentering and SNV data sets showed good classification accuracy as compared to other preprocessing data sets as revealed in Table 2. e classification result of the PLS-DA model for a different preprocessing method is shown in Figure 6(b).
For classification purposes, both LDA and QDA are also used and achieved good accuracy results. In the LDA model, the accuracy of classification for all the cases (10 lines, 27 lines, and all lines) cannot achieve the 100% for FFPE tissues differentiation. e highest and lowest accuracy values for LDA classification model are 98% and 77%, respectively. While in QDA model classification, normalization by total area preprocessed method achieved 98% accuracy in the 27line input variable data set. e minimum accuracy for QDA model classification is calculated for the whole spectra input variable data set in SNV preprocessed method, which is 77%. e percentage accuracy rate of LDA and QDA models are shown in Figures 6(c), and 6(d), respectively. e accuracy results of ANN, PLS-DA, LDA, and QDA models along with suitable and appropriate preprocessing methods are illustrated in Table 2, which showed the highest accuracy for different input variable data sets and suitable preprocessed method. In the ANN model for the 10-line input variable data set, only the autoscaling preprocessed method showed the highest 100% accuracy, while for the 27line input variable data set only the mean-centering showed the highest 100% accuracy. For the whole spectral input data set, only the norm-area preprocessing method showed the highest 98% accuracy, while the other preprocessed methods showed lower accuracy results. Similarly, in the PLS-DA model for 10-line intensity input variable data set, three preprocessing methods (autoscaling, SNV, and mean-centering) showed the highest 100% accuracy; on the other hand, for 27-line intensity and whole spectral data set, the preprocessed methods (mean-centering, normalized by the area, and SNV) showed the highest 100% accuracy, respectively. Furthermore, the LDA model classification for 10-line input variable data set showed the highest 98% accuracy for the autoscaling preprocessed method, while for 27-line intensity, the only normalization by area (normarea) revealed 98% accuracy result and for the whole input variable data set and also only the autoscaling preprocessed method showed 98% accuracy result. In the QDA model classification for 10 lines, input variable data set, only autoscaling, and normalization preprocessed method showed the highest 94% accuracy; similarly, for 27-line input variable data set, normalization by area showed 100% accuracy as well, and for whole data input variable, only autoscaling revealed the highest 98% accuracy result for melanoma FFPE and normal tissue differentiation. In comparison, ANN and PLS-DA is the best classification e ROC curves of the ANN, PLS-DA, LDA, and QDA model showed the lowest accuracy of 98% for the 10-line input variable dataset. Similarly, for the ROC curve for 27line input variable data set, the highest sensitivity 100%, specificity 100%, and accuracy 100% for ANN, PLS-DA, and QDA model are shown, while LDA showed the same result as for 10-line input variable data set. On the other hand, for the whole input variable data set, the highest sensitivity 100%, specificity 100%, and accuracy 100% are revealed for PLS-DA models while for LDA and QDA classification models, the whole input variable data set showed the highest sensitivity 96%, specificity 100%, and accuracy 98%. It is concluded that the PLS-DA models are the best and useful for FFPE tissue classification as compared to LDA and QDA models. (minimum) accuracy percentage is shown 52% by SNV preprocessed data in whole spectral input variable data set. Similarly, for PLS-DA model classification, the highest accuracy is 100%, while the lowest is 94% for normalization by area (norm-area) in 10-line input variable data set. Furthermore, for the LDA model classification, the highest accuracy is 98% while the lowest (minimum) accuracy is 77% for SNV preprocessed method in the whole spectral input variable data set. On the other hand, for QDA classification model, the highest accuracy is 100%, while the lowest is 77% for SNV preprocessed method in the whole spectral input variable data set, the same as the LDA model result for the whole input variable data set. All model results revealed that the 27-line intensity was the average highest accuracy rate of 97.6% as compared to others; autoscaling data set showed the highest average accuracy 97.8% of all the models, and PLS-DA showed the averaged best performance model in all of them; average accuracy is 99.16%.
Conclusion
e purpose of the research was to discriminate melanoma skin cancer and normal skin by using laser-induced breakdown spectroscopy (LIBS) combined with the chemometric methods. e melanoma and normal FFPE tissues were used as samples. In the LIBS spectra, the intensities of the lines of several elements showed a significant difference between the melanoma and normal FFPE samples and were regarded as feature variables. e lines intensities of calcium (Ca), magnesium (Mg), phosphorus (P), potassium (K), sodium (Na), and oxygen (O) in melanoma samples were higher than the normal samples. Phosphorus (P) line, especially, showed the highest intensity for malignancies. erefore, phosphorus (P), calcium (Ca), magnesium (Mg), and potassium (K) were defined as biomarkers for discrimination in this study. e chemometric models, such as ANN, PLS-DA, LDA, and QDA were used to analyse the spectral data from melanoma and normal tissue in FFPE. e best performance of the model (sensitivity, specificity, and accuracy) has been achieved by the ANN and PLS-DA models (all were 100%). e results indicated that LIBS combined with the chemometric models could be used as a quick discrimination method for human malignancies.
Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
e authors declare that there are no conflicts of interest related to this article. | v3-fos-license |
2017-09-07T07:11:14.855Z | 2012-07-28T00:00:00.000 | 36247234 | {
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} | pes2o/s2orc | Phytochemical and Anticonvulsant Studies on the Aqueous Ethanol Extract of the Root-Back of Ficus Abutilifolia ( Miq . ) Miq . ( Family : Moracea )
Ficus abutilifolia, belonging to the family Moraceae is a small to medium sized tree that grow mostly in the African continent. It was reported to be used traditionally, in promoting fertility in humans and in the treatment of skin wart and management of epilepsy. Preliminary phytochemical investigation of the powdered root revealed the presences of flavonoids, saponins and tannins among others. The intraperitoneal LD50 of the 70% aqueous ethanol extract was found to be 2154.1 mg/Kg in mice. The anticonvulsant studies of the extract revealed that a single administration (at the dose of 100 – 400 mg/Kg) produced a dose-dependent protection against MEST; however, the extract did not offer significant protection against pentylenetetrazoleand 4amino pyridine–induced seizures. These finding suggest some level of protection by the aqueous ethanol extract against MES induced seizure in chicks, thereby giving support to the traditional claim for the use of the plant in the treatment and/or management of convulsion and epilepsy.
INTRODUCTION
Plants and their products have been in use in the treatment of chronic ailments, pains and infectious diseases before the modern civilization.And to date, they still remain the almost exclusive source of drugs for more than 60% of the world's population, especially in developing countries, and also serve as important source of new drugs, new drug leads, and new chemical entities.It is in record that the potential of plants in general and higher plants in particular as a source of new drugs has not been fully explored.Some individual plant extract may have been subjected to specific pharmacological test (e.g. for cardiac activity only) however, the same extract may be examined for other types of activities such as pain reliving, anti-inflammation, antidiarrhea etc (Balunas and Kinghorn, 2005;Li and Vederas, 2009;Phillipson, 2001).Ficus abutilifolia (Miq.)Miq. is commonly called large-leaved rock fig or rock wild fig and belongs to the family Moraceae.It is a small to medium sized, deciduous to semi-deciduous tree that may grow up to 15 m high (though it seldom exceeds 5 m).The bark of the plant is yellowish-white, smooth and flaking.The trunk is usually twisted.Leaves are broadly ovate and cordate at base, they usually measured 7.5 -20.0 X 6.5 -18.0 cm.Fruits are 1.5 -2.5 cm in diameter usually borne singly or in pairs at the leaf axils, smooth or slightly hairy.
A decoction of the leaf of the plant is reported to be used in promoting fertility in humans and the milky latex is used to remove skin warts.Bark decoction is taken by men as a strengthening tonic (Burring, 2006).
Information made available to us (personal communication, 2004) showed that the root of F. abutilifolia is also used as part of a preparation in the management of epilepsy.We therefore, investigate and report here the preliminary phytochemical screening and for the first time the anticonvulsant properties of the F. abutilifolia root-bark.
Plant Material
The plant specimen was collected in October, 2006 around Basawa-Zaria, Kaduna State Nigeria.It was identified by the herbarium keepers of the Department of Biological Sciences, Ahmadu Bello University, Zaria-Nigeria, where a specimen (voucher specimen number 900, 742) was deposited for future reference.The root of the plant was cleaned, air dried and ground to powder using pestle and mortar.
Extraction of the Plant Material
Powdered plant (150 g) was macerated with 750 ml of 70% aqueous ethanol for four days after which, it was filtered and evaporated to dryness on water bath to give a brownish residue.The residue, subsequently referred to as the extract was stored in an air tight container until required for further use.
Animals
Swiss albino mice of both sexes, weighing 18 -25 g were obtained from the Animal House facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria.One day old Ranger cockerels were obtained from National Animal Production Research Institute (NAPRI) Shika-Zaria, Nigeria.The mice were kept under well-ventilated conditions, 12 hours light/dark cycle, temperature of 25 ± 2 0 C and fed on standard laboratory animal Feeds and had access to water ad-libitum.
Preliminary Phytochemical Screening
The powdered root of F. abutilifolia was subjected to preliminary phytochemical analysis to test for the presence of phytochemical constituents using the following methods: Anthraquinones [Borntrager's test: 100 mg of powdered plant in 5 ml of chloroform, filtered.2 ml filtrate + 2 ml 10% NH 4 OH.A bright pink colour indicates the presence of anthraquinones; Modified Borntrager's test: 200 mg plant material boiled in 5ml 10% HCl, filtered.Filtrate extracted with 5ml benzene, and benzene layer shaken with 5 ml 10% NH 4 OH.A rose pink or cherry red colour indicates the presence of anthraquinone derivatives (Evans, 1996).
Acute Toxicity Study
LD 50 determination was conducted using the method of Lorke (1983).Nine mice were divided into 3 groups of 3 mice each.The first group received the extract (i.p.) at a dose of 1000mg/kg; group 2 received the extract at a dose of 100mg/kg (i.p.), while the last group received the extract at the dose of 10mg/kg body weight.Animals were observed for general signs and symptoms of toxicity including mortality over a period of 24 hours.
In the second phase, 4 mice were divided into 4 groups of one mouse each.The extract was administered at the dose of 600, 1000, 1600, and 2900 mg/Kg (i.p.) to animals respectively, based on the result of the first phase.LD 50 was calculated as the square root of the geometrical mean of highest non lethal dose for which the animal survived and the lowest lethal dose for which the animal died.
Anticonvulsant Activity Maximal Electroshock-Induced Seizures in Chicks (MEST)
The methods previously described by Swinyard and Kupferberg (1985) and Browning, (1992) were employed.Fifty one day old cockerels were randomly divided into 5 groups of 10 chicks per group.The first group received normal saline i.p.; groups 2 -4 received the extracts (100, 200 and 400 mg/kg, i.p. respectively).While the fifth group received phenytoin 20 mg/kg, (i.p.).Thirty minutes later, maximal electroshock was delivered to induce seizures in the chicks using Ugo basile electroconvulsive machine (model 1801) with corneal electrodes placed on the upper eyelid of the chick after dipping them in normal saline.The current, shock duration, frequency and pulse width were set and maintained at 90mA, 1.0 sec, 200 Hz and 1.0 ms -1 respectively.An episode of tonic extension of the hind limbs of the chicks was considered as full convulsions.Lack of tonic extension of the hind limbs was regarded as protection.The recovery time was taken for the unprotected animals.
Pentylenetetrazole-Induced Seizures in Mice (Sc-PTZ)
The method of Swinyard et al. (1952) was employed.Twenty-five mice were randomly divided into 5 groups of five mice per group.The first group which served as negative control was treated with normal saline (i.p.).Groups 2 -4 received different doses of the aqueous ethanol extract reconstituted in water (100, 200 and 400 mg/kg, i.p. respectively).Group 5 which served as positive control was treated with 200 mg/kg i.p. valproic acid.
Thirty minutes later, 85mg/Kg of freshly prepared solution of pentylenetetrazole was administered subcutaneously to all the mice.The mice were observed for 30 minutes for the onset and incidence of seizures.An episode of clonic spasm of at least 5 seconds was considered as seizure.Lack of threshold convulsion during 30 minutes of observation was regarded as protection.The number of mice protected was noted and the anticonvulsant properties of the extract expressed as percentage protection.
4-Amino Pyridine-Induced Seizure in Mice
The method of Yagamuchi and Rogawski, (1992) was adopted.Twenty-five mice were randomly divided into 5 groups of five mice per group.The first group which served as negative control was treated with normal saline.Groups 2 -4 received different doses of the aqueous ethanol extract reconstituted in water (100, 200 and 400 mg/Kg, i.p. respectively).Group 5 which served as positive control was treated with 20 mg/Kg (i.p.) phenobarbitone.
Thirty minutes later, 15mg/Kg of freshly prepared solution of 4-aminopyridine was administered subcutaneously to all the mice.The mice were observed for 30 minutes for characteristic behavioral signs such as hyperactivity, trembling, intermittent forelimb extension, tonic seizure and death.Lack of threshold convulsion during 30 minutes of observation was regarded as protection.The number of mice protected was noted and the anticonvulsant properties of the extract expressed as percentage protection.
Statistics Analysis
Data were expressed as Mean ± Standard Error of Mean.Statistical analysis was carried out using one-way ANOVA, followed by Dunnett's test and Chi-square for percentage protection and P<0.05 was considered significant.
RESULTS AND DISCUSSION
Preliminary phytochemical screening tests for the powdered root of F. abutilifolia (table 1) revealed the presence of saponins, flavonoids, tannins, terpenoids and/or steroids.However, alkaloids were not detected in the root.The presence of the above chemical compounds may alone or in combination contribute to the observed anticonvulsant effect of the root extract.The median lethal dose of the aqueous ethanol extract of F. abutifolia was estimated to be 2, 154.1 mg/Kg body weight.The acute toxicity index (LD 50 ), serve as a means of giving an idea about the toxic effect of any potential drug substances.The result of the present study shows that the 70% aqueous ethanol extract of F. abutilifolia has an LD 50 of over 2,000 mg/Kg body weight in mice when administered through intraperitoneous route.The extract can be regarded as slightly toxic but the risk of acute intoxication is minimal (Lorke, 1983).
The aqueous ethanol root extract of F. abutifolia dose dependently protected the animals against maximal electroshock seizure with the highest protection of 40% produced at the dose of 400 mg/Kg.However, the extract did not offer any significant protection against pentylenetetrazole and 4-aminopyridine induced seizures in mice.The hind limb tonic extension (HLTE) produced electrically as in the maximum electroshock test (MEST), is a common feature in many animal species including humans.And the response of the brains of the animals to anticonvulsant is similar to that of humans.Extracts of F. abutilifolia afforded dose depended protection to the laboratory animals against the HLTE showing the ability of the extract to inhibit or prevent seizure discharge within the brainstem seizure substrate (Browning, 1992).This suggests that the 70% aqueous extract of the plant under study contain some compounds that may be beneficial in the treatment of generalized tonic-clonic and partial seizure.The chemically-induced seizure using PTZ test usually identifies drugs that raise seizure threshold in the brain (White et al, 1998).The standard convulsant PTZ has also been shown to interact with γ-amino butyric acid (GABA) -a neurotransmmiterand the GABA receptor complex (Bum et al, 2001).Drugs that inhibit the PTZ activity such as diazepam and valproic acid exert their effect by enhancing GABA mediated inhibition in the brain.The inability of the aqueous ethanol extract of F. abutilifolia to protect the animals against PTZ-induced seizure suggests that it may not be effective in the treatment of absence and/or myoclonic seizures.The potassium channel blocker -4-aminopyridine -is a convulsant that penetrates the blood-brain barrier (Yagamuchi and Rogawski, 1992), and causes convulsion by enhancing spontaneous and evoked neurotransmitters.The inability of the extract to afford any protection to the laboratory animals against the chemically-induced seizure by 4-aminopyridine indicates that the extract does not produce its activity via potassium channel.
CONCLUSION
F. abutilifolia root extracts afforded protection to laboratory animals against maximum electroshock, indicating that it may be useful in the management of grand mal epilepsy.However, the absence of any significant protection to the animals against the chemically induced seizures of PTZ-and 4AP suggests that, at this dose the extracts may not be beneficial in the management of petit mal epilepsy.
Table . 1
: Preliminary Phytochemical Screening Result of Powdered root-bark of F. abutilifolia.
Table . 2
: Effects of different doses of aqueous ethanol extract of F. abutilifolia root-bark on the convulsive activities of electroshock .
Table . 3
: Effects of aqueous ethanol extract of F. abutilifolia root on pentylenetetrazole-induced seizure in mice.
Table . 4
: Effects of different doses of aqueous ethanol extract of F. abutilifolia root on the convulsive activities of 4-aminopyridine. | v3-fos-license |
2018-04-03T04:08:45.094Z | 1996-07-26T00:00:00.000 | 37828572 | {
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} | pes2o/s2orc | The serpin-enzyme complex receptor recognizes soluble, nontoxic amyloid-beta peptide but not aggregated, cytotoxic amyloid-beta peptide.
There is now extensive evidence that amyloid-β peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-β 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of α1-antitrypsin (α1-AT) and other serine protease inhibitors, also recognizes the amyloid-β 25-35 domain. In fact, by recognizing the amyloid-β 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-β peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-β peptide. A series of peptides based on the sequences of amyloid-β peptide and α1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-β peptide 25-35 and amyloid-β peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-β 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric amβ/α1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-β 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-β peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-β peptide, if soluble amyloid-β peptide proves to be an in vivo precursor of the insoluble, toxic peptide.
The serpin 1 -enzyme complex receptor (SEC-R) was identified originally as a receptor of human mononuclear phagocytes and liver cells which recognized ␣1-antitrypsin (␣1-AT)-elastase complexes and mediated feedback up-regulation of ␣1-AT biosynthesis (1). The receptor was characterized with a radioiodinated synthetic peptide, 125 I-peptide 105Y (SIPPEVKFNK-PFVYLI), based on the sequence of a candidate receptor binding domain in the carboxyl-terminal tail of ␣1-AT. This peptide mediated an increase in synthesis of ␣1-AT in monocytes and human hepatoma HepG2 cells, and binding studies showed that it described a receptor with a K d of ϳ40 nM and ϳ450,000 plasma membrane receptors per cell (1). Binding of radioiodinated peptide 105Y was blocked by unlabeled ␣1-ATelastase complexes, and binding of radioiodinated ␣1-AT-elastase complexes was blocked by unlabeled peptide 105Y (1), thus providing evidence that SEC-R was indeed a binding site for ␣1-AT-elastase complexes.
SEC-R is now known to have a ligand-binding subunit of ϳ84 kDa (2) which is expressed on a diverse array of cell types. It mediates endocytosis and intracellular degradation of ␣1-ATelastase complexes (3) and mediates directed migration of neutrophils toward this ligand (4). In studies designed to define the minimal requirements for binding, we showed that a pentapeptide at the carboxyl-terminal aspect of peptide 105Y was sufficient for binding to SEC-R (5). This pentapeptide is highly conserved among members of the serpin supergene family. Several of the other serpins, such as ␣1-antichymotrypsin, antithrombin III, heparin cofactor II and, to a lesser extent, C1 inhibitor and plasminogen activator inhibitor I, when in complex with their cognate serine protease, compete for binding to SEC-R (1,6). A similar pentapeptide sequence was identified in several tachykinins and amyloid- peptide. In fact, competitive binding studies have shown that: (i) soluble amyloid- peptide binds to SEC-R on HepG2 cells (2); (ii) soluble amyloid- peptide binds to SEC-R on neutrophils, mediating chemotactic activity and conferring homologous desensitization to the chemotactic activity of peptide 105Y (4); (iii) soluble amyloid- peptide binds to SEC-R on neuronal cells (7); (iv) the region corresponding to amyloid- peptide 25-35, and particularly amyloid- 31-35, is critical for binding to SEC-R on any of these cell types (7); (v) amyloid- 1-39, amyloid- 1-40, and amyloid- 1-42, when prepared in soluble form in Me 2 SO, present this binding domain to SEC-R to an equivalent extent (7); (vi) SEC-R mediates endocytosis and catabolism of soluble amyloid- peptide in hepatocytic and neuronal cell types (7).
The data regarding amyloid- binding to SEC-R are particularly interesting in that neurotoxic effects have been attributed to this peptide and have been considered in the common final pathway for neuronal degeneration and presenile dementia of Alzheimer's disease and Down's syndrome (8,9). Although the minimal peptide sequence for the neurotoxic effect of amyloid-, amyloid- 25-35 correlates with the minimal peptide sequence for binding to SEC-R, several recent studies have suggested that amyloid- 25-35 must be aggregated to induce its toxic effects (9 -16). In this study we compared monomeric, soluble amyloid- peptide with its aggregated form to determine the correlation between SEC-R binding and cytotoxic activities.
Cell Lines-Maintenance of HepG2 cells has been described previously (3). PC12 cells were kindly provided by Drs. Eugene Johnson and Karen O'Malley (St. Louis, MO). Human glioblastoma U373MG cells, HeLa cells, and Chinese hamster ovary cells were purchased from ATCC.
Murine Cortical Cultures-Dissociated murine cortical cultures containing both neurons and glia were prepared from fetal (E15) Swiss Webster mice and subcultured in 24-well plates on an established monolayer of glial cells (15-30 days in vitro) as described previously (18). Glial cell division was inhibited after 3-5 days by a 48-h incubation in medium supplemented with cytosine arabinoside (10 M).
Determination of Cell Surface Receptor Binding-Peptides 105Y and Yam 25-35 were radioiodinated by the chloramine-T method. 125 I-Peptide 105Y and 125 I-Yam 25-35 (YGSNKGAIIGLM) were purified on Sephadex G-10. Specific radioactivity varied from 25,000 to 35,000 cpm/ng. For binding studies in HepG2 cells, separate monolayers in 24-well plates were dunked in phosphate-buffered saline and incubated for 2 h at 4°C in 125 I-labeled ligand in the absence or presence of competitor and diluted in binding medium (Dulbecco's modified Eagle's medium containing 10 mM Hepes, 0.1 mg/ml cytochrome c, 0.01% Tween 80, 1 mg/ml bovine serum albumin). The cells were then rinsed in phosphate-buffered saline and cell-associated radioactivity determined in 1 N NaOH homogenates. For binding studies in PC12 cells, cells were detached from tissue culture flasks, washed with phosphatebuffered saline supplemented with 3 mM EDTA, and then, in suspension, incubated for 2 h at 4°C with 125 I-labeled ligand with and without competitors diluted in suspension binding medium (phosphate-buffered saline, 3 mM EDTA, 1 mg/ml bovine serum albumin, and 0.01% Tween 80). At the end of this time interval, cells were washed, lysed in 1 N NaOH and cell-associated radioactivity was determined. Specific binding was defined as the difference between total binding in the absence of unlabeled competitor and nonspecific binding in the presence of unlabeled competitor. Nonspecific binding did vary from 8 to 30% depending on individual radioligand and individual radioligand preparations.
Our previous studies have shown that binding characteristics and specificity of binding of 125 I-peptide 105Y and 125 I-Yam 25-35 are virtually identical in HepG2 cells and PC12 cells (7). Because binding to HepG2 cells in monolayer more easily permits multiple competitors and replicates to be examined in a single experiment than binding to PC12 cells in suspension, this cell line was used as the model for most of the experiments reported here. Moreover, because binding of 125 I-peptide 105Y has been most extensively characterized in our previous competitive binding studies (1-7), 125 I-peptide 105Y was used as the labeled ligand in most of the experiments reported here. For each of the experiments, however, results were identical when 125 I-Yam 25-35 was used as a labeled ligand in HepG2 cells or when 125 I-peptide 105Y or 125 I-Yam 25-35 was used in PC12 cells. Cytotoxicity Assays-To assess toxic effects of peptides in PC12 cells, we used the MTT reduction assay (19,20). In this assay a tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide (MTT), is used as a colorimetric substrate for measuring cell viability. When cells are injured there is an alteration of cellular redox activity such that cells are not able to reduce the dye. Alterations in cellular redox activity, as measured by the MTT assay, correlate well with other measures of cytotoxicity in amyloid--induced neurotoxicity (19) as well as in other types of cytotoxicity (20).
For our MTT assay, PC12 cells were plated at density of 2 ϫ 10 4 cells/ml in 24-well tissue culture plates and incubated with peptide in RPMI supplemented by 10% horse serum and 5% fetal calf serum. Unless otherwise indicated, this incubation was 4 h in duration. At the end of this time interval, MTT was added to a final concentration 0.5 mg/ml. The incubation was continued overnight for 16 -18 h. Cell lysis buffer (20% SDS, 50% N,N-dimethylformamide, pH 4.7) was added (100 l/well) and the incubation continued for another 2 h. Colorimetric analysis at 570 nM was then completed. Values in the presence of vehicle alone were considered 100% control response, and values after cell lysis in phosphate-buffered saline, 0.5% Triton X-100 were considered 0% control response. This means that cytotoxicity is indicated by a reduction from 100 for the percent control response. Results are reported as the mean Ϯ 1 S.D. for four measurements at each point.
To assess toxic effects of peptides in murine cortical neurons, we used conventional morphologic and lactate acid dehydrogenase (LDH) release assays. After 9 days in culture, the cortical cell monolayers were incubated for 48 h in medium (modified Eagle's medium with 25 mM glucose and 26 mM bicarbonate) alone or supplemented with specific synthetic peptides. Neuronal cell death was assessed by microscopic examination and quantified by release of LDH into the medium (18).
SDS-PAGE-Synthetic peptides were dissolved in Laemmli sample buffer and subjected to electrophoresis on 16% gels under reducing conditions and next to molecular mass markers exactly as described previously (2).
DNA Fragmentation Assays-Cells were lysed in 0.5% Triton X-100, 5 mM Tris buffer, pH 7.4, 20 mM EDTA for 20 min at 4°C as described previously (21). After centrifugation at 27,000 ϫ g for 15 min, supernatants were extracted with phenol-chloroform and precipitated in ethanol. Samples were subjected to electrophoresis on 1.2% agarose gels. The gels were incubated with RNase A (20 g/ml) at 37°C for 3 h before staining with ethidium bromide.
RESULTS
Toxic Effect of Amyloid- Peptide in PC12 Cells-We examined the possibility of measuring the toxicity of amyloid- peptide 25-35 in a cell line of neuronal origin which also expresses SEC-R. The neuronal cell line PC12, which expresses SEC-R (7), and which undergoes programmed cell death in response to amyloid- 25-35 (19), was selected. Toxicity was measured by the MTT reduction assay. Inhibition of MTT reduction by PC12 cells has been shown to be an early indicator of cell death as defined by an increase in LDH release, decrease in [ 3 H]thymidine incorporation, and abnormal morphology (19,20). First, we examined the effect of amyloid- 25-35 and other amyloid peptides on MTT reduction in PC12 cells (Fig. 1). In each case the peptides were prepared in water and incubated at 37°C for 5 days to optimize formation of aggregates. The results showed a concentration-dependent inhibition of MTT reduction by amyloid- 25-35 and amyloid- 1-40, but not by amyloid- 1-16 or amyloid- 1-28 (Fig. 1A). The effect of amyloid- 25-35 on MTT reduction by PC12 cells is half-maximal at 8 -80 nM. Next, we examined the possibility that the neurotoxic effect of amyloid- 25-35 involved an apoptotic mechanism. PC12 cells were incubated with actinomycin D or cycloheximide and then with amyloid- 25-35. The toxic effect was inhibited by both actinomycin D and cycloheximide in concentration-dependent manner (Fig. 1B). The mechanism of amyloid-induced neurotoxicity was also examined by agarose gel electrophoresis (Fig. 1C). There was evidence for intranucleosomal DNA fragmentation in response to amyloid- 25-35 (left lane). This DNA fragmentation was similar to that induced by a positive control, serum starvation (middle lane). For a negative control, we examined glutamate (right lane). Glutamate is known to induce a toxic, nonapoptotic effect in primary culture of cortical neurons (22). At 1 M it also has a toxic effect on PC12 cells as reported in Ref. 23 and determined by us in the MTT assay (data not shown). However, it did not cause DNA fragmentation in PC12 cells (right lane). Thus, the effect of amyloid- 25-35 on DNA fragmentation in PC12 cells is specific. These data provide evidence for a specific apoptotic effect of amyloid- 25-35 in a neuronal cell line using a relatively simple and reproducible assay. We could now examine whether this effect was mediated by the SEC-R.
Cell Type Specificity of Amyloid--induced Cytotoxicity-We examined whether the toxicity of amyloid- 25-35 was confined to cell types that express SEC-R (Fig. 2). Three cell lines that express SEC-R, neuronal cell line PC12, hepatoma cell line HepG2, and glial cell line U373MG, and two cell lines that do not express SEC-R, HeLa and Chinese hamster ovary, were subjected to MTT assay after incubation in amyloid- 25-35 under conditions associated previously with toxicity in PC12 cells, as shown in Fig. 1. The results show some variability in susceptibility to the neurotoxic effect among these cell lines with PC12 cells being most susceptible. However, there was inhibition of MTT reduction in all five cell lines, suggesting that the toxic effect is independent of SEC-R expression.
Correlation among SEC-R Binding, Toxicity, and Aggregation of Amyloid- Peptides-We used a panel of assays to examine the correlation of SEC-R binding, toxicity, and aggregation of amyloid- peptides. For SEC-R binding, we examined the competition of unlabeled amyloid- peptides for binding of 125 I-peptide 105Y to HepG2 cells. In each case, with a more limited number of replicate samples, the results were almost identical for binding of 125 I-Yam 25-35 to HepG2 cells or for binding of 125 I-peptide 105Y or 125 I-Yam 25-35 to PC12 cells (data not shown). For toxicity we used the MTT reduction assay in PC12 cells. For assessment of peptide aggregation, we used SDS-PAGE analysis. Each peptide was examined in presumed soluble form by dissolving it in Me 2 SO and in presumed aggregated form by aging it in water for 5 days at 37°C as described by previous studies (7)(8)(9)(10)(11)(12)(13)(14)(15).
First, we examined amyloid- 25-35 (Fig. 3A). The data showed that amyloid- 25-35 prepared in Me 2 SO was much better than amyloid- 25-35 aged in water as a competitor for binding to SEC-R (left panel). However, amyloid- 25-35 aged in water, but not amyloid- 25-35 prepared in Me 2 SO, was toxic (right panel). The results were almost identical when amyloid- 1-40 was subjected to this comparison (Fig. 3B). Amyloid- 1-40 prepared in Me 2 SO was a much better competitor than amyloid- 1-40 aged in water for binding to SEC-R (left panel). However, amyloid- peptide prepared in Me 2 SO had minimal toxicity, and that minimal toxicity was elicited at a significantly higher concentration than the potent toxic effect of amyloid- 1-40 aged in water (right panel). There was ϳ15% inhibition of MTT reduction at 10 M amyloid- 1-40 prepared in Me 2 SO but almost 60% inhibition of MTT reduction at 1 M amyloid- 1-40 aged in water. For each of these experiments the amyloid- peptide was prepared in Me 2 SO in such a way that the final concentration of Me 2 SO was 0.2% or less. Me 2 SO at 0.2% by itself did not alter MTT reduction by PC12 cells. Moreover, Me 2 SO at 0.2% did not reverse or enhance the inhibition of MTT reduction by PC12 cells which was induced by aged amyloid- 25-35 or amyloid- 1-40 (data not shown).
To confirm that peptide that was prepared in Me 2 SO and said to be soluble was truly in a monomeric state compared with peptide that was aged in water and said to be aggregated, aliquots of each peptide preparation were subjected to SDS-PAGE/Coomassie Blue staining (Fig. 3C). The results showed that amyloid- 25-35 prepared in Me 2 SO migrated predominantly as a single band at the leading edge, and soluble amyloid- 1-40 migrated predominantly as a single band at ϳ4.5 kDa. Almost all of the amyloid- 25-35 prepared in Me 2 SO was retained at the top of the gel. A significant proportion of the amyloid- 1-40 preparation that had been aged in water was retained at the top of the gel or spread at slower electrophoretic mobility. These results are similar to those shown in many previous studies (9,10,12,14,16). It was therefore possible to call the peptides prepared in Me 2 SO predominantly soluble or predominantly monomeric and the same peptides aged in water predominantly aggregated. It was also possible to conclude that these peptide preparations were similar to those of previous studies in which amyloid- peptide neurotoxicity has been characterized. Taken together, these data suggest that soluble, predominantly monomeric amyloid- peptide is recognized by SEC-R and is nontoxic, whereas aggregated amyloid- peptide is poorly recognized by SEC-R and is toxic.
Previous studies have shown that human amylin, a peptide that is unrelated to amyloid- peptide in primary sequence but can form a -helical fibrillar structure, is similar to amyloid- peptide in cytotoxic effects (11,15). We examined the capacity for human amylin to compete for SEC-R binding and to mediate a cytotoxic effect in our system. The results showed that amylin does not compete for binding of peptide 105Y, even when prepared soluble in Me 2 SO, but was indeed as toxic as amyloid- peptide when aged in water or prepared soluble in Me 2 SO (data not shown).
We also compared amyloid- 25-35 with the original ␣1-AT peptide, peptide 105Y, in terms of SEC-R binding properties and toxicity (Fig. 4A). When prepared in Me 2 SO both are effective competitors for SEC-R binding (left panel) and are nontoxic (right panel). When aged in water, however, there is a marked reduction in SEC-R binding for amyloid- 25-35 (left panel), even though it is now quite toxic (right panel). When aged in water, there is no reduction in the capacity of peptide 105Y to compete for binding to SEC-R (left panel) and no development of toxic properties (right panel).
These results could be interpreted in two ways. The most likely explanation is that the toxic effect of amyloid- 25-35 requires that it assume a particular conformation that is not recognized by SEC-R and thus is not mediated by SEC-R. Because previous studies have shown that the carboxyl-terminal pentapeptides of peptide 105Y and soluble amyloid- 25-35 are essential for SEC-R binding (5, 7), there was still a remote possibility that toxicity required both the binding properties of the carboxyl-terminal pentapeptide and the signal transduction properties conferred by the amino-terminal domain of amyloid- 25-35, but not by the corresponding domain of peptide 105Y. To exclude this possibility we generated two chimeric am/␣1-AT peptides in which the amino-terminal domain of amyloid- was followed by the carboxyl-terminal domain of ␣1-AT (am/␣1-AT-GSNKGAFVFLM) and vice versa (␣1-AT/am-VKFNKPIIGLM). The results show that the ␣1-AT/am chimeric peptide competed effectively for SEC-R binding (Fig. 4A, left panel) but was nontoxic (right panel) when aged in water or prepared in Me 2 SO. The am/␣1-AT chimeric peptide did not compete for SEC-R binding when aged in water and was a poor competitor when prepared in Me 2 SO (left panel). However, the am/␣1-AT chimeric peptide was as toxic as amyloid- 25-35 when aged in water and was almost as toxic when prepared in Me 2 SO (right panel). To determine whether there was a correlation among loss of recognition by SEC-R, development of toxic properties, and formation of aggregates, we subjected peptide 105Y, ␣1-AT/am, and am/␣1-AT chimeric peptides to SDS-PAGE (Fig. 4B). The results show that the nontoxic peptides ␣1-AT/am and 105Y migrate predominantly as monomers, whereas the toxic peptide am/␣1-AT is retained predominantly at the top of the gel in a manner identical to that of the amyloid- 25-35 peptide. The am/ ␣1-AT chimeric peptide was the only peptide that was toxic when prepared in Me 2 SO. It was also retained predominantly at the top of SDS gels, whereas peptide 105Y Me 2 SO, amyloid- 25-35 Me 2 SO, and ␣1-AT/am Me 2 SO migrated predominantly as monomers (Fig. 4C). The results of this SDS-PAGE analysis are similar to results for typical predominantly monomeric and predominantly aggregated peptides in previous studies (9,10,12,14,16).
We also examined the amino-terminal and carboxyl-terminal domains of amyloid- 25-35 as isolated peptides (Fig. 4D). The results show that amyloid- 31-35 competes for binding to To exclude the possibility that these results were unique to the PC12 neuronal cell line and the MTT assay, we examined the same peptides for toxicity in primary cultures of murine cortical neurons using morphology and LDH release assays (Fig. 5). After 9 days in culture, separate cultures were incubated for 48 h in medium alone or medium supplemented with specific synthetic peptides. By morphologic criteria there was degeneration of neurons mediated by amyloid- 25-35 aged in water and by the am/␣1-AT chimeric peptide aged in water, but not by medium alone, by the ␣1-AT/am chimeric peptide aged in water, peptide 105Y aged in water or amyloid- 25-35 prepared in Me 2 SO (panels A-F). By LDH assays, there was neuronal cell death mediated in a concentration-dependent manner by amyloid- 25-35 aged in water and by the am/ ␣1-AT chimeric peptide aged in water, but no cell death in response to amyloid- 25-35 prepared in Me 2 SO, peptide 105Y aged in water, or the ␣1-AT/am chimeric peptide aged in water (panel G). The concentration of peptide required for toxic effects (half-maximal at ϳ20 M) is very similar to that reported previously by other laboratories using morphology and LDH assays in primary culture (8, 10 -16). These results pro-vide further evidence for the high degree of correlation between the MTT assay in PC12 cells and more conventional assays for toxicity in neurons in primary culture. In fact, the MTT assay is apparently more sensitive than either morphologic or LDH assays. In all three assay systems the am/␣1-AT chimeric peptide has the most potent toxic effect on neuronal cells.
Taken together, these data are most likely explained by the capacity of amyloid- 25-35, when aged in water, and am/␣1-AT, when aged in water or prepared in Me 2 SO, to aggregate into a conformation that is not recognized by SEC-R and, thus, not delivered into the endocytic pathway for catabolism. This conformation also confers cytotoxic properties on the peptide. The amino-terminal domain, amyloid- 25-30, is critical but not sufficient for conferring the aggregating/toxic properties.
To understand further the differences in toxic properties of ␣1-AT-derived peptide 105Y and amyloid- 25-35, we again examined the sequences of these two peptides (Fig. 4A, top). First, compared with the native ␣1-AT sequence (shown in the top line), there are two substitutions in peptide 105Y: Phe at amino acid 372 was replaced by Tyr for iodination, and Met at amino acid 372 was replaced by Ile for ease of synthesis. However, neither of these substitutions affected the properties of the peptide because a new peptide, which was identical to the ␣1-AT sequence in the corresponding region, had identical properties in binding, toxicity, and SDS-PAGE assays (data not shown). Second, compared with amyloid- 25-35 (Fig. 4A, top), peptide 105Y has five additional amino acids (SIPPE) at its amino terminus. These additional amino acids do not affect the properties of peptide 105Y because peptide 105BC, which is identical to peptide 105Y except that it is missing the five amino-terminal amino acids (Fig. 6, top), had properties identical to those of peptide 105Y in binding, toxicity, and SDS-PAGE assays ( Fig. 6 and data not shown). Peptide 105BC prepared in Me 2 SO or aged in water competes for binding to SEC-R, is nontoxic, and migrates as a monomer in SDS-PAGE.
Next, we compared the sequences in the amino-terminal domains of peptides 105BC and amyloid- 25-35 (Fig. 6, top) and noticed an NK sequence in both. In amyloid- 25-35 it is separated from the carboxyl-terminal pentapeptide by 2 amino acids, GA, whereas in ␣1-AT it is separated by one amino acid, P. We examined the effect of swapping these two domains on SEC-R binding, toxicity, and aggregation (Fig. 6). In the case of ␣1-AT, the insertion of GA for P (swap 2) did not affect its properties. The swapped peptide competed effectively for SEC-R binding (left panel), was not toxic (right panel), and migrated as a monomer on SDS-PAGE (data not shown). However, for amyloid- 25-35, the substitution of P for GA (swap 1) made the peptide an effective competitor for SEC-R (left panel), abrogated toxicity (right panel), and it migrated as a monomer when in Me 2 SO or aged in water (data not shown). These data indicate that the GA sequence at amyloid- 29 -30 is important for the toxic/aggregating properties of amyloid- 25-35. We also examined the importance of the II sequence at amyloid- 31-32. Replacement of these two residues by TT (amyloid- 25-35 TT) had interesting effects. There was a marked reduction in both competitive binding efficacy of amyloid- 25-35 TT (left panel) and in its toxic properties (right panel) whether prepared in Me 2 SO or aged in water. In SDS-PAGE analysis, there was no evidence for aggregation of amyloid- 25-35 TT even when aged in water (data not shown). These data suggest that amyloid- 31-32 II is important for SEC-R binding of peptide dissolved in Me 2 SO and important for toxic/aggregating properties of peptide aged in water.
Next, we examined the effect of deletions on the cell surface binding properties and toxicity of amyloid- peptide (Fig. 7A). The results show that deletion of two or three carboxyl-terminal residues, as exemplified by peptides amyloid- 25-33 and Yam 22-32, is associated with loss of both SEC-R binding and toxicity. This effect is not due to reduction in the length of the peptide as shown by peptide Yam 22-32, in which there is deletion of three carboxyl-terminal residues, but inclusion of four additional residues, derived from the sequence of amyloid- peptide, at the amino terminus. This peptide does not compete for binding to SEC-R and does not have toxic effects. Deletion of two amino-terminal residues in peptide amyloid- 27-35 has no effect on binding to SEC-R but is associated with loss of toxic effects. Amyloid- 27-35 prepared in water and aged to optimize aggregate formation still competes for binding to SEC-R (left panel), has minimal toxicity (right panel), and migrates as a monomer on SDS-PAGE (data not shown). These data suggest that deletion of residues at either end of amyloid- 25-35 prevents aggregate formation and toxicity, but only deletion of the carboxyl-terminal residues, within the SEC-R binding pentapeptide domain, affects SEC-R binding.
We also examined the effect of substitutions on the cell surface binding properties and toxicity of amyloid- peptide (Fig. 7B). The results show that substitution of alanine for the carboxyl-terminal methionine (amyloid- 25-35 35A) reduces competitive binding efficacy, but substitution of alanine for synthetic peptides as specified on the top right. Each point represents the mean Ϯ S.E. of four independent cultures expressed as percentage of complete neuronal death induced by 500 M NMDA. LDH release by cultures incubated with medium alone (controls) was considered 0% neuronal death. Values Ͼ100% induced by the am/␣1-AT chimeric peptide at high concentrations probably represent additional injury to glial cells. DMSO, dimethyl sulfoxide. These results are again consistent with the importance of the carboxyl-terminal domain, and particularly the carboxyl-terminal methionine, in recognition by SEC-R (2, 5). There is a marked reduction in SEC-R binding of all these substituted peptides when they are prepared in water and aged (left panel). All three substituted peptides retain some toxicity when they are prepared in water and aged (right panel), and this correlates with marked slowing of electrophoretic mobility on SDS-PAGE (data not shown). The rank order of cytotoxic potency is amyloid- 25-35 Ͼ amyloid- 25-35 25A Ͼ amyloid- 25-35 26A Ͼ amyloid- 25-35 35A. Toxicity is reduced markedly when these peptides are prepared soluble in Me 2 SO (right panel), and this correlates with migration as monomers on SDS-PAGE (data not shown). These data again show that toxicity correlates most closely with aggregating ability as determined by SDS-PAGE. The carboxyl-terminal methionine residue is critical for SEC-R binding of amyloid- 25-35. Substitution of the carboxyl-terminal methionine and the amino-terminal glycine and serine residues by alanine has minimal effects on aggregating and toxic properties.
Finally, we examined the SEC-R binding and toxicity of All-D-amyloid- 25-35 (Fig. 8). The results show that D-amyloid- 25-35 does not compete for binding to SEC-R in either Me 2 SO or water but remains a potent toxin when aged in water and, to a lesser extent, when prepared in Me 2 SO. Substitution of the II sequence of D-amyloid- 25-35 residues 31-32 with TT completely abrogated toxicity. The toxicity of All-D-amyloid- 25-35 peptide correlated exactly with aggregation as determined by SDS-PAGE (data not shown). Results of studies in murine cortical neurons using morphologic and LDH assays also indicated that the All-D-amyloid- 25-35 peptide was toxic, although somewhat less toxic than the L-enantiomer. Substitution of the II sequence at residues 31-32 with TT completely abrogated toxicity (data not shown).
These data provide strong evidence that SEC-R does not mediate the toxic effect of amyloid- 25-35. Rather, the toxic effect is conferred by amyloid- peptide, which is capable of forming aggregates and, in this state, is no longer recognized by SEC-R. Although the D-enantiomer of amyloid- 25-35 is ca- pable of conferring toxic/aggregating properties, there is sequence specificity required for this effect in that replacement of amyloid- 31-32 II by TT completely reverses these properties.
Effect of Blocking SEC-R on the Toxic Properties of Amyloid- 25-35-Next, we examined the effect of blocking SEC-R with nontoxic ligand peptide 105Y on the cytotoxicity of aggregated amyloid- peptide. Initially, we found that preincubation and/or coincubation of PC12 cells with peptide 105Y did not decrease the toxic effect of amyloid- 25-35 aged in water. If anything, peptide 105Y potentiated the toxic effect of amyloid- 25-35 (data not shown). To provide further evidence for this potentiation effect, we used the MTT assay (Fig. 9) on PC12 cells that were preincubated with peptide 105Y, irrelevant peptide, or no peptide. The PC12 cells were then incubated with the am/␣1-AT chimeric peptide prepared in Me 2 SO or the amyloid- 25-34A peptide aged in water. Each of these peptide preparations is suboptimal for toxicity and, therein, would allow potentiation of toxicity to be detected. The results showed that preincubation with peptide 105Y, but not irrelevant control peptide, potentiated the toxicity in each case even though it had no toxic effect by itself. The mechanism for this potentiation is not yet known.
Binding to SEC-R in an Amyloid- Peptide-resistant PC12 Cell Line-Behl et al. (11) have shown that PC12 cells become resistant to the toxic effect of amyloid- peptide if they are grown in the presence of aggregated amyloid- 25-35 for a number of passages. We examined the possibility of establishing such a cell line and determining its SEC-R binding characteristics. PC12 cells were incubated with 20 M amyloid- 25-35 aged in water for 10 passages. An aliquot of these cells was then grown in the absence of amyloid- 25-35 for one passage. MTT assays showed that these cells were, indeed, resistant to the toxic effects of aggregated amyloid- 25-35 (Fig. 10A). The same cells were then subjected to SEC-R binding studies (Fig. 10B). In the amyloid--resistant subclone there was specific and saturable binding of 125 I-peptide 105Y with a plateau reached at 60 nM (left panel). Binding of 125 Ipeptide 105Y to the resistant subclone was then compared with that of the parent cell line (right panel). The results show that SEC-R binding does not decrease in the PC12 cell line that has become resistant to the toxic effect of aggregated amyloid- 25-35. In fact, Scatchard plot analysis indicates that there are ϳ1.1 ϫ 10 6 SEC-R molecules/cell in the amyloid--resistant subclone, 2.5-3-fold higher than the 4.33 ϫ 10 5 SEC-R mole-cules/cell in the parental PC12 cells. The K d for binding of 125 I-peptide 105Y in the amyloid--resistant PC12 subclone was 48.3 nM compared with 43.7 nM for the parental PC12 cells (7). There was no change in the specificity of SEC-R binding in the amyloid--resistant subclone of PC12 in that binding of 125 I-peptide 105Y to these cells was blocked by amyloid- 25-35 prepared in Me 2 SO, but not by amyloid- 25-35 aged in water (data not shown).
We also examined the effect of human amylin on cytotoxicity in the amyloid--resistant subclone of PC12 compared with the parent PC12 cell line (Fig. 10C). Although amylin had a potent toxic effect on the parent, it did not alter the subclone at all. Taken together, these data provide initial evidence that the development of resistance to amyloid- cytotoxicity is a general rather than a specific phenomenon. DISCUSSION A number of studies have shown that amyloid- peptide is toxic to neurons and that its toxic properties may be fundamentally important in the common final pathway for neuronal degradation in Alzheimer's disease (8,9). Moreover, the amyloid- 25-35 region and an aggregated state appear to be required for full neurotoxic potential (9 -16). In this study, we examined the role of SEC-R in the neurotoxic effects of amyloid- peptide. SEC-R is expressed on many cell types, including neurons and glial cells. It mediates endocytosis and degradation of soluble amyloid- peptide by recognizing the amyloid- 25-35 region in a sequence-specific manner (7). Using an assay for cellular redox activity which is indicative of amyloid- toxicity and an assay for DNA fragmentation in the neuronal cell line PC12 as well as conventional morphologic and LDH release assays in primary cultures of murine cortical neurons, our results confirm previous studies indicating that amyloid- peptide must be aggregated to induce its neurotoxic effects. SEC-R does not recognize amyloid- peptides that are aggregated and toxic and, therefore, cannot be implicated in mediating the neurotoxicity of amyloid- peptide. SEC-R does recognize the same peptides when in a soluble, nontoxic state.
Our conclusions are based on several major results. First, aggregated amyloid- 25-35 is cytotoxic in two cell lines that do not express SEC-R, or at most, have negligible levels of SEC-R expression (Fig. 2). Second, amyloid- 25-35 and amyloid- 1-40 are recognized by SEC-R, are nontoxic, and migrate in SDS-PAGE as monomers when prepared in Me 2 SO, but are not recognized by SEC-R, are toxic, and are aggregated when aged at 37°C for 5 days in water (Fig. 3). Third, the D-enantiomer of amyloid- 25-35 is not recognized by SEC-R when prepared in either Me 2 SO or water but is toxic when prepared as an aggregate in water (Fig. 8).
Substitutions, deletions, and swapping of domains within the amyloid- 25-35 peptide also demonstrate multiple examples of dissociation of SEC-R binding from toxic properties. For example, substitution of the amino-terminal domain of amyloid- 25-35 with the corresponding domain of ␣1-AT results in a peptide, the ␣1-AT/am chimeric peptide, which is now well recognized by SEC-R, is no longer toxic, and behaves as a monomer in SDS-PAGE even when aged in water at 37°C for 5 days (Figs. 4 and 5). The opposite, the am/␣1-AT chimeric peptide, is poorly recognized by SEC-R because it aggregates and induces a potent toxic effect on PC12 cells. A second example, the ␣1-AT peptide 105Y, is soluble and behaves as a monomer in either Me 2 SO or aged in water. It binds well to SEC-R but is not toxic (Figs. 4 and 5). Third, deletion of the amino-terminal domain of amyloid- 25-35 (peptide IIGLM) minimally reduces SEC-R binding but completely abrogates toxicity (Fig. 4D). Fourth, swapping of the central glycinealanine domain of amyloid- 25-35, amyloid- 29 -30, for the corresponding domain of ␣1-AT, proline 369, results in a peptide that is recognized by SEC-R but nontoxic and migrates as a monomer in SDS-PAGE (Fig. 6). Fifth, deletion of amyloid- 25-26 results in a peptide, amyloid- 27-35, which still competes for SEC-R binding but is nontoxic and migrates as a monomer on SDS-PAGE (Fig. 7). Sixth, replacement of amyloid- 35 methionine by alanine markedly reduces SEC-R binding but only minimally affects toxicity. The effect of this substitution is highly specific since replacement of amyloid- 25 or 26 by alanine has minimal effects on SEC-R binding or toxicity (Fig. 7).
Several other experimental results show that SEC-R does not recognize aggregated amyloid peptide and does not mediate its cytotoxic effect. Blocking of SEC-R with the nontoxic ligand peptide 105Y does not block the toxic effect of aggregated amyloid- 25-35 (Fig. 9). In fact, it potentiated the toxic effect of amyloid- peptide preparations. Finally, when compared with the parental PC12 cell line there was an increase in the number of SEC-R molecules/cell for a subclone of PC12 cells which had developed resistance to the cytotoxic effect of amyloid- 25-35 (Fig. 10).
The results of this study also provide further evidence that the neurotoxic effects of amyloid- peptide depend on its capacity to form aggregates. There is excellent correlation between toxicity as defined by the MTT assay and migration as an aggregate in SDS-PAGE. Moreover, acquisition of toxic and aggregating properties, as defined by these assays, is closely correlated with loss of recognition by SEC-R. By virtue of the correlation between these different types of assays, the results also provide internally consistent information about the structural requirements for the toxic/aggregating properties of amyloid- peptide. For example, deletion of two amino acids at either the amino or carboxyl terminus is associated with a loss of toxic/aggregating properties (Fig. 7). This is not simply an issue of length, however, since Yam 22-32 does not aggregate or have toxic effects. Replacement of amyloid- 25, 26, or 35 with alanine is associated with a mild reduction in toxic/aggregating properties. Second, replacement of amyloid- 31-32 II with TT is associated with loss of toxic/aggregating properties (Fig. 6). Third, replacement of amyloid- 25-30 (GSNKGA) with the ␣1-AT sequence VKFNKP is associated with a loss of aggregation, whereas replacement of amyloid- 31-35 (IIGLM) with the ␣1-AT sequence FVFLM does not affect toxic/aggregating properties (Figs. 4 and 5). In fact, the am/␣1-AT chimeric peptide has an even greater tendency toward aggregation/toxicity than amyloid- 25-35 itself. The amyloid- 29 -30 GA sequence may be particularly important. Replacement by P, which is in the corresponding position in ␣1-AT, abrogates toxic/aggregating properties. However, insertion of GA in the middle of the corresponding ␣1-AT sequence did not confer new aggregating/toxic properties on it. The structural requirements for the toxic/aggregating properties of amyloid- peptide defined by our data using the MTT assay in PC12 cells are similar to those defined by morphometry and LDH assays in primary cultures of neurons in this study (Fig. 5) and in the studies of FIG. 9. Effect of preincubation with peptide 105Y on toxicity of amyloid- peptide preparations. PC12 cells were preincubated for 1 h with no peptide, peptide 105Y, or irrelevant peptide control (IL-8 receptor 9 -23 peptide) to achieve a final concentration of 80 M. Am/␣1-AT chimeric peptide prepared in Me 2 SO or amyloid- 25-34A aged in water was then added to achieve a final concentration of 8 M or 0.8 M as indicated. In separate cell aliquots no peptide was added at this time. After 2 h at 37°C the cells were then subjected to the MTT assay exactly as described in the legend to Fig. 1. Results of triplicate determinations are reported as mean Ϯ 1 S.D. for percent toxicity. 0% toxicity represents the value in the absence of any potential toxin and 100% toxicity the value in cells which had been lysed in phosphate-buffered saline, 0.5% Triton X-100. A paired Student's t test was used to show that the difference between peptide 105Y and no peptide or between peptide 105Y and peptide control was statistically significant to p Ͻ 0.001 in each case.
Pike et al. (16). Several lines of evidence suggest that these only represent the requirements for formation of the aggregated/ fibrillar conformation and not for the toxic interaction with cells. For example, the observation that All-D-amyloid- 25-35 forms aggregates and elicits a toxic effect implies that the toxic interaction with cells is not sequence-specific. Lorenzo et al. (15) showed similar toxic effects in neurons and pancreatic islet cells using amylin, a peptide with a completely different sequence but with a tendency to aggregate. Our data also suggest that the toxic effect of amyloid- peptide is not cell type-specific. So far there is evidence for toxic effects in every cell type that we have exposed to aggregated amyloid- 25-35.
Using model cell systems and conditions designed to exaggerate the soluble and aggregated states of amyloid- peptide in this and our previous study (7), we could clearly distinguish the fate of soluble and aggregated forms of this peptide. The soluble form of amyloid- peptide is recognized by SEC-R and delivered by endocytosis to an intracellular vesicular compart- ment for degradation. The same peptide, when aggregated, is not recognized by SEC-R but can interact with the plasma membrane of the target cell in a relatively specific manner to elicit a completely different biologic effect, apoptosis. However, these studies do not address the relationship between soluble and aggregated forms of amyloid- peptide and SEC-R under physiologic and pathophysiologic conditions, which are likely to be far more complex. In future studies, it will be essential to provide quantitative information about the conversion of soluble amyloid- peptide to its aggregated form, about the relative reversibility of this conversion, about the relationship of this conversion to the kinetics (on-rate and off-rate) with which soluble amyloid- peptide binds to SEC-R, and about the relationship of this conversion to the kinetics and specificity with which aggregated amyloid- peptide interacts with the target cell membrane in a SEC-R-independent manner. Finally, it will be essential to consider other factors such as apolipoprotein E (24,25), ␣1-antichymotrypsin (26,27), transthyretin (28), zinc (29), and the nonamyloid components of Alzheimer's disease amyloid (30), which are likely to regulate these relationships in physiologic and pathophysiologic states in vivo. | v3-fos-license |
2018-12-11T05:42:57.060Z | 2009-07-02T00:00:00.000 | 97527218 | {
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} | pes2o/s2orc | Novel 1-hydroxy-1,1-bisphosphonates derived from indazole: synthesis and characterization
Bisphosphonates (BPs) are an important class of drugs used in the treatment of abnormal calcium metabolism diseases. The first syntheses of bisphosphonates derived from indazole, substituted at the N-1, N-2 and C-3 positions are reported. The 1-hydroxy-1,1-bisphosphonates were synthesized from the corresponding carboxylic acid or acyl chloride compounds, by two different methods. These BPs have a side chain with different lengths ((CH 2 ) n , n = 0-5) between the indazole ring and the bisphosphonate group
Introduction
Bisphosphonates (BPs) are an important class of drugs known by their broad spectrum of therapeutic applications in the treatment of diseases characterized by abnormal calcium metabolism, such as hypercalcemia, osteoporosis and tumor-associated osteolysis. 1 BPs were first developed as pharmaceuticals in the mid-1960´s and have been used as an effective treatment for Paget's disease. 2 Since then, many BPs were synthesized and made commercially available due to their value in the treatment of disorders of bone mineral. 3These compounds have high affinity for calcium and therefore to target the bone mineral, where they appear to be internalized selectively by bone-reabsorbing osteoclasts and induce their apoptosis. 4BPs offer several advantages in treating osteoporosis since they are bone-tissue specific, have minimal side effects, have no known risk of carcinogenesis and have antiresorptive efficacy equivalent to, or even greater than estrogens. 5n addition, functional BPs showed anti-tumour properties which includes activity against bone metastases secondary to breast cancer 6 and prostate cancer, 7 inhibition of cell proliferation, invasion and adhesion to bone. 8They also showed to have applications in the inhibition of angiogenesis 9 and as anti-inflammatory agents. 10BPs are also useful as novel ligands in welldefined radioactive metal complexes that can be used in magnetic resonance imaging and imagiology, scintigraphy and radiotherapy applications. 11Another property of these compounds under investigation is their chelating efficiency toward metal ions; several laboratories are developing BPs as chelating agents for the treatment of human metal intoxications. 12hemically, BPs are structural analogues of pyrophosphonates in which the oxygen atom of P-O-P is replaced by a carbon atom, resulting in P-C-P bonds which are stable toward heat and chemical reagents, completely resistant to enzymatic degradation, and usually have low toxicity. 1,13he biological activities of these compounds are determined by the nature of the alkyl moiety bound to the bisphosphonic structure as well as the functional groups located on the alkyl chain, with the nitrogen-containing homologues, such as risedronate and zoledronate, being amongst the most potent BPs in the treatment of several bone diseases.The most potent BPs contains 1 or 2 nitrogen atoms in a heteroaromatic moiety linked via a small side chain to a geminal bisphosphonate group.Some of the heterocyclic-BPs are drugs commercially available, such as Actonel ® (risedronate sodium) and Zometa ® (zoledronic acid). 14,15The BPs usually present a very low oral bioavailability due to the high hydrophilicity of their phosphonate groups. 16ndazole derivatives are pharmacologically important compounds and the indazole ring system forms the basis of several drug molecules. 17However, the chemistry of indazole remains less studied in comparison with other heteroaromatic compounds, such as indole or benzimidazole.
The indazole ring has two nitrogen atoms, like the 3 rd generation BPs, which can lead to an increased effect of BPs and can be functionalized with high selectivity at different positions.The length of the side chain and the different positions at the indazole ring, where it could be bonded, can afford a large number of indazolebisphosphonates, presenting a promising field to achieve new BPs with biological and therapeutical properties.
To the best of our knowledge, no BPs derived from indazole were synthesized.The aim of this work is to obtain new BPs derived from indazole with high potential biological/therapeutical activities.
Here, we report the synthesis and structural characterization of new indazole BPs substituted at different C-or N-positions of indazole rings, and with a side chain with different lengths (CH 2 ) n n=0-5 from the bisphosphonate function to the nitrogen or carbon atom of the ring.
Synthesis
The new 1-hydroxy-1,1-indazolebisphosphonates were prepared from the corresponding carboxylic acid derived from indazole, substituted at N-1, N-2 and C-3 positions, with a side chain with different length 1a-e, 4a-e, 6 and 8.The synthesis of carboxylic acids 1a-e and 4a-e, and their characterization were reported previously. 18everal methods for the synthesis of 1-hydroxy-1,1-bisphosphonates have been reported. 19he most used method involves the reaction of a carboxylic acid with phosphorus trichloride and phosphorous acid or phosphoric acid, followed by acidic hydrolysis (Method A). 15,20 Recently, a modified Arbuzov reaction method was proposed by Lecouvey et al. 21This method involves the reaction of an acyl chloride with tris(trimethylsilyl)phosphite, followed by methanolysis (Method B). 21-Hydroxy-1,1-indazolebisphosphonate derivatives 2a-e were prepared in moderate yield from the corresponding carboxylic acid derived from an indazole substituted at the N-1 position 1a-e (n=1-5), by treatment with a mixture of phosphoric acid and phosphorus trichloride, followed by acid hydrolysis (Table 1).The separation and purification of BPs were done by precipitation in acetone and methanol, being always a difficult and tedious process, as previously described by other authors.15 In the case of the compound 2a, with a single methylene group, the yield of the reaction was very low.So, the Lecouvey's alternative method (Method B) was used.The acyl chloride 3a was prepared in situ, by reaction of the carboxylic acid 1a with thionyl chloride, which showed to be very unstable.The acyl chloride 3a reacted with tris(trimethylsilyl)phosphite, followed by methanolysis, to afford BP 2a, in 71% yield (Scheme 1).
Scheme 1
The 1-hydroxy-1,1-indazole-bisphosphonate derivatives 5a-e were also prepared by reaction of the corresponding carboxylic acid derived from indazole substituted at N-2 position 4a-e (n = 1-5) with phosphorus trichloride and phosphoric acid, followed by acidic hydrolysis (Method A).However, the reactions afforded the BPs in low yields (exception for BP 5e, with n=5) (Table 2).In order to increase the yields of these syntheses, the Lecouvey's method (Method B) was used and the synthesis of compound 5b afforded these BP in 77% yield, but compounds 5a and 5c were always isolated in low yields.The lower solubility of compounds 4a and 4c, in the solvent used to prepare the corresponding acyl chloride and the lower stability of the acyl chloride should explain the low yield of BP 5a and BP 5c (Table 2).1-Hydroxy-1,1-indazolebisphosphonate 7, prepared from the carboxylic acid 6, substituted at the indazole C-3 position, was synthesized following the method of Lecouvey (Method B). 21The reaction of commercially available 1H-indazole-3-carboxylic acid 6 with SOCl 2 , to produce in situ the corresponding acyl chloride, followed by reaction with tris(trimethylsilyl)phosphite and hydrolysis, afforded the BP 7, as white powder, in 67% yield (Scheme 2).i) SOCl 2 (4 eq.) To increase the length of the alkyl side chain at the C-3 position, the carboxylic acid derivative 8 was synthesized according to literature procedures, from o-nitrobenzaldehyde, malonic acid and ammonium acetate in acetic acid. 22BP 9 was obtained by Lecouvey's method (Method B) from the corresponding carboxylic acid with 16% yield (Scheme 3).
Spectroscopic characterization
Compounds 2a-e, 5a-e, 7 and 9 were fully characterized by NMR (including bidimensional techniques), IR spectroscopy, mass spectrometry (low and high resolution) and elemental analysis.
The BPs were submitted to Electron Impact Ionization (EI) Mass Spectrometry, but most did not show the molecular ion.Fast Atom Bombardment (FAB) or Electrospray Ionization (ESI) methods were used to show the molecular ion of all BPs, which confirmed their proposed molecular formulae.The mass spectra showed characteristic fragment ions corresponding to the loss of H 2 O and H 3 PO 3 fragments. 23he IR spectra of indazolebisphophonates showed the disappearance of the strong C=O stretching band from the carboxylic acid (starting material) and the appearance of two strong characteristic bands at approximately 1250-1100 and 1100-900 cm -1 , due to the ν(P=O) (1200-1160 cm -1 ), ν(P-OH) (≈ 1000 and ≈ 925 cm -1 ) and δ(POH) (around 1080 cm -1 ) bands of bisphosphonate group. 24A broad band with a maximum near 3400 cm -1 is attributed to the O-H stretching band, in agreement with the presence of the bisphosphonate group.C-H(Ar) and CH 2 stretching bands are observed over this ν(O-H) band.The ν(O-H) band also overlaps with the very large and weak ν(PO-H) and δ(POH) bands, with maximum around 2600-2800 cm -1 . 24he bisphosphonate structure of BPs was readily identified by analysis of NMR data.The unambiguous identification of isomers of indazolebisphosphonates derivatives substituted at N-1, N-2, and C-3, was carried out by 1 H and 13 C NMR spectroscopy, including DEPT and APT, and two-dimensional NMR techniques (HETCOR, COSY, HSQC and HMBC).
The 1 H NMR and 13 C NMR spectra of N-1 and N-2 isomers of indazolebisphosphonates 2a-e and 5a-e are sufficiently different to be used as diagnostic tools for the position of substitution.The main resonances in the 1 H NMR spectra of BPs 2a-e are: (i) the resonance of CH 2 protons, which appears at δ 1.23-2.35ppm, (ii) the resonance of NCH 2 protons, which appears at δ 4.36-4.85ppm, (iii) four resonances in the region of δ 7.11-7.75ppm, relative to the aromatic ring protons, usually as two one proton doublets (4-H and 7-H) and two one proton triplets (5-H and 6-H), (iv) a singlet (or a doublet, with a very small coupling constant, J 0.6 Hz) at δ 7.99-8.12,which corresponds to the 3-H proton.
The 1 H NMR spectra of BPs 5a-e also showed four resonances in the region δ 7.01-7.68ppm, attributed to the aromatic ring protons, usually as two one proton doublets (4-H and 7-H) and two one proton triplets (5-H and 6-H); these resonances are generally 0.10 ppm upfield from the corresponding signals of the BPs 2a-e.The 3-H proton is observed as one proton singlet or doublet (J 0.6 Hz) at δ 8.30-8.42ppm, which are generally 0.30 ppm downfield from the signals of the BPs 2a-e.Both BPs 2a and 5a (all with n=1) present the 3-H proton resonance 0.12 ppm downfield relatively to the BPs 2b-e and 5b-e (with aliphatic side chains with more than one methylene group).Compounds 2a and 5a showed a triplet ( 3 J H,P ≈10 Hz), at 4.85 and 4.90 ppm, respectively, due to the coupling of NCH 2 protons with two phosphorous atoms, supporting the proposed structures of two equivalent phosphonate groups attached to the same carbon.
The indazoleBPs 7 and 9, derived from indazole substituted at C-3, showed four resonances in the region of δ 7.01-7.28ppm relative to the aromatic ring protons, as two one proton doublets (4-H and 7-H) and two one proton triplets (5-H and 6-H).The BP 7, which has the 1-hydroxy-1,1-bisphosphonate group bonded to the pyrazole ring of the indazole, with no aliphatic side chain, present the doublet, relative to H-4, 0.5 ppm downfield relative to indazoleBP 9 (with a methylene group at the side chain).The 1 H NMR spectrum of BP 9 has a CH 2 triplet ( 3 J H,P 14 Hz) at δ 3.57 ppm, which confirmed the presence of two equivalent phosphonate groups attached to the same carbon. 13C NMR spectroscopy also confirms the presence of the bisphosphonate group.The appearance of a quaternary carbon triplet (disappearing in DEPT-135 13 C NMR spectra) at δ 71.1-74.3ppm (with 2 J C,P 143-146 Hz) supported the proposed structures with two phosphonate groups attached to the same carbon (P-COH-P) (Tables 3-5). 13C NMR is also the best spectroscopic method for assigning the position of substitution, since the C-3, C-7 and C-7a showed different chemical shifts between the N-1, N-2 and C-3 isomers (Table 3-5).In N-1 substituted indazoleBPs spectra, C-3 was generally shifted downfield 10 ppm, and C-7 and C-7a were shifted upfield, relatively to the corresponding carbons in N-2 substituted indazoleBPs.The C-3 substituted isomers (BPs 7 and 9) present C-7 and C-7a with similar chemical shifts to the N-1 isomers but the C-3 quaternary carbon atom was observed further downfield, at approximately 8 ppm.The assignment of the C-3 isomers was supported by HSQC and HMBC spectra (Table 5).The connectivities found in these spectra allowed the full assignment of proton and carbon atoms, including the three quaternary carbon resonances.We may note that C-3 and C-7a have similar chemical shifts, but C-3, relative to C-7a, is observed upfield in BP 7 and downfield in BP 9.
The proton-decoupled 31 P NMR spectra of indazoleBPs 2, 5, 7 and 9 showed a single signal in the range of 15.4-21.1 ppm, which confirmed that two phosphorus atoms are magnetically equivalent.The chemical shifts of phosphorus atoms are similar between the corresponding isomers.The lower chemical shift is observed for indazoleBP 7, with no aliphatic side chain, with the phosphorus atoms connected to a carbon atom directly bonded to the aromatic indazole ring.The chemical shift generally increases with the increasing number of methylene groups in side chain.* These assignments may be reversed.
Conclusions
We report the first synthesis of 1-hydroxy-1,1-indazolebisphosphonates substituted at N-1, N-2 and C-3 positions of indazole ring, with the side chain with different (CH 2 ) n groups (n=1-5 for N-1 and N-2 isomers and n=0,1 for C-3 BPs), in moderated to good yields, starting from their corresponding carboxylic acids or acyl chlorides.In order to increase the synthetic yields, two different methods were used.Despite the difficulty in the separation and purification of these bisphosphonates, they were fully characterized using the usual spectroscopic methods, especially NMR spectroscopy, including two dimensional NMR techniques.NMR spectroscopic characterization allowed the assignment of 1 H-and 13 C-atoms of different BPs.The presence of a quaternary carbon triplet confirmed the presence of the bisphosphonate group with two equivalent phosphorous atoms bonded to the same carbon atom.
This work also showed that NMR spectroscopy allowed the easy identification of the N-1, N-2 and C-3 isomers since they showed spectra with different chemical shifts, especially the C-3, C-7 and C-7a 13 C NMR signals.The N-1 indazole BPs showed C-3 carbon atom generally shifted downfield (≈10 ppm) relative to the corresponding carbon atom in N-2 indazole BPs, while C-7 and C-7a were shifted upfield.The C-7 and C-7a carbon atoms of C-3 substituted BPs (BPs 7 and 9) have similar chemical shifts relative to N-1 isomers but the C-3 quaternary carbon atom was observed further downfield, at approximately 8 ppm.Connectivities in HMBC spectra of BPs 7 and 9 also showed that those compounds present inversion in the chemical shift of C-3 and C-7a quaternary carbon atoms.
These synthesized BPs will be submitted to in vitro and in vivo studies to assess their potential therapeutic applications (studies currently in progress).
Experimental Section
General Procedures.NMR spectra were recorded on Bruker AMX 300 and on a Bruker Avance II 300 ( 1 H 300 MHz, 13 C-75 MHz, 31 P 121 MHz) and on a Bruker Avance II 400 ( 1 H 400 MHz, 13 C-100 MHz, 31 P 162 MHz) spectrometers.Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. 1 H-and 13 C NMR chemical shifts were assigned using DEPT and APT sequences, and bidimensional COSY, HETCOR, HSQC and HMBC techniques.Assignments were made by comparison of chemical shifts, peak multiplicities and J values, and were supported by bidimensional heteronuclear HMBC and HSQC correlation techniques.Infrared spectra were recorded on a Perkin Elmer FT-IR 1725xIR Fourier Transform spectrophotometer using KBr discs.The bands are quoted in cm -1 .Low resolution and high resolution (HRMS) mass spectra analyses were performed at the 'C.A.C.T.I. -Unidad de Espectrometria de Masas' at the University of Vigo, Spain, on a VG AutoSpect M, MicroTOF (Bruker Daltonics) or APEX-Q (Bruker Daltonics) instrument.Elemental analysis were performed on a CE instrument EA 1110CHNS O and a Fisons EA-1108 elemental analyzer.Melting points were determined on a Reichert Thermovar melting point apparatus and are uncorrected.All the reactions involving air sensitive reagents were performed under an atmosphere of dry nitrogen and all solvents were degassed before use.All solvents were distilled under a nitrogen atmosphere.THF were distilled from sodium benzophenone ketyl.CHCl 3 was distilled from calcium hydride.1H-Indazole-3-carboxylic acid 6 is commercially available (Aldrich).Carboxylic acids 1a-e and 3a-e derived from indazole substituted at N-1 or N-2 position, with side chains with different lengths (n=1-5), were prepared as already reported in the literature. 18 from indazole substituted at C-3 were synthesized in a two step synthesis according to reported synthetic procedures. 22
General procedure 1 (Method A)
A mixture of a carboxylic acid (1 eq.), H 3 PO 4 (85%) (2 eq.) and chlorobenzene was heated to 100-110 °C with stirring.After all solids were dissolved, PCl 3 (3 eq.) was added slowly and the reaction mixture was vigorously stirred at 100-110 °C for 5 h.After cooling, chlorobenzene was decanted and 9 N HCl was added to the residue.The resulting solution was refluxed for 3 h and the solvent was removed under reduced pressure.Acetone and methanol were added to precipitate the 1-hydroxy-1,1-indazolebisphosphonate as a white powder, which was washed with acetone and dried.
General procedure 2 (Method B)
A mixture of a carboxylic acid (1 eq.) in CHCl 3 and thionyl chloride (4 eq.) was kept under reflux for 2 h.Solvents were removed under reduced pressure to give the corresponding acyl chloride, which was immediately used without further purification.The crude acyl chloride was dissolved in dry THF and tris(trimethylsilyl)phosphite (2 eq.) was added.Then, the mixture was stirred at room temperature for 30 min.The excess solvent was removed under reduced pressure and methanol was added and the mixture was stirred for 1 h.After solvent removal under reduced pressure, the residue was washed with ethyl ether and precipitated with acetone.
Table 2 .
Preparation of bisphosphonates 5a-e derived from indazole substituted at N-2 position
Table 5 .
13C NMR data of compounds 7 and 9 in DMSO-d 6 | v3-fos-license |
2019-02-09T15:02:16.087Z | 2019-02-08T00:00:00.000 | 59618113 | {
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} | pes2o/s2orc | Combination treatment with highly bioavailable curcumin and NQO1 inhibitor exhibits potent antitumor effects on esophageal squamous cell carcinoma
Background Esophageal squamous cell carcinoma (ESCC) is one of the most intractable cancers, so the development of novel therapeutics has been required to improve patient outcomes. Curcumin, a polyphenol from Curcuma longa, exhibits various health benefits including antitumor effects, but its clinical utility is limited because of low bioavailability. Theracurmin® (THC) is a highly bioavailable curcumin dispersed with colloidal submicron particles. Methods We examined antitumor effects of THC on ESCC cells by cell viability assay, colony and spheroid formation assay, and xenograft models. To reveal its mechanisms, we investigated the levels of reactive oxygen species (ROS) and performed microarray gene expression analysis. According to those analyses, we focused on NQO1, which involved in the removal of ROS, and examined the effects of NQO1-knockdown or overexpression on THC treatment. Moreover, the therapeutic effect of THC and NQO1 inhibitor on ESCC patient-derived xenografts (PDX) was investigated. Results THC caused cytotoxicity in ESCC cells, and suppressed the growth of xenografted tumors more efficiently than curcumin. THC increased ROS levels and activated the NRF2–NMRAL2P–NQO1 expressions. Inhibition of NQO1 in ESCC cells by shRNA or NQO1 inhibitor resulted in an increased sensitivity of cells to THC, whereas overexpression of NQO1 antagonized it. Notably, NQO1 inhibitor significantly enhanced the antitumor effects of THC in ESCC PDX tumors. Conclusions These findings suggest the potential usefulness of THC and its combination with NQO1 inhibitor as a therapeutic option for ESCC. Electronic supplementary material The online version of this article (10.1007/s00535-019-01549-x) contains supplementary material, which is available to authorized users.
Introduction
Esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancer [1,2], which is the sixth leading cause of cancer-related mortality and the eighth most common cancer worldwide [3,4]. Despite recent progress in systematic therapeutics, ESCC remains one of the most intractable cancers, having an extremely low 5-year survival rate [5,6]. Therefore, the development of novel treatment options has been needed to improve the outcomes for ESCC patients.
Curcumin is a naturally occurring polyphenol derived from the root of Curcuma longa that is recognized as a generally safe compound by the Food and Drug Administration [7,8]. Curcumin demonstrates various biological benefits including antimicrobial and anti-inflammatory actions, and is involved in the regulation of programmed cell death and survival pathways by modulating transcription factors such as nuclear factor-jB, growth factors, inflammatory cytokines, and receptors [9]. Curcumin has been shown to have antitumor effects on several types of cancer cells including lung cancer [10], glioblastoma [11], colon cancer [12], pancreatic cancer [13], prostate cancer [14], and ESCC [15][16][17].
Despite the demonstration of the promising antitumor effects of curcumin in preclinical studies, its clinical use is currently limited because of its poor bioavailability in humans [18]. Curcumin is not easily soluble in water [19], and oral administration of curcumin does not achieve sufficient blood concentrations to exert therapeutic efficacy [20][21][22]. To overcome this limitation, various strategies of drug development have been attempted to improve the bioavailability of curcumin [23][24][25][26][27].
Theracurmin Ò (THC, curcumin content 30% w/w) is an effective preparation of curcumin dispersed with colloidal submicron particles, making it easily disperse in water [22]. Consequently, the bioavailability of curcumin in THC is much improved, and the area under the blood concentration-time curve (AUC) after the oral administration of THC is more than 40-fold higher than that of curcumin in rats and 27-fold higher than that of curcumin in humans [22]. In fact, THC has been reported to be clinically useful for treating osteoarthritis [28], muscle damage [29], and atherosclerotic hyperlipidemia [30]. With regard to experimental cancer research, the cytotoxicity or antitumor effects of THC have been reported using several cancer cell lines [31,32], but the effectiveness of THC against ESCC has not been fully clarified.
The purposes of our study were to investigate the antitumor effects of THC on ESCC cells and to compare the effects of curcumin and THC in vivo. Here, we found that induction of NAD(P)H quinone dehydrogenase 1 (NQO1), which is the enzyme that scavenge reactive oxygen species (ROS) [33], plays an antagonistic role in THC-induced antitumor effects, and we, therefore, examined the effects on ESCC of a combination treatment with THC and NQO1 inhibitor.
Assessment of bioavailability and antitumor effects of curcumin and THC in vivo
All animal experiments conformed to the relevant regulatory standards and were approved by the Institutional Animal Care and Use Committee of Kyoto University (Med Kyo 18284). C57BL/6 male mice (CLEA Japan, Inc., Tokyo, Japan) were given either a control diet (without curcumin or THC), a curcumin diet (containing 0.6 g/kg curcumin), or a THC diet (containing 2 g/kg THC that included 0.6 g/kg curcumin). After 1 week, blood was taken from the heart of mice and placed into heparinized tubes. Plasma was immediately prepared by centrifugation at 1000g, 4°C for 10 min and stored at -80°C until use. The plasma concentration of curcumin was measured using high-performance liquid chromatography-tandem mass spectrometry (MS)/MS as described previously [22].
To compare the tumor growth-inhibitory effects of curcumin and THC, xenografted tumors derived from TE-11R cells were used. TE-11R cells (1.5 9 10 6 cells) were suspended in 50% Matrigel (BD Biosciences, San Jose, CA), followed by subcutaneous implantation into the left flank of 6-week-old hairless SCID male mice (Charles River Laboratories Japan Inc. Yokohama, Japan) (n = 15, day 0). The mice were randomly assigned to three groups (n = 5 each) and received either control, curcumin, or THC diet from day 0 to day 70.
The tumors were measured with a caliper, and tumor volume (mm 3 ) was calculated using the following formula: (length) 9 (width) 2 9 0.5.
Assessment of antitumor effects of THC and NQO1 inhibitor in vivo
Patient-derived xenograft (PDX) ESCC tumors were utilized to assess the therapeutic effects of THC and NQO1 inhibitor in vivo. All experiments conformed to the relevant regulatory standards and were approved by the Institutional Animal Care and Use Committee of Kyoto University (Med Kyo 18284) and the Ethics Committee of Kyoto University (G0770).
To establish PDX tumors, biopsy specimens taken from human ESCC tissue of primary site were placed in a subcutaneous pocket created by a 5-mm incision in the left flank of 6-week-old hairless SCID male mice (n = 20), which was then closed by suturing. Mice were randomly assigned to one of the four groups at day 21 (n = 5 each), and given either normal water or THC-containing water (5000 ppm) for drinking from day 21 to day 70. Either DMSO (mock) or 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) (sc-362737, Santa Cruz Biotechnology, Inc., CA, USA) (5 mg/kg) was administered intraperitoneally every other day from day 21 to day 70.
The tumors were monitored with a caliper, and tumor volume (mm 3 ) was calculated using the following formula: (length) 9 (width) 2 9 0.5.
Immunohistochemical staining
Immunohistochemical staining was performed as described previously [34]. Additional information is given in Supplementary materials and methods.
Statistical analyses
Data are presented as the mean ± standard error (SE) of triplicate experiments unless otherwise stated. Differences between two groups were analyzed using the 2-tailed Student's t test, and *P \ 0.05 and **P \ 0.01 were considered significant. All statistical analyses were performed using SPSS 21 for Windows (SPSS Inc., Chicago, IL, USA).
Next, we measured caspase 3 and caspase 7 activity in those cells treated with THC. Treatment with THC significantly increased the caspase activity as well as the cleavage of Poly (ADP-ribose) polymerase (PARP) protein, showing the capability of THC to induce apoptosis (Fig. 1b, c).
In addition, we investigated whether THC influences cell cycle and/or senescence in ESCC cells. As shown in Supplementary Collectively, these results demonstrated the cytotoxic and antiproliferative effects of THC on ESCC cells.
Inhibitory effects of THC for spheroid and colony formation in ESCC cells
We performed spheroid and soft agar colony formation assays to investigate the effect of THC on the stem cell-like properties and anchorage-independent cell growth activities of ESCC cells. THC showed a strong dose-dependent inhibition of spheroid formation (Fig. 1d) and colony formation (Fig. 1e) in both TE-8 and TE-11R cells.
Bioavailability and antitumor effects of THC against ESCC xenografts in vivo
To examine the difference in bioavailability between curcumin and THC in vivo, we fed mice either a curcumin diet (curcumin group) or a THC diet (THC group) containing equal amounts of curcumin for a week. There was no significant difference in the dietary intake of the groups. In addition, there were no obvious abnormalities in their general condition (e.g., adverse hematological effects and body weight loss) (data not shown). We examined plasma curcumin concentrations in these mice, and the THC group showed markedly higher plasma levels of curcumin than the curcumin group (1110.4 ± 199.8 ng/mL vs 191.1 ± 64.4 ng/mL) (Fig. 2a). To investigate whether this better bioavailability of THC led to a stronger antitumor effect in vivo, we compared the tumor growth inhibition in ESCC (TE-11R) xenograft mice treated with THC or curcumin. As shown in Fig. 2b, the THC group demonstrated significantly greater tumor growth inhibition than the curcumin group (43% vs 11% at day 70).
Upregulation of NMRAL2P after THC treatment
Next, we performed microarray gene expression analysis to identify the changes in gene expression patterns in ESCC cells (TE-5 and TE-8 cells) after THC treatment. We identified several genes that showed differential RNA expression after THC treatment. Among these, expression levels of NmrA-like redox sensor 2 pseudogene (NMRAL2P, also known as Loc344887) were elevated in THC-treated TE-5 and TE-8 cells ( Fig. 3). We confirmed by RT-PCR that THC upregulated NMRAL2P expression time-dependently in TE-5, TE-8, and TE-11R cells (Fig. 3a). Fig. 1 Cytotoxic effects of Theracurmin Ò on ESCC cells. a Cell viability: ESCC cells were cultured with the indicated concentrations (0-50 lM) of Theracurmin Ò (THC) for 96 h. Cell viability was measured using the WST-1 assay. Viability of each cell type treated with THC relative to that of untreated cells is indicated (*P \ 0.05, **P \ 0.01, vs untreated cells; n = 3). b Caspase 3/7 activity: ESCC cells were cultured with the indicated concentrations (0-50 lM) of THC for 12 h (TE-5) or 24 h (TE-8 and TE-11R). Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay. Relative Caspase 3/7 activity of each cell type treated with the indicated concentrations of THC relative to that of untreated cells is indicated (*P \ 0.05, **P \ 0.01, vs untreated cells; n = 3). c Assessment of apoptosis: ESCC cells were cultured with the indicated concentrations (0-50 lM) of THC for 24 h. The cleavage of PARP was investigated by western blotting. b-actin served as a loading control. d Spheroid assay: Spheres were generated by TE-8 and TE-11R cells treated with the indicated concentrations (0-50 lM) of THC for 2 weeks. Spheroid formation level was measured using the CellTiter-Glo 3D Cell Viability Assay. Spheroid formation by each cell type treated with THC relative to that of untreated cells is indicated (**P \ 0.01, vs untreated cells; n = 3). e Colony formation assay: Colonies were generated by TE-8 and TE-11R cells with the indicated concentrations (0-50 lM) of THC for 2 weeks (TE-11R) or 3 weeks (TE-8). Colony numbers per high-power field (HPF) were counted in 15 random fields to determine the mean colony-forming units for each sample (*P \ 0.05, **P \ 0.01, vs untreated cells) Fig. 2 Comparison of blood curcumin concentration and antitumor effect on ESCC after ingestion of curcumin and Theracurmin Ò diet. a Plasma concentration of curcumin: C57BL/6 mice received control, curcumin (0.6 g/kg) or Theracurmin Ò (THC) (2 g/kg: containing 0.6 g/kg curcumin) diet for 1 week. Plasma concentration of curcumin was measured (**P \ 0.01, vs curcumin diet; n = 5). b Xenografted-tumor growth: TE-11R cells (1.5 9 10 6 cells) were injected into hairless SCID mice that received control, curcumin, or THC diet for 70 days. The tumor volume was measured with a caliper [*P \ 0.05, **P \ 0.01, n.s. (non significant, P [ 0.05), vs control or curcumin diet; n = 5] Activation of NRF2-NMRAL2P-NQO1 pathway by THC Because NMRAL2P has been shown to be involved in the nuclear factor erythroid 2 like 2 (NRF2) pathway, in which NRF2 regulates NMRAL2P expression and NMRAL2P subsequently regulates expression of NQO1 [36][37][38], we next evaluated whether THC affected NRF2 activation and NQO1 expression in ESCC cells. As shown in Fig. 3b, THC increased nuclear, but not cytosolic, NRF2 protein levels, suggesting that THC treatment activated the NRF2 transcription factor. THC also increased mRNA and protein levels of NQO1 (Fig. 3c, d).
Kelch-like ECH-associated protein 1 (KEAP1) is a repressive partner of NRF2 that suppresses activation of the NRF2 pathway [39]. We next performed siRNA-mediated inhibition of KEAP1, NRF2, and NMRAL2P in TE-5 and TE-8 cells, and confirmed the reduced expression levels of the respective genes ( Supplementary Fig. 4a-d). Knockdown of KEAP1 resulted in an increased expression of NMRAL2P, while knockdown of NRF2 caused a reduction of NMRAL2P levels in both TE-5 and TE-8 cells with and without THC treatment (Supplementary Fig. 4e). As shown in Supplementary Fig. 4f, g, knockdown of NMRAL2P or NRF2 attenuated NQO1 mRNA and protein induction by THC. These results suggested that THC promoted the NRF2-NMRAL2P-NQO1 pathway via the activation of NRF2 transcription factor.
In addition, we examined the basal KEAP1 mRNA expression level or nuclear NRF2 protein level in ESCC cells, and we assessed the correlation between their expression levels and their differentiation status, morphological phenotype, and sensitivity to THC (Supplementary Tables 1, 2 and Supplementary Fig. 5a, b). Although nuclear NRF2 expression level is considered to be associated with the sensitivity to chemoradiotherapy in ESCC [40] and the expression of NRF2/KEAP1 might be associated with oncogenic characteristics in ESCC cells, there were no significant correlations between them in this study (data not shown).
Increased intracellular and mitochondrial ROS levels induced by THC
Because NRF2 plays an important role in the response to oxidative stress [39], we measured the ROS levels in TE-5, TE-8, and TE-11R cells after THC treatment. As shown in Fig. 4a, THC significantly increased intracellular ROS levels in a dose-dependent manner. In addition, 8-OHdG, a marker of oxidative DNA damage, was increased by THC treatment (Fig. 4b), and mitochondrial ROS levels also showed a dose-dependent elevation after THC treatment (Fig. 4c).
Inhibitory effect of THC on the TCA cycle
Because THC increased mitochondrial ROS levels in ESCC cells, we investigated whether THC affected mitochondrial functions such as the tricarboxylic acid (TCA) cycle. Metabolome analyses revealed that THC sharply decreased the levels of cis-aconitic acid, isocitric acid, and 2-oxoglutaric acid, components of the TCA cycle, indicating the inhibitory effects of THC on the TCA cycle (Fig. 4d).
The role of NQO1 in ESCC cells with THC treatment
To investigate how NQO1 influences the cytotoxic effect of THC on ESCC cells, we created NQO1 knockdown TE-11R cells using three types of shRNAs. As shown in Fig. 5a, b, NQO1 expression levels were remarkably reduced by all shRNAs at both mRNA and protein levels. NQO1 knockdown resulted in an increase of 8-OHdGindicated oxidative damage in THC-treated cells (Fig. 5c). Although NQO1 knockdown alone did not affect cell growth (data not shown), susceptibility to THC in NQO1 knockdown cells was significantly higher than that in control cells (Fig. 5d). Conversely, when we overexpressed NQO1 in TE-11R cells via lentivirus infection (Fig. 5e, f), NQO1 overexpression resulted in a decrease of 8-OHdGindicated oxidative damage after THC treatment (Fig. 5g) and was associated with resistance to THC treatment (Fig. 5h).
Combination effects of THC and NQO1 inhibitor in vitro
Because of the additive effects of THC and NQO1 inhibition, we next examined whether NQO1 inhibitor enhanced the cytotoxic effects of THC. ESCC cells were treated with THC and ES936, which is a mechanism-based inhibitor of NQO1 [41]. ES936 has been shown not to decrease NQO1 protein expression [42]. The combination of THC and NQO1 inhibitor resulted in an increase in ROS production, 8-OHdG-indicated oxidative damage, and the cleavage of PARP protein (Fig. 6).
Combination effect of THC and NQO1 inhibitor against ESCC PDX tumors in vivo
To assess the antitumor effects of THC and NQO1 inhibitor in vivo, we evaluated the tumor growth inhibition in ESCC PDX tumors. First, to determine the optimal dose of THC, mice were given either normal water or THC-containing water (2500, 5000, or 10000 ppm), and plasma curcumin concentrations were examined. As shown in Fig. 7a, plasma curcumin levels increased THC dose-dependently. When 5000 ppm of THC was administered, the plasma concentration of curcumin reached 3973.8 ng/mL (about 11 lM), which was similar to the concentration we used in in vitro experiments. Therefore, we used water containing 5000 ppm THC for subsequent treatments. As shown in Fig. 7b, treatment with the combination of THC and NQO1 inhibitor resulted in a significant inhibition of PDX tumor growth compared with that after control or monotherapy (inhibition at day 70: NQO1 inhibitor 1.9%, THC 40.7%, THC plus NQO1 inhibitor 72.6%). There was no significant difference in the water intake between groups, and no significant adverse hematological effects or weight loss were detected (data not shown). Combination treatment with THC and NQO1 inhibitor significantly decreased Ki67 expression, a marker of cellular proliferation, compared with vehicle control and/or monotherapy with THC (Fig. 7c, d). In addition, combination treatment with THC and NQO1 inhibitor and/or THC monotherapy significantly increased single-stranded DNA (ssDNA), a marker of apoptosis, compared with vehicle control (Fig. 7c, e). Moreover, 8-OHdG, nuclear NRF2, and NQO1 levels were increased by treatment with THC alone as well as combination treatment with THC and NQO1 inhibitor (Fig. 7c). As a note, THC treatment did not cause any histological damages in normal esophageal tissues, and it did not increase ROS levels (8-OHdG levels) in normal esophageal tissues (data not shown).
Discussion
Highly bioavailable curcumin (Theracurmin Ò , THC) showed antitumor effects on various types of ESCC cells and xenografted tumors. THC increased ROS levels in accompany with the activation of NRF2-NMRAL2P- NQO1 pathway. Since NQO1 revealed to play an antagonistic and antioxidative role in THC-induced cytotoxicity, we proposed a combination treatment with THC and NQO1 inhibitor. As a result, such a treatment strategy exhibited potent antitumor effects on ESCC PDX tumors. In this study, biological effects (e.g., cytotoxic effects, ROS production, and sensitization of NQO1 inhibitor) of THC were similar to those of curcumin in vitro (Figs. 1a, 4a, 6c and Supplementary Fig. 6). We suggest that this is because THC is identical with curcumin as a component [22]. However, the antitumor effect of THC in vivo was much higher than that of an equal dose of curcumin. We suggest that the difference in the antitumor effect between THC and curcumin in vivo is caused by their different bioavailability (Fig. 2a).
The peak plasma curcumin levels following oral THC administration (5000 ppm) in mice reached 3973.8 ng/mL (Fig. 7a), which is roughly equivalent to 11 lM. In our in vitro experiments, we used 5-50 lM THC and the IC 50 values of THC in various ESCC cells were between 7.03 and 34.98 lM. Because the plasma curcumin concentration after THC administration (400 mg curcumin/day) in humans was reported to be at about 3.7 lM [43], we presume that the dose of THC used in our experiments does not greatly exceed the achievable physiological range.
In the present study, NMRAL2P was upregulated in ESCC cells treated with THC. NMRAL2P is a long noncoding RNA that acts as a coactivator of NQO1 [38].
Although its precise function remains to be clarified, potential roles for NMRAL2P as a prognostic factor and/or a therapeutic target for cancer have been reported [44,45]. Our results showed that KEAP1 knockdown increased expression of NMRAL2P, while NRF2 knockdown reduced it. Moreover, NMRAL2P knockdown inhibited NQO1 induction by THC. These results are consistent with the previous reports [36][37][38], and indicate that NMRAL2P is acting downstream of NRF2 and upstream of NQO1 in the NRF2-related signal cascade.
We showed that THC treatment increased intracellular and mitochondrial ROS levels in ESCC cells. These results were consistent with the previous reports showing curcumin-mediated ROS generation [46][47][48] that led to mitochondrial damage [49]. In this study, we also demonstrated that THC treatment affected the TCA cycle, which may retain a role in cancer cell metabolism. In particular, 2-oxoglutaric acid plays critical roles as a precursor of glutamine formation, as a nitrogen transporter for the urea cycle and/or ammonia detoxification, and as a cosubstrate for dioxygenases [50]. In addition, 2-oxoglutaric acid-dependent dioxygenases mediate the demethylation of DNA and histones, which is involved in regulation of the expression of many genes [51,52], and depletion of 2-oxoglutaric acid was able to cause epigenetic changes [53]. Thus, the decrease in 2-oxoglutaric acid caused by THC may induce a range of aberrations in cellular metabolism. Fig. 7 Antitumor effect of Theracurmin Ò and NQO1 inhibitor against ESCC PDX tumors. a Plasma concentration of curcumin after ingesting Theracurmin Ò (THC)containing water: C57BL/6 mice received water-containing 2500, 5000, or 10000 ppm THC for 2 days. Plasma concentration of curcumin was measured (**P \ 0.01, vs control; n = 5). b PDX tumor growth: Growth kinetics of subcutaneous ESCC PDX tumors treated with or without THC and/or NQO1 inhibitor (*P \ 0.05, **P \ 0.01, vs DMSO/water or DMSO/THC; n = 5). c Hematoxylin and eosin (HE) and immunohistochemical staining: HE and immunohistochemical staining for Ki67, ssDNA, 8-OHdG, NRF2, and NQO1 in ESCC PDX tumors treated with or without THC and/or NQO1 inhibitor. Scale bar = 100 lm (HE, Ki67, and 8-OHdG) and 50 lm (ssDNA, NRF2, and NQO1). Arrowheads indicate nuclear NRF2-positive cells. d Ki67 positively stained nuclei rate: Ki67 positively stained nuclei rate were counted in 6 random fields (*P \ 0.05, **P \ 0.01, vs DMSO/water or DMSO/THC). e ssDNA positively stained nuclei rate: ssDNA positively stained nuclei rate were counted in 6 random fields (*P \ 0.05, **P \ 0.01, vs DMSO/water) We showed that THC increased nuclear NRF2 as well as NQO1 expressions. As NRF2 is upregulated by the ROS production [39] and NQO1 is a downstream factor of NRF2 [39], we suggest that nuclear NRF2 is increased via THCmediated ROS production and NQO1 is upregulated via NRF2 activation. To examine the role of NQO1 in THCmediated cytotoxicity in ESCC cells, we performed the experiments using cells with NQO1 gene modification and/ or NQO1 inhibitors. Knockdown of NQO1 or administration of NQO1 inhibitor resulted in an increase of ROS and/ or 8-OHdG levels in THC-treated ESCC cells, while overexpression of NQO1 resulted in a decrease of 8-OHdG in THC-treated ESCC cells. These results suggest that NQO1 plays an antioxidative role in THC-mediated cytotoxicity. As NQO1 acts as a reductase [33,[54][55][56], cells undergoing oxidative stress are considered to induce NQO1 to protect cells from those stresses. Accordingly, we thought that inhibition of NQO1 could enhance the antitumor effects of THC and we revealed that the strategy is effective.
We showed that basal levels of NQO1 protein in ESCC cells tended to be high, compared with those in normal esophageal epithelial cells (Supplementary Fig. 5c). Therefore, cytotoxic effect of THC may occur efficiently on ESCC cells in the presence of NQO1 inhibitor. As NQO1 inhibitor has not been used clinically, future studies to develop NQO1 inhibitors are warranted.
A limitation of this study is that we could not determine whether NMRAL2P regulates NQO1 directly or indirectly. Moreover, it remains unclear whether the antitumor effect of THC and NQO1 inhibitor is due to the direct effect on tumor cells or indirect effect on microenvironment. Further study will be required to address these questions.
In conclusion, THC exhibits in vitro and in vivo antitumor effects, and showed remarkably higher bioavailability and stronger antitumor effects than curcumin in vivo. THC induced ROS in accompany with the activation of NRF2-NMRAL2P-NQO1 pathway. NQO1 played an antioxidative role in THC-mediated cytotoxicity. Importantly, NQO1 inhibitor enhanced the THC-induced antitumor effects (Supplementary Fig. 7). These results suggest the potential usefulness of combination therapy with THC and NQO1 inhibitor for the treatment of ESCC. | v3-fos-license |
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} | pes2o/s2orc | Strategies for Reuse of Skins Separated From Grape Pomace as Ingredient of Functional Beverages
Wine grape pomace, the by-product of wine making, is a source of polyphenols, metals, and organic acids, and may be exploited for the production of functional beverages. Among red wines, Primitivo and Negramaro varieties possess an interesting amount of polyphenolic compounds and other chemicals. Consequently, study of the biological activity of Primitivo and Negramaro vinification by-products is of great interest as well as optimizing the extraction of its bioactive components. In order to stabilize the grape pomace, different methods of drying grape pomace were tested. After stabilization of the pomace, the grape skins were manually separated from the seeds and any woody parts. The chemical characterizations of acidified alcoholic (methanol/ethanol) and water extracts and either microwave-assisted or ultrasound-assisted extractions of separated grape skins were compared. Besides that, the in vitro antioxidant activity of wine pomace skin extracts was also investigated as Trolox equivalents antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC). Overall, the alcoholic extractions were found to be the most effective for recovering phenolic compounds, when compared with those in water. Ultrasound- and microwave-assisted extraction of pomace skin using acidified water allowed the highest TEAC value. Taking into account the water extraction result, in order to reuse grape pomace skins to produce a functional beverage, we utilized them in combination with black tea, karkadè (Hibiscus sabdariffa L.), or rooibos (Aspalathus linearis Burm.) to produce an infusion. The combination of grape skins and black tea showed the highest ratio of total phenol content to antioxidant activity. Moreover, skin isolated from pomace, with or without black tea infusions, were shown to have anti-inflammatory capacity in human cell culture. Our results raise the value of grape skin pomace as a rich source of bioactive compounds with antioxidant and anti-inflammatory activity and suggest its exploitation as an ingredient for functional beverages.
INTRODUCTION
Grape pomace is the by-product of the winemaking process, representing 20% of the processed grape weight (Beres et al., 2017). Grape pomace can be exploited as a source of various ingredients for the human diet, such as antioxidants, dietary fibers, and phenolic extracts, to produce novel foods. The byproduct can thus re-enter the food cycle, avoiding environmental complications, due to the phenols (Tang et al., 2018). There is growing interest in the potential food applications of phenolic compounds therefore, the reuse of by-products could provide a new food chain. The vast amounts of winemaking by-products and the seasonality of their production force the development of fast and economical methods that allow their stabilization and storage for later use. Winemaking by-products must be dried for further utilization. Drying methods have a great impact on the stability of bioactive compounds, directly influencing changes in the physicochemical properties of food ingredients.
The most utilized drying methods include, for example, hotair/oven drying, low temperature-air drying, and freeze drying (Barba et al., 2016;Medina-Torres et al., 2017). Except for freeze drying, these conventional drying methods are easier and have lower production costs. The optimal drying conditions for the storage of grape pomace for further application need further investigation. Moreover, optimization of the experimental conditions for extraction of phenolics from grape pomace is a key issue for future industrial developments.
Industrial extraction from the grape pomace is a process that combines water with other organic solvents (Chemat et al., 2017). In order to meet the ongoing demands for minimally processed by-products and meet the requirements of a green extraction concept, alternative extraction methods have been pointed out. Microwave-and ultrasound-assisted extraction have been described as good alternatives to conventional techniques due to some advantages such as shorter extraction time, lower solvent requirement, and a higher extraction rate (Chemat et al., 2017). UAE treatment offers the advantage of greater permeation of the solvent into dried tissues, shorter incubation, higher yields and reproducibility, high processing throughput, extraction of heat labile components, and less energy needed for extraction (Roselló-Soto et al., 2015). The MAE extraction process is another green method based on the polarity of the compounds. Due to the faster heating for the extraction of bioactive plant substances, the reduction of thermal gradients and the increase of the yield of the extracts, the MAE process is widely studied for the extraction of bioactive molecules.
The most sustainable approach to exploitation of pomace could be its use as a raw material to produce ingredients for functional foods, and pharmaceuticals (Meral and Dogan, 2013;Wittenauer et al., 2016). Our previous studies on grape skin and data from the literature show that the composition and amounts of polyphenols in grape berries can be influenced by different factors, among which genotype usually has the greatest impact (Giovinazzo and Grieco, 2015;Calabriso et al., 2016b). Moreover, viticulture and vinification practices also affect the polyphenol extractability in wine and grape pomace (Grieco et al., 2019). Previous studies have shown that red grape skin polyphenols counteract the atherosclerotic process through reduced expression of endothelial adhesion molecules, chemokines, and matrix metalloproteinases (Calabriso et al., 2016a,b). This study aimed to: (i) investigate the influence of various drying conditions on the antioxidant activity and polyphenol stability of grape pomace skin for potential application in functional foods and beverages; (ii) compare the antioxidant capacity of pomace skin from different varieties, their phenolic and flavonoid quantification and characterization after organic solvent and water extractions; (iii) evaluate the phenolic and flavonoid content and antioxidant activity of grape pomace skin infusion alone or combined with black tea, karkadè (Hibiscus sabdariffa L.), or rooibos (Aspalathus linearis Burm.); (iv) assess the anti-inflammatory capacity in human cell culture of grape pomace skin infusion alone or combined with black tea. Our findings aimed to raise the value of grape skin pomace as a rich source of bioactive compounds with health properties and suggest its exploitation as an ingredient for functional beverages.
Raw Material and Sample Preparation
Four batches of wine pomace, Vitis vinifera varieties Primitivo (P), Negramaro (N), Negramaro/Lambrusco (N/L) (achieved after fermentation for red wine making), and Verdeca (B) (without fermentation, as it is used in white wine making) were obtained from a commercial winemaking facility located in Salento (Cantine Due Palme; southern area of the Apulia Region, Italy). The pomace was dried (a) in an oven at 50 • C, (b) at room temperature, or (c) by a Freezone R 2.5 model 76530 lyophilizer (Labconco Corp., Kansas City, MO, USA) until constant weight. Subsequently, the skins were manually recovered from the pomace samples and stored at −20 • C until further processing. A sample of wet skins was stored at −20 • C and utilized for comparison experiments. Commercial black tea leaves, dried hibiscus petals (Hibiscus sabdariffa L.), and rooibos tea (Aspalathus linearis Burm.) were purchased from a local supermarket. Three blends were prepared by mixing 50% dried skin from grape pomace with 50% black tea, hibiscus petals, or rooibos tea.
Extraction of Polyphenol Compounds in Organic Solvent
Polyphenol compounds were extracted from wet grape skins and skins separated from dried grape pomace by freezing the samples in liquid nitrogen and grinding with a blender until a fine powder was obtained. The samples (1 g) were treated with 10 mL of methanol:ethanol (80:20, v:v) and extracted at room temperature for 16 h in the dark under continuous stirring. Extraction mixtures were centrifuged (4,000 × g) for 5 min and the supernatants stored at −20 • C until analysis.
Extraction of Polyphenol Compounds in Acidified Water
Skins isolated from grape pomace and homogenized as described above (5 g and 10 g) were extracted in distilled water (100 mL) acidified with citric acid (0.001 M, final concentration) (acidified water) at room temperature for 16 h in the dark under continuous stirring. After centrifugation (4,000 × g for 5 min) of the extraction slurry, the supernatants were stored at −20 • C until analysis.
Ultrasound-Assisted Extraction
Ultrasound-assisted extraction (UAE) was carried out in an ultrasound bath (Labsonic Falc, LBS1-H3) at 35 kHz frequency and 88 W, at room temperature for 15 min. Samples of grape skin from dried pomace (5 g) were extracted with 100 mL of distilled water acidified as described above. Samples were centrifuged at 4000 × g for 5 min after ultrasound treatment or were left at room temperature for 16 h in the dark under continuous stirring and then centrifuged to remove cell debris as described above.
Microwave-Assisted Extraction
Microwave-assisted extraction (MAE) was carried out in a Multiwave 3000 SOLV Microwave Reaction System (Anton Paar, Austria). Samples of dried skin pomace (5 g) were extracted by adding 100 mL of acidified water at room temperature and using fluctuating radiation to keep the temperature steady at a set value of 50 • C and 200 W, as described by Drosou et al. (2015). After microwave treatment, the samples were centrifuged at 4,000 × g for 5 min or were left at room temperature for 16 h in the dark under continuous stirring and then centrifuged to recover supernatants.
Infusion
Primitivo grape skins isolated from pomace as described above (P), black tea (T), karkadè (K), and rooibos (R) infusions were formulated as follows: 2 g of P; 1 g of P and 1 g of T (P/T); 1 g of P and 1 g of K (P/K); 1 g of P and 1 g of R (P/R). The formulations (2 g total) were infused in 100 mL of drinking water at a temperature of 90 • C for 5 min; the infusions were acidified using 1 mL of lemon juice. The infusions were stored at −20 • C until characterization analyses. The infusions were freeze-dried by a Freezone R 2.5 model 76530 lyophilizer (Labconco Corp., Kansas City, MO, USA) and stored at −20 • C until biological activity analysis.
High Performance Liquid Chromatography (HPLC) Characterization of Anthocyanins
To quantify the anthocyanin molecules in both alcoholic and aqueous extracts from skins isolated from grape pomace, we performed HPLC analysis using an Agilent-1100 liquid chromatograph equipped with a DAD detector as described by Gerardi et al. (2015). Chromatograms were acquired at 520 nm. Quantification of total anthocyanins was performed by HPLC/DAD using a five-point regression curve (r 2 ≥ 0.99) generated through the use of malvidin 3-O-glucoside (oenin) as a reference compound and expressed as oenin equivalents (OEs).
HPLC Characterization of Phenolic Acids, Stilbenes, Flavanols, and Flavonols
Different phenolic compounds present in alcoholic and aqueous extracts were separated by RP-HPLC DAD (Agilent 1100 HPLC system, Santa Clara, CA, USA). The separation was performed as described by Calabriso et al. (2016b). The chromatographic analysis was based on the comparison of peak retention time with the retention time of external standards.
HPLC Characterization of Organic Acids and Alcohols
Organic acids and alcohol were identified and quantified by the HPLC system (Agilent 1100 series) equipped with a refractive index detector (RID) for alcohol analysis, and a UV/Vis detector for analyzing organic acids at 210 nm onto an Aminex HPX-87H column (300 × 7.8 mm, 9 µm) (Bio-Rad, Hercules, CA, USA), at 55 • C. The analytical method was the same as that reported by Gerardi et al. (2019).
Metal Characterization
Element concentrations in grape pomace samples were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The spectrometer was an ICAP 6300 with Dual view, empowered by iTEVA software (Thermo Scientific, Waltham, MA, USA).
One gram of dried grape skin pomace or lyophilized infusion was treated and analyzed as described by Bruno et al. (2018), whereas the liquid samples were analyzed as they were. A blank sample was also prepared with the same solvents and preparation procedures used for the pomace samples: 4 mL of H 2 O 2 and 6 mL of 69% HNO 3 , treatment at 180 • C for 10 min in a START D microwave digestion system (Milestone, Italy), and dilution with superpure water to a final volume of 25 mL.
The spectrometer was previously calibrated for quantitative analysis with five standard solutions containing known concentrations (0.001, 0.01, 0.1, 0.5, and 1.0 mg/L) of the elements. The calibration lines showed correlation coefficients (r) >0.99 for all the measured elements. The results were expressed as the average of three different measurements, and the element concentrations were expressed as ppm (mg/kg of sample weight) for solid samples, and as mg/L for liquid samples. Std for blank sample: 0.003 (0 ± 0.003).
ANTIOXIDANT ACTIVITY OF ALCOHOLIC AND AQUEOUS EXTRACTS Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The TEAC assay was performed by the method described by Re et al. (1999), modified as reported by Scarano et al. (2018). Briefly, the ABTS radical cation was diluted in PBS (pH 7.4) to an absorbance of 0.40 at 734 nm. After the addition of 200 µL of diluted ABTS to 10 µL of Trolox standard or extract, the absorbance reading at 734 nm was taken 6 min after initial mixing using an Infinite 200 Pro plate reader (Tecan, Männedorf, Switzerland). The percentage inhibition of absorbance at 734 nm was calculated and plotted as a function of the concentration of Trolox, and the TEAC value expressed as Trolox equivalents (µmol) using Magellan v7.2 software.
Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC procedure was carried out following the procedure established by Dávalos et al. (2004). The assay was performed in 75 mM phosphate buffer (pH 7.4) at 37 • C using 96-well plates and an Infinite 200 Pro plate reader (Tecan, Männedorf, Switzerland). The antioxidant capacity of 20 µL of extract from dried grape skin isolated from pomace was assayed by recording for 80 min the decay curves of fluorescein (70 nM final concentration) after the addition of a generator of radical species (AAPH, 12 mM final concentration). The antioxidant Trolox was used to make a standard curve (1-6 µM) and final ORAC values were expressed as µmol Trolox equivalents (TE)/g of dried weight of grape skins or µmol TE/L of aqueous extractions.
Folin-Ciocalteu Assay
A rapid method (Magalhães et al., 2010) was used to assess the total phenols in alcoholic and water extracts from dried skins isolated from pomace in 96-well plates (Corning) using a microplate reader (Tecan, Infinite M200). Folin-Ciocalteu reagent (1:5, v/v) (50 µL) was placed in each well, and then 100 µL of sodium hydroxide solution (0.35 M) was added. The absorbance at 760 nm of the blue complex formed was monitored after 5 min. Gallic acid was used to obtain a calibration curve in the range from 2.5 to 40.0 mg/L (R ≥ 0.9997). The total phenol content of the samples was expressed as gallic acid equivalents.
BIOLOGICAL ACTIVITY OF AQUEOUS EXTRACTS Cell Culture and Treatment
Human microvascular endothelial cell line (HMEC-1) was obtained from Dr. Thomas J. Lawley and cultured as described by Ades et al. (1992). Briefly, microvascular endothelial cells were cultured in MCDB-131 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 µg/mL hydrocortisone (Sigma), 10 ng/mL human epidermal growth factor (Sigma), 50 U/mL penicillin, and 50 µg/mL streptomycin (Gibco BRL, Life Technologies, Paisley, UK). For treatments, confluent endothelial cells were shifted to MCDB-131 medium supplemented with 3% fetal bovine serum for 3 h and then treated for 2 h with aqueous extracts of grape skins infusion of Primitivo pomace (P) at several concentrations (1-5 mg/mL). Then, endothelial monolayers were stimulated with the cytokine tumor necrosis factor-α (TNF-α) (10 ng/mL) for an additional 16 h, after which cellular toxicity, inflammatory markers, and adhesion assays were evaluated.
Monocytoid Cell Adhesion Assays
Monocytoid U937 cells were obtained through the American Tissue Culture Collection (Rockville, MD) and grown as described by Carluccio et al. (2003). Adhesion assays were performed as follows: HMEC-1 were grown to confluence in sixwell tissue culture plates, after which TNF-α was added (16 h) to induce the expression of VCAM-1, in the presence or absence of aqueous extracts of grape skin infusion of Primitivo pomace (P) at several concentrations (1-5 mg/mL). Adhesion assays were performed as described by Carluccio et al. (2003). After 10 min, non-adhering cells were removed by gentle washing with cell culture medium, and the monolayers were fixed with 1% paraformaldehyde. The number of adherent cells was obtained by counting six different fields by using an ocular grid and a 20× objective (0.16 mm 2 /field).
Detection of Endothelial Cell Surface Molecules
HMEC-1 were grown to confluence in 96-well tissue culture plates. Then, the cultures were incubated in the presence or absence of aqueous extracts of grape skin infusion of Primitivo pomace (P), tea (T), or P/T (1:1) for 2 h and then stimulated with TNF-α for 16 h.
Statistical Analysis
The experiments with skin pomace extracts were conducted in three independent tests, and data were presented as mean ± standard deviation (SD). For bioactivity analyses, multiple comparisons were carried out by one-way analysis of variance (ANOVA) and by Tukey's test with p = 0.05. Dissimilarities between means from at least three independent tests (p < 0.05) were judged statistically significant.
Comparison of Different Pomace Drying Methods
In order to stabilize grape pomace (GP), we utilized different drying methods and then compared several parameters in the dehydrated matrix. Wet fresh pomace of Negramaro cv. was dried by oven at 50 • C for 48 h (N50 • ), subjected to natural drying at room temperature for 72 h (N r.t.), or freeze-dried for 48 h (N f.d.). Subsequently, the skins were manually recovered from the grape pomace and stored at −20 • C until further processing. As shown in Figure 1, the antioxidant activity and phenol content (Folin) of skin pomace dried with different methods (N50 • , N r.t., N f.d.) showed similar results for all dehydration treatments. When compared to the wet material, the antioxidant activity of the skin pomace samples assayed by TEAC and ORAC methods was reduced by up to 50% in lyophilized and air-dried samples. The mean reduction in the total phenolic content was 49.3% for all drying treatments. Therefore, we selected the dehydration method by oven at 50 • C because it is cheaper, faster, and more reliable than drying with air at room temperature (only 48 vs. 72 h).
Extraction by Organic Solvents
In this study, we characterized the polyphenolic pattern and antioxidant properties of three different red GP, from Primitivo (P), Negramaro (N), and Negramaro/Lambrusco (N/L), and one white GP from Verdeca (B). Grape skin pomaces, separated from raw pomace dehydrated by oven at 50 • C for 48 h, were analyzed for antioxidant activity (TEAC and ORAC) and total phenol content and compared.
Both the total phenols (TP) and antioxidant activity (TEAC and ORAC) showed significant differences among GP skins (Figure 2). Indeed, N skin pomace showed higher values for both antioxidant activity (TEAC: 114.00 µmol TE/g DW; ORAC: 148.53 µmol TE/g DW) and TP content (8.94 g GAE/kg DW). TEAC, ORAC, and Folin-Ciocalteu assays indicated slightly lower values for P skin pomace, while the N/L blend showed the lowest values, similar to those for white GP skin (B), and for this reason was discarded from further analyses.
Polyphenolic composition was determined by solid-phase extraction in methanol/ethanol (8 : 2, v/v). Notably, anthocyanins were the most representative polyphenols in all the red extracts, followed by phenolic acids, flavonols, flavanols, and stilbenes ( Table 1). Conversely, in white GP skin, a higher content of flavonols was recorded, followed by flavanols and phenolic acids ( Table 1).
Climatic conditions, viticulture practices, and the winemaking process can determine important variations in the mineral content of wine pomace more than for other components (Lachman et al., 2013). For example, the type and principally the duration of maceration processes have a strong impact on the extraction and reabsorption of minerals during winemaking. Table 2 reports the mineral content in different GP skins. Potassium (K) and calcium (Ca) were the most abundant microelements detected in all samples. More differences were found in white pomace (B) for magnesium (Mg) content, and in N samples for iron (Fe) content.
Water Extraction
With the aim of utilizing the pomace skin powder to produce a functional drink, we set up a method to extract different polyphenol classes and other chemical compounds with functional activity using citric acid-acidified water. The three grape skin pomaces were treated for phytochemical recovery after water extraction. Two concentrations of skin powder (5 and 10%) were used to extract phytochemicals overnight (16 h) at 26 • C. The results were similar for 5 and 10% grape skin powder (data not shown), suggesting a saturation effect during extraction. For this reason, further experiments were carried out using 5% grape skin concentration.
Preliminary results suggest that 5% pomace skin powder dissolved in water might be a good GP powder/water ratio. This water extract was used to characterize total anthocyanins, flavonols, and soluble acids among polyphenols together with organic acids, alcohol, and essential metals. To increase the polyphenol extraction rate in water, several different methods using ultrasound and/or microwave treatment were also used.
Antioxidant capability, TP, characterization of main polyphenol classes, organic acids, and metal analyses are reported in Tables 3-5. Table 3 shows that the aqueous extracts of B showed the highest content of flavonols but the lowest content of the remaining polyphenol classes, and lowest antioxidant activity and total polyphenol content. The N aqueous extract showed the highest antioxidant capacity, Polyphenols (± SD) of grape skin from pomace of Primitivo cv (P), Negramaro cv (N), Negramaro/Lambrusco cv (N/L), and one white grape skin pomace of Verdeca cv (B).
Results are average ± SD. and total polyphenol and phenolic acid content, while the anthocyanin content of P was the highest for the pomaces extracted here in water. As reported in Table 4, tartaric acid was the most abundant organic acid in aqueous extracts of P, N, and B, followed by citric acid. The content of tartaric and citric acid was quite similar in the three analyzed extracts. Ethanol was not detectable in aqueous extracts. Characterization of the mineral contents in aqueous extracts of grape skins recovered from GP ( Table 5) showed a high and similar content of both Ca and K and a higher content of Mg in B and of Na in P aqueous extract.
Comparison of Different Extraction Treatments in Water
We utilized traditional extraction methods generally based on a low heating process that, although enabling mass transfer among different phases of the extraction system, consume little energy, and prevent degradation of heat sensitive molecules.
To increase the polyphenol extraction yield and shorten extraction time, ultrasound and microwave methods have been investigated (Galanakis, 2013). These treatments aim to avoid/reduce the use of organic solvents, shorten the treatment Minerals (± standard deviation) from 5% w/v, skin of Primitivo pomace (P), skin of Negramaro pomace (N), and skin of Verdeca white pomace (B). Cu detection limit: 0.001 mg/L. std for blank sample: 0.003 (0 ± 0.003). Results are average ± SD.
time, decrease the temperature and energy consumption, increase the extraction yields, and preserve the final extract quality (Medina-Torres et al., 2017). The TP content and antioxidant activity of extracts were determined to evaluate the effects of different extraction methods (Figure 3). Our results indicated a similar TP content (Folin in Figure 3) in samples subjected to UAE, MAE, and H 2 O extraction; these results confirmed that TP are extracted well in water, and UAE or MAE extraction treatments do not improve extraction in any case. An interesting result was obtained for the antioxidant activity recorded after MAE of all the skin pomace samples. Indeed, the antioxidant activity of MAE aqueous extracts was significantly higher (P: 68% more; N: 52% more; and B: 90% more). These data parallel with the amount of anthocyanins extracted, which was higher in the samples extracted by MAE in comparison to the controls extracted overnight at 26 • C (P: 61% more and N: 71% more). It can be assumed that skin pomace samples with a higher anthocyanin content have a higher antioxidant activity.
PRODUCTION AND CHARACTERIZATION OF INFUSIONS Antioxidant Activity, and Polyphenol, Organic Acid, and Essential Metal Content of Skin Pomace Infusions and Skin Pomace-Enriched Infusions
Despite the number of reports in the literature describing the anti-inflammatory, anticancer, and antioxidant activity of herb infusions, there are no clinical studies confirming their healthincreasing properties. Probably, due to lower complexity, herbal infusions are a less suitable delivery vehicle compared to extracts of bioactive ingredients consumed in a controlled dosage (Yin et al., 2017). Therefore, the production of an infusion with a customary and reliable biological effect is a challenge (Tschiggerl and Bucar, 2012).
With the aim to reuse skin pomace powder as an ingredient for infusions and taking into account the results obtained using water extraction, skin pomace powder was incubated in hot water for 5 min. The infusion obtained was analyzed for TP content and related antioxidant properties. Table 6 shows the results of the analyses carried out on infusion of Primitivo skin pomace (P, 2 g) in 100 mL hot water, and infusion of blends of 1 g P and 1 g of tea (P/T), 1 g P and 1 g of rooibos (P/R), and 1 g of P and 1 g of hibiscus dried flowers (P/K) in 100 mL of hot water. The results indicated that the total polyphenol amount and antioxidant capability were significantly different among samples, with highest levels recorded for the blend P/T followed by P/R ( Table 6). The ratios of antioxidant activity (AA) to TP content for P and the blends are 0.012 except for P/T for which the ratio is 0.016 ( Table 6).
Moving to mineral content, Table 7 reports the content in different GP skins infusion blends. Significant amounts of potassium were found in all infusions analyzed. Due to their relatively high mineral content, infusions obtained from wine pomace are interesting alternatives to increase the intake of minerals in the diet, especially potassium, which has essential functions in human health with particular beneficial effects on cardiovascular diseases.
Anti-inflammatory Properties of GP Skin Infusions
Several studies have reported that red grape polyphenols possess desirable biological actions, including cardiovascular protection due to their anti-inflammatory effects and endothelial protection (Castaldo et al., 2019).
The vascular endothelium plays a crucial role in the formation and development of atherosclerotic plaque. Under the influence of inflammatory and atherosclerotic stimuli, the endothelium acquires new functional and phenotypic properties; it becomes adhesive against monocytes due to the overexpression of leukocyte adhesion molecules such as vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1.
With the aim to investigate the potential anti-inflammatory effect of aqueous extracts of red grape skin infusion of Primitivo pomace (P), we analyzed endothelial-monocyte adhesion, the first step essential in the development of atherosclerosis, as well as the stimulated expression of endothelial adhesion molecules in cultured human endothelial cells challenged with the proinflammatory cytokine TNF-α.
To evaluate the effects of P infusion on endothelial-monocyte adhesion, HMEC-1 cells were pre-exposed to aqueous extracts of grape skin infusion of Primitivo pomace (P) at several concentrations (1, 2.5, and 5 mg/mL) for 2 h before stimulation with TNF-α (10 ng/mL).
Since the adhesion of monocytes to endothelium is related to the increased expression of endothelial adhesion molecules, we analyzed the effect of aqueous extracts of red grape Primitivo pomace infusion on the TNF-induced expression of VCAM-1 and ICAM-1, by cell-surface EIA ( Figure 4B). P infusion reduced, in a concentration-dependent manner (1, 2.5, and 5 mg of freeze-dried infusion/mL), the TNF-stimulated expression of endothelial adhesion molecules ICAM-1 and even more of VCAM-1 ( Figure 4B), with 1 mg/mL the lowest effective concentration.
We analyzed the anti-inflammatory properties of infusion obtained with grape skin Primitivo pomace (P) and tea (T) (1:1), named P/T. We compared the effect of the same concentration (2.5 mg/mL) of P/T with P and T on VCAM-1 expression in TNFstimulated endothelial cells. We showed that the P/T blend is more effective in reducing endothelial VCAM-1 expression than P and T separately, lowering TNF-induced VCAM-1 expression by about 70% (Figure 4C).
This study has shown that the infusion of GP skin retains the endothelium-protective properties. The anti-inflammatory effect is even enhanced by the P/T infusion, suggesting its possible use as a functional beverage.
DISCUSSION
The valorisation of by-products is the priority to improve the sustainability of production chains through the reuse, recycling and recovery of energy. Moreover, the final use of a by-product should be integral, positively affecting human health or the environment (Galanakis, 2013). The direct use of wine chain byproducts could be useful in the formulation of functional foods in order to improve consumer health. Mineral composition is also relevant to food production. For example, enzymes that are crucial in the quality of cereal products were stabilized by grape seed flours reach in calcium (Mironeasa et al., 2016). Furthermore, the exploitation of seasonings from wine pomace could improve the mineral content of foods, as it allows a reduction in the salt content. Products derived from grape and grape skin pomace provide interesting alternatives to increase the intake of minerals. For instance, sufficient potassium intake can contribute to control blood pressure and neuromuscular excitability (Rüdel et al., 1984;He and MacGregor, 2010).
To produce a sustainable beverage rich in GP skins, we have determined the conditions (time and temperature) that allow the extraction of the phenolic compounds using water as a solvent. The extracted bioactive molecules can find a wide range of applications in human nutrition, while the remaining organic residues in the extracts are not of concern.
Therefore, the drying process is important to stabilize and storage grape pomace. This drying step could enhance the potential use of pomace as a powerful natural antioxidant ingredient in functional foods. To avoid losing of bioactive compounds, due to their thermal instability, freeze drying is considered to retain higher levels of polyphenols than oven drying (Tseng and Zhao, 2012). As reported by Larrauri et al. (1997), no significant differences in total phenols were found either from freeze dryed or from oven dried (50 • C) GP skin. Moreover, in our conditions, heat treatment over long-lasting periods may favorites the extractability of different low molecular weight compounds, increasing the level of phenolic compounds (Pedroza et al., 2012; FIGURE 4 | (A) Endothelial effect of grape pomace infusions. (A) Aqueous extracts of grape skins infusion of Primitivo pomace (P) decrease monocytoid cell adhesion to endothelial cells. U937 cells greatly adhere to endothelial cells challenged with TNF. P infusion decreases U937 cell adhesion in a concentration dependent manner. (B) Aqueous extracts of grape skin infusion of P inhibits the expression of TNF-stimulated expression of VCAM-1, and ICAM-1. (C) Aqueous extracts of grape skin infusion of blend P/Tea 1:1 (P/T) (2.5 mg/mL) reduces the surface expression of VCAM-1 in TNF-stimulated endothelial cells more than P or tea (T). Data are expressed as the percentage of TNF (mean ± S.D.) and are representative of three different experiments. *p < 0.05 vs. TNF alone. Planinić, 2015). Drying by oven at 50 • C was preferred because it allows the storage of grape skin from pomace and is faster and reproducible (Figure 1). The GP skins of two red grape varieties, Primitivo (P) and Negramaro (N), one blend of Negramaro/Lambrusco (N/L), and one white grape variety Verdeca (B) were stabilized at 50 • C and their extraction in methanol/ethanol was characterized as reported in Figure 2 and Tables 1, 2. N and P extracts showed the highest AA and total polyphenol content, and an interesting composition of phenolic acids, stilbenes, and anthocyanins.
The metal content of GP skins is reported in Table 3 and shows that K is the most abundant metal in all varieties analyzed; similar findings have been reported by Pérez Cid et al. (2019) for grape skins isolated from the pomace of five Galician grape varieties.
The aim of the present work was to extract a significant amount of bioactive molecules, modifying the extraction solvent, finding a good compromise thus between quantity and quality. With the aim to utilize the pomace skin powder to produce a functional beverage, we set up a method to extract different polyphenol classes and other chemical compounds with functional activity in acidified water. Preliminary results suggest that 5% pomace powder dissolved in water is the right ratio of powder to water. As reported in Tables 3-5, we characterized total anthocyanins, flavonols and soluble acids among polyphenols, and four organic acids, alcohol, and metals in this water extract. As reported by Ferri et al. (2016) a lower amount of polyphenols are recovered from dried pomace in comparison to fresh pomace and organic solvents have a positive effect on their recovery.
Although it is necessary to improve the extractability of grape skin pomace in water, the content of these bioactive compounds and AA confirm grape skin pomace as a rich source of bioactive compounds with antioxidant and anti-inflammatory activity and suggest its exploitation as a functional ingredient.
In this study, grape skin extraction with acidified water was investigated in terms of maceration vs. UAE vs. MAE, to verify the differences and increase in the amount of polyphenols in water extract with the aim to produce a "ready-to-drink" functional beverage formulation. Actually, MAE and UAE extraction, even though considerably reducing the processing time, did not increase the amount of polyphenols in water, but the MAE process caused an increase in the AA and anthocyanin content in acidified water extracts. We conclude that these alternative techniques are interesting approaches that require study in more depth.
Several preclinical and clinical studies suggested that chemical synthesized or purified polyphenols do not provide the same biological activity as food matrix rich in the same compounds (Calabriso et al., 2016a,b;Scarano et al., 2018). With the aim to use grape skin isolated from GP to prepare infusions, in this study, hot water extracts were investigated. The AA, polyphenol content, mineral content, and anti-inflammatory effects of infusions formulated from Primitivo pomace skins alone or with added black tea, rooibos, or karkadè were evaluated (Tables 6, 7). As reported by Bekhit et al. (2011), the highest AA and polyphenol content were found in the infusion of grape skin pomace with added tea; moreover, the highest ratio of AA/TP points out that the P/T blend shows a higher AA of the TP fraction. This result is suggestive of a synergistic effect of the different classes of polyphenols contained in the blend. The polyphenols present in skin pomace water extract being rapidly absorbed, metabolized, and excreted, they exhibit low bioavailability. Nevertheless, accumulation of evidence that the beneficial activity of polyphenols occurs in humans is increasing. The potential synergism among different polyphenol metabolites could explain this argumentative result. For this reason, we have analyzed the health-promoting effects of infusions containing different types of bioactive molecules such as those present in grape skin pomace alone and with tea added. This study has shown that infusion of GP skin retain the endothelium-protective properties. The anti-inflammatory effect is even enhanced by the infusion of grape skin pomace and tea, suggesting a possible use as a functional beverage. Functional food/beverages development goal is to increase levels of health-promoting compounds human intake in order to deliver an efficacious amount. In a previous paper we demonstrated, by in vitro study, that polyphenols extracted from red grape skins exhibited beneficial effects at concentrations that are likely to be achieved in the plasma of subjects after moderate red grape skins consumption (Calabriso et al., 2016a). Moreover, the observed inhibitory effects of pure polyphenols, both flavonols and stilbenes, occurred at concentrations higher than those that can be found in red grape skin extracts, this suggests the occurrence of a synergism among different polyphenols in the extracts and that low doses of extracts could exhibit, also in vivo, synergic bioactive health-promoting effects. Moreover, factors that may improve polyphenols bioavailability is the glycosylation of the compound. Therefore, since grape skin pomace beverages contained stilbenes and flavonols mainly as glycosilated forms, it could be expected that they could be effective in vivo also at low doses.
CONCLUSIONS
Studies on wine pomace demonstrate the potential exploitation of this by-product and suggest a new production chain for functional food production. Winemaking by-products can be reused as different food ingredients, such as fibers, polyphenol extracts, grape seed oil, and applied to produce new foods. However, investment costs for new food chain products are often high and a recovery strategy for the use of value products in functional food results in supplementary regulatory concerns. Thus, further scientific research is necessary to achieve significant advances in economic and regulatory problems. This work, substituting organic solvents with acidified water and using alternative techniques like UAE and MAE, meets the sustainable development concept since it reduces time and energy consumption. Our results clearly indicate the good quality of skin pomace acidified water extracts in term of polyphenol, organic acid, and mineral content. Moreover, infusions of grape skins alone and in combination with black tea were tested in cell models, showing anti-inflammatory activity. These attributes, together with its valuable AA, render grape skin pomace a noble by-product with great potential as an ingredient in healthpromoting beverages and foods.
DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author. | v3-fos-license |
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} | pes2o/s2orc | UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition.
Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.
101 cross-links in alternating purine-pyrimidine copolymers. We reasoned that in these polymers there should be a great reduction in UV photoproduct formation since there are no adjacent pyrimidines in the same strand, thus facilitating the identification of any pyrimidine-containing cross-linking photoproducts. However, in studying the alternating copolymer d(GT),.d(CA), this approach did not appear fruitful as we found no evidence of UV-induced cross-linking or DNA photoproduct formation when the polymer was irradiated in Bor A-form.
Both Zimmer et al. (2) and Vorlickova et al. (1) have reported a transition o f d(GT),.d(CA), characterized by a negative maximum of the circular dichroism spectrum in the region of 275-280 nm. They found a variety of solution conditions which produced this spectral alteration, including high concentrations of ethanol, manganese, cesium chloride, cesium fluoride, or ammonium fluoride. These authors speculated that this transition may represent a B-DNA to Z-DNA conversion. Additional investigations of d(GT),.d(CA), under these solution conditions have been carried out in other laboratories. Jenkins et al. ' using NMR and Raman analysis of the polymer at low and high cesium chloride concentrations found a conformational rearrangement; however, the high salt conditions appeared to produce a variation of B-form DNA. Sutherland and Mugavero' (11) examined this transition induced by ethanolic conditions using CD and vacuum ultraviolet CD. They found that the CD alteration at 275-280 nm was not accompanied by the characteristic Z-DNA spectrum in the 180-200 nm region; both the B-form and the alternative form showed a large positive peak at 187 nm, characteristic of right-hand duplex DNA. Given these additional studies it seems likely that the transition first detected by Zimmer et al. ( 2 ) and Vorlickova et al. (1) represents a conversion of Bform polymer to a modified B-form.
We UV irradiated d(GT),.d(CA), in this alternative conformation induced by either ethanolic solution or high manganese concentrations. Under these conditions there was a dose-dependent cross-linking of d(GT), strands with d(CA), strands. We have also detected a thymine containing photoproduct in acid hydrolysates of the polymer. These findings suggest that a property of this alternative conformation is that pyrimidines from different strands are closely approximated, allowing for photodimerization. (12). Alkaline CszS04 equilibrium sedimentation yielded two peaks (AzM)), p = 1.416 and p = 1.492, the expected values for d(GT), and d(CA),, respectively (12). The standard was d(AT),, p = 1.416 (12). Neutral gel analysis (0.3% agarose) revealed that most of the polymer did not migrate out of the sample loading well after lengthy electrophoresis; by extrapolation from X-HindIII markers, we conservatively estimate the size of the double-stranded polymer to exceed 100,000 base pairs. Alkaline gel electrophoresis showed fragments ranging from 200 to 4000 nucleotides with a maximum staining intensity at about 1100 bases. The polymer was visualized in the alkaline gel, following a neutralization step, with ethidium bromide (which binds to doublestranded DNA) or acridine orange (which binds to both single-and double-stranded DNA); results were identical. From this analysis we concluded that the polymer was double-stranded d(GT),.d(CA), with overlapping d(GT), strands and d(CA), strands forming long concatemers with intermittent single-stranded nicks or gaps. The polymer was stored at 1 mg/ml in water or 0.1 M NaCl. The order of addition of the components of the ethanolic solution used in this study was as follows: to an aliquot of polymer solution (1 mg/ ml), first CaClZ (1 mM stock), then CsCl(100 mM stock), then water, and finally 95% ethanol was added. The reason for this protocol is that we had earlier found that addition of 60% ethanol (no salts) to the polymer resulted in a CD spectrum typical of denaturation (13). Subsequent addition of the salts did not alter this CD spectrum.
Ultraviolet Irradiation-Ultraviolet irradiation was performed at 23 "C under a General Electric germicidal bulb with a maximum output at 254 nm. The dose rate was 10 J/mz s as determined by a UVX Digital Radiometer (Ultra-Violet products Inc.).
Circular Dichroism-CD spectra were measured on a Jasco J-500A spectropolarimeter at 23 "C. Polymer concentration was 10 pg/ml in all samples.
Alkaline Agarose Gel Electrophoresis-Following UV irradiation the polymer was ethanol precipitated. It was necessary to ethanol precipitate several times if the polymer had been in MnCIz solution, since any residual Mn2+ precipitated in alkali. Following precipitation the polymer was dissolved in loading solution of 10% glycerol, 0.02% bromocresol green, 100 mM NaOH. Electrophoresis was at 2 V/cm for 3.5 h in 0.5% agarose gels submerged in 30 mM NaOH, 2 mM Na2EDTA. Gels were neutralized and stained in 89 mM Tris-OH, 89 mM boric acid (pH 8.3), 2.5 mM NazEDTA, 0.5 pg/ml ethidium bromide. All lanes contain 5 pg of polymer.
Thin Luyer Chromatography-d(GT),-d(CA), was radioactively labeled by nick translation in the presence of [methyl-3H]WP. The final specific activity was 2-5 X lo6 cpm/pg. Following nick translation the polymer was eluted through a Sephadex G-75 column. The elutant was either 100 mM NaCl, 10 mM Tris (pH 7.5), 2 mM EDTA, 10 mM MnC12, or 4.5 mM CsCl and 0.06 mM CaClZ. In the case of 10 mM MnClZ, following elution the polymer solution was adjusted to final MnCIZ concentrations using a 3.0 M stock. In the case of 4.5 mM CsCl, 0.06 mM CaClZ the polymer solution was adjusted to final concentrations by the addition of water and ethanol. Precipitation of the polymer at the ethanol and salt concentrations used in this study did not occur, as assessed by lack of turbidity and sedimentability, even upon storage at -20 "C overnight. Precipitation could be achieved by the following combination: increasing the ethanol concentrations to 70% (v/v) and adding additional salt (0.5 M NH, acetate, or 0.5 M NaCl final). We have noted that ethanol precipita-tion of the polymer at anytime prior to ultraviolet irradiation decreased photoproduct yield.
Following ultraviolet irradiation, yeast tRNA was added to a final concentration of 1 mg/ml and the sample ethanol precipitated, resuspended in 90% formic acid, and hydrolyzed in combustion vials at 180 "C for 60 min. The formic acid was then removed by vacuum centrifuge and the dried hydrolysate dissolved in a small volume of water and spotted on the TLC plates (silica gel G-60; Merck). The running solvent was 80% ethyl acetate, 20% 1-propanol (v/v) which was water saturated. Plates were developed to 12 or 17.5 cm and processed as described by Reynolds et al. (16). Fractions were 0.5 cm each. Finally, Fig. 1D shows inhibition of the ethanol-induced CD inversion when CaC1, or CsCl was omitted or when 0.1 M NaCl was added. This latter alteration generates a CD spectrum typical of A-form DNA (13). These various solution Conditions were employed in the following experiments designed to assess UV-induced interstrand cross-links.
Fig
Alkaline agarose gel electrophoresis ( Fig. 2) separates single-stranded fragments on the basis of size; the larger the fragment, the slower it migrates (17). If a double-stranded fragment becomes cross-linked, the strands will not separate A€ ' 1 completely upon denaturation and consequently will migrate at the combined molecular weight. There was a dose-dependent increase in molecular weight of the polymer when irradiated in 2 M MnC12 (Fig. 2, left). Examination of the gel shows cross-linked DNA fragments in excess of the 23-kilobase Mind111 marker; indeed some of the polymer was so large it did not move out of the sample loading well. This enhancement of molecular weight may be accounted for as follows: the native polymer is of high molecular weight (greater than 100 kilobase pairs; see "Experimental Procedures"); however, it contains frequent nicks or gaps on both strands yielding fragments averaging about 1100 bases upon alkaline gel electrophoresis (Fig. 2, left, truck 2). Hence, as the number of covalent cross-links increases the denatured polymer approaches native size. It was possible that cross-linking observed in Fig. 2 (left) was not related to conformational alterations in the polymer (e.g. perhaps Mn2+ acts as a catalyst). We therefore assessed UV cross-linking when CD inversion was induced by another condition: 60% ethanol, 1.5 mM csc1, 0.02 mM CaC12. Fig. 2 (middle) shows an increase in cross-linking as a function of UV irradiation in the presence of the ethanolic solution. The enhancement of molecular weight was greater with MnCI2 than with 60% ethanol, 1.5 mM CsC1, 0.02 mM CaCI2. Irradiation in the latter condition appeared to favor the formation of a discrete band at about 2000 bases, probably representing the cross-linking of a single fragment of d(GT), to a single fragment of d(CA),. UV irradiation of d(GT),.d(CA), in B-DNA forming conditions showed no evidence of cross-linking (Fig. 2, right). We considered that the degree of cross-linking may be underestimated in alkaline gel electrophoresis due to alkaline labile sites in the polymer. However, we found that similar experi- Additional experiments were performed in an effort to correlate CD alteration with susceptibility to UV cross-linking. The omission of CaClz or CsCl from the ethanolic solution greatly diminished the ethanol-induced CD spectrum inversion (Fig. 1D). UV irradiation of polymer in the solutions lacking CaClz did not result in detectable cross-linking; however, when CsCl was omitted there was detectable, although diminished cross-linking. Finally, when 0.1 M NaCl was added to polymer in the complete ethanolic solution there was a reversal of the CD spectrum with a large positive At (270 nm) (Fig. 1D). When we UV irradiated the polymer in this environment, we did not detect any cross-linking (Fig. 2, right).
The degree of CD inversion could be controlled by the concentration of ethanol added to the polymer and trace salt solution (see Fig. 1C). The polymer, exhibiting various degrees of CD inversion was irradiated with 2500 J/m2 and subjected to alkaline gel electrophoresis (Fig. 3, left). Below about 45% ethanol there is little or no cross-linking, correlating closely with the lack of CD inversion. This suggests that the effect of ethanol is not that of a reactant or catalyst, otherwise one might expect that the reduction in ethanol concentration from 60 to 40% would have produced only a modest reduction in UV cross-linking. Fig. 3 (right) shows the results of irradiation at the relatively low dose of 500 J/m2 in the presence of varying concentrations of MnC12. The degree of cross-linking reached maximum at 1.0 M MnClz and did not increase at higher concentrations of MnC12 (including 2 M MnC12, not shown). Note that the corresponding CD achieves maximal inversion by 1.0 M (Fig. 1B). Is the reduced cross-linking seen at low manganese concentrations overcome at higher UV doses? Fig. 4 shows the dose dependence of cross-linking in 0.5 MnClz solution. Extensive cross-linking occurs at high doses; however, the maximal degree of cross-linking achieved (2000 and 5000 J/m2 lanes) is less than that seen in 2 M MnClz (Fig. 2, left).
Since Following irradiation or mock irradiation samples were ethanol precipitated 1 to 2 times and then resuspended in alkaline Cs2S04 solution for centrifugation. Because of tube to tube variation in polymer content the optical scans are normalized in each panel such that total area under the curves are equal.
in alkaline Cs2S04 (12), interstrand cross-linking should result in intermediate density material. Fig. 5 shows the results of experiments similar to previous ones, but utilizing alkaline Cs2S04 gradients for analysis of cross-links. d(CA), bands near the top of the gradient (to the left as shown) and d(GT), bands near the bottom (right). When we UV irradiated the polymer in 60% ethanol, 1.5 mM csc1, 0.02 mM CaClz (Fig. 5A), or in 2 M MnClz (Fig. 5B), we saw an intermediate density peak that was dose dependent. The intermediate density peak, indicating the presence of cross-linked polymer, showed slight run to run variation in density, falling within the range of p = 1.460-1.466. The cross-linked polymer in alkaline gradients banded at a greater density than the native polymer in neutral gradients ( p = 1.423). We attribute this difference to the known enhancement of bouyant density that occurs upon alkaline titration of G and T residues (12,18). UV of the polymer in B-form did not show any evidence of cross-link formation (Fig. 5C). Fig. 6 shows a representative thin layer chromatography profile of polymer which had been 3H-labeled in thymine by nick translation, then irradiated, and formic acid hydrolyzed. The hydrolysis degrades DNA to its constituent bases and other by-products (17). The large peak is [3H]thymine; the small peak is evident when irradiation occurred in the setting of inverted CD spectrum. This peak co-migrated with thymine-containing dimer marker obtained from Escherichia coli DNA (irradiated under conventional solution conditions). This TLC system has a low resolution for different dimeric photoproducts, and the presence of a single photoproduct peak does not imply that the novel photoproduct is homogeneous. Fig. 7 shows a typical experiment measuring photoproduct as a function of UV dose using the TLC system. The maximal level of photoproduct varied from experiment to experiment, within the range of 0.8-1.8%. Polymer irradiated in MnClz did not consistently show greater photoproduct formation than polymer irradiated in the ethanol solution. By 5000 J/m2 the formation of photoproduct had consistently achieved maximal values; irradiation up to 25,000 J/m2 did not result in increased photoproduct formation (not shown).
The finding of a plateau value for photoproduct formation, well below the expected available number of potential dimer forming sites, suggests the possibility that UV not only induces photoproduct, but also induces photoreversal of the photoproduct to monomers. This is a typical feature of cyclobutane dimer formation in wild-type DNA (19). We tested this hypothesis by eluting the photoproduct containing TLC fractions in water and then irradiating the photoproduct solution for doses up to 24,000 J/m2. This irradiation condition strongly favors the photomonomerization reaction, since two bases, once cleaved by UV irradiation rapidly diffuse away from each other in solution. We rechromatographed the photoreversed material. Fig. 8 shows an experiment assessing photoreversal of photoproduct originally generated in polymer under MnC12 or ethanol conditions. As with photoproduct formation there was experimental variation, but photoreversal was always greater than 50%. In all experiments photomonomerization was accompanied by quantitative regeneration of the thymine peak (not shown).
We investigated the possibility of cross-link formation in the self-complementary Z-DNA forming polymers d(GC), and d(GmeC),. Fig. 9 (left and middle) (2,20); however, we found that the ethanolic solution employed in these studies (60% ethanol, 1.5 mM CsC1, 0.02 mM CaC12) resulted in a CD spectrum of d(GC), typical of B-DNA and identical with the CD spectrum found in 0.1 M NaCl. In all solution conditions there was no increase in molecular weight of the d(GC), polymer following irradiation of 1500 or 5000 J/m2. It is possible that lack of enhanced molecular weight might be due to hairpin conformation of all the d(GC), strands; however, this possibility was excluded by a comparison of alkaline (Fig. 9, left and middle) and neutral (Fig. 9, right) agarose gel electrophoresis of unirradiated polymer. If the polymer is hairpinned there should be an increase in size (relative to markers) upon alkaline denaturation (e.g. a 500-base pair double-stranded fragment would denature to become a 1000 base single-stranded fragment). Instead of an increase in size we found a large decrease in the size of the polymer upon denaturation, suggesting that the d(GC), polymer was a concatemer under neutral conditions, similar to the d(GT),. d(CA), polymer (see "Experimental Procedures"). This experiment suggests that d(GC), in Z-conformation (4 M NaCl, 2 M MnC12) or in our standard ethanolic solution conditions is not subject to cross-linking by ultraviolet irradiation.
In addition, we assessed cross-linking of d(GmeC), (not shown) in experiments identical to those in Fig. 9, except that 1. any indication of increased molecular weight of polymer upon ultraviolet irradiation. As with d(GC),, comparison of alkaline and neutral agarose gel electrophoresis did not show evidence of hairpin conformation of d(GmeC),.
DISCUSSION
We have investigated the UV photochemistry of d(GT),. d(CA), under a variety of solution conditions. In B-form the polymer is not subject to UV cross-linking or production of photoproduct. However, in ethanolic or manganese solutions that produce a negative peak in the 275 nm region of the CD spectrum, UV produces both interstrand cross-linking and thymine-containing photoproduct detectable in acid hydrolysates of the polymer.
Cross-linking is demonstrated by two different criteria: 1) an increase in polymer size as detected in alkaline agarose gels, and 2) the production of intermediate density material in alkaline cesium sulfate isopycnic gradients. The degree of cross-linking, assessed by alkaline gel electrophoresis, correlates with the degree of CD peak inversion at 275 nm in ethanol and manganese solutions. The CD alterations in ethanolic solution suggest a cooperative transition between 40 and 60% ethanol (1); this is accompanied by susceptibility to UV cross-linking (Fig. 3, left). Alterations in the CD spectrum due to the addition of NaCl or the omission of CsCl or CaC12 from the ethanolic solution are also accompanied by decreased susceptibility to cross-linking (Fig. 2, right). It is noteworthy that although omission of either CsCl or CaC12 from the ethanolic solution produces a similar diminution of negative peak height at 275 nm, the omission of CaC12 reproducibly induces a greater inhibition of cross-linking than omission of CsCl. The concentration of MnCl, in the polymer solution renders the polymer susceptible to UV cross-linking in a fashion that also correlates with CD spectral alterations (Figs. 3, right, and 4). Finally, when the polymer is UV irradiated in B-form no photoproduct is detectable in acid hydrolysates; however, when irradiated in the alternative conformation there is a dose-dependent formation of thymine-containing photoproduct (Fig. 7). A substantial fraction of the photoproduct is photoreversible (Fig. 8).
The correspondence between UV cross-linking and the production of thymine-containing photoproduct in the alternative solution conditions suggests that the photoproduct may be the cross-link. However, the alternative DNA structure may permit formation of multiple photoproducts, one or more of which may be cross-links. In preliminary studies (data not shown) we have chromatographically resolved the photoproduct, as found in trifluoroacetic acid hydrolysates of the polymer, into three peaks. One of these peaks, comprising about 15% of the overall photoproduct, has been tentatively identified as thymine-cytosine cyclobutane dimer. This assignment is based on the following: 1) migration of the photoproduct on two paper chromatographic systems capable of resolving the thymine-cytosine cyclobutane dimer from other photoproducts; and 2) detection of this photoproduct in polymer which has been labeled by nick translation either in [methyl-3H]thymine or [U-'4C]cytosine. This photoproduct should be a covalent cross-link between a d(GT), strand and a d(CA), strand. One may speculate that its isomeric conformation may be other than the usual cis-syn type (10). The other two peaks have not yet been assigned.
A variety of dipyrimidine photoproducts other than crosslinks of d(GT), to d(CA), are possible. Although the isopycnic gradients (Fig. 5) indicate that substantial cross-linking of d(GT), to d(CA), occurs, cross-linking of polymer strands to like polymer strands may also occur. Even more interesting is the possibility of dimerization of nonadjacent pyrimidines within the same strand. This phenomenon was recently discovered by Brown et al. (21) who showed that cytosines, separated by an intervening thymine may be dimerized. This finding supports a model these authors previously proposed for a self-complementary double-stranded conformation of the polymer d(CT), in which there is a core of stacked protonated C'C' base pairs with thymidyl residues being looped out into the solvent. Obviously, the presence of a similar photoproduct in the current studies would have implications for the novel d(GT),. d(CA), structure occurring in the alternative solution conditions.
We have considered the hypothesis that the formation of UV-induced interstrand cross-links in d(GT),-d(CA), may result in stabilization of the alternative conformation, the cross-links serving to "lock" this conformation into place. As a test of this hypothesis we UV irradiated d(GT),.d(CA), up to 5000 J/m2 in the ethanolic or manganese solution conditions, and then dialyzed the polymer into 0.1 M NaCl. CD spectroscopy was then performed. The CD spectra did not suggest retention of the alternative conformation, and in all cases appeared similar to B conformation spectra (data not shown). We speculate that the frequency of cross-links within the polymer is insufficient to restrict the transition to B-form DNA. If one assumes that all photoproduct detected in the thin layer chromatographic assay is indeed cross-link (see Fig. 7), then one would expect only about one cross-link/lOO base pairs at saturating doses of UV.
The current findings suggest that the geometry of the alternative d(GT),. d(CA), structure is such that pyrimidines from the d(GT), strand overlap pyrimidines from the d(CA), strand. One structure that allows for the approximation of pyrimidine residues from different strands is Z-DNA, in which there is partial base stacking of pyrimidines from opposite strands (22). As Zimmer et al, (2) and Vorlickova et al. (1) point out, the negative CD peak at 275 nm is reminiscent of the CD spectrum of Z-DNA. However, the lack of a large positive deflection in the 260 nm region suggests that the structure is other than Z-DNA (23). Also, the studies discussed in the Introduction suggest that the transition is to a variation of B-DNA. In the current work we have investigated the possibility of cross-link formation in the self-complementary 2-DNA forming polymers d(GC), and d(GmeC), (Fig. 9). In a variety of solution conditions producing either B-DNA or Z-DNA conformations, we UV irradiated these polymers up to doses of 5000 J/m2. In no case did we see any increase in molecular weight upon alkaiine gel electrophoresis. It occurred to us that this might be due to a hairpin conformation of all the d(GC), and d(GmeC), strands; however, this latter possibility was excluded by a comparison of neutral and alkaline agarose gel electrophoresis, which showed a large decrease in the size of both polymers upon alkaline denaturation. These experiments suggest that Z-DNA conformation does not predispose to UV cross-linking. Consistent with this is work by Doetsch3 who found that Z-conformation d(GC), did not predispose to formation of UV-induced pyrimidinepyrimidone(6-4) products using the hot alkali assay (24).
A possible tertiary structural change of d(GT),.d(CA), is aggregation, perhaps predisposing towards pyrimidine dimer formation between strands of different helices. However, both sedimentability of the polymer. At our usual working concentrations of polymer, 10 @g/ml, manganese chloride solution conditions also did not yield detectable precipitable polymer. Higher concentrations of polymer (25-50 pg/ml) in manganese solutions of concentrations rl M resulted in both turbidity and precipitable material; however, the UV dose dependence of both interstrand cross-linking and photoproduct formation was independent of polymer concentration (data not shown). In addition, at 0.5 M MnCl,, in which we never detected precipitable material, the polymer was easily cross-linked (Fig. 4). These findings argue against, but do not exclude, a role for aggregation in cross-link and photoproduct formation.
Another structure which would facilitate cross-linking of pyrimidines from opposite strands is the intercalation of bases, rather than Watson-Crick base pairing. Viswamitra and Pandit (25) have proposed that such a structure is stereochemically reasonable and may occur in antiparallel DNA in stretches of base mismatch. It is possible that the solution conditions employed in the current study permit small regions of intercalation without a large rearrangement of the B-DNA conformation. This hypothesis is consistent with the current structural findings about d(GT),. d(CA), in alternative conformation (see Introduction), and may also be consistent with the findings of Glisin and Doty (7) that focal denaturation (but not complete denaturation) of wild-type DNA enhances UV cross-link formation. However, these latter findings have been difficult to reproduce.' Resolution of the structures of the two unidentified photoproducts mentioned above may provide further insight concerning the structural alterations of d(GT),.d(CA),. | v3-fos-license |
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} | pes2o/s2orc | MiRNA Profiling in Plasma Neural-Derived Small Extracellular Vesicles from Patients with Alzheimer’s Disease
Small extracellular vesicles (EVs) are able to pass from the central nervous system (CNS) into peripheral blood and contain molecule markers of their parental origin. The aim of our study was to isolate and characterize total and neural-derived small EVs (NDEVs) and their micro RNA (miRNA) cargo in Alzheimer’s disease (AD) patients. Small NDEVs were isolated from plasma in a population consisting of 40 AD patients and 40 healthy subjects (CTRLs) using high throughput Advanced TaqMan miRNA OpenArrays®, which enables the simultaneous determination of 754 miRNAs. MiR-23a-3p, miR-223-3p, miR-100-3p and miR-190-5p showed a significant dysregulation in small NDEVs from AD patients as compared with controls (1.16 ± 0.49 versus 7.54 ± 2.5, p = 0.026; 9.32 ± 2.27 versus 0.66 ± 0.18, p <0.0001; 0.069 ± 0.01 versus 0.5 ± 0.1, p < 0.0001 and 2.9 ± 1.2 versus 1.93 ± 0.9, p < 0.05, respectively). A further validation analysis confirmed that miR-23a-3p, miR-223-3p and miR-190a-5p levels in small NDEVs from AD patients were significantly upregulated as compared with controls (p = 0.008; p = 0.016; p = 0.003, respectively) whereas miR-100-3p levels were significantly downregulated (p = 0.008). This is the first study that carries out the comparison between total plasma small EV population and NDEVs, demonstrating the presence of a specific AD NDEV miRNA signature.
Background
Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by cognitive decline and memory impairment. The neuropathological hallmarks include the deposition of senile plaques resulting from the excessive accumulation of β-amyloid protein (Aβ) and the formation of neurofibrillary tangles (NFTs) derived from hyperphosphorylated tau proteins leading globally to neural dysfunction [1]. Increasing epidemiological evidence shows that there is a presymptomatic long-lasting period for clinical incubation of the disease that could take several years before leading to cognitive dysfunction. The long preclinical period of AD makes it necessary to seek robust and reliable diagnostic biomarkers for early intervention. At present, diagnostic research criteria for AD [2] include emission tomography (PET) with amyloid tracer, which is a complex and very expensive tool, and the cerebrospinal fluid (CSF) pathogenic protein detection, that implies the invasive procedure of sample collection by lumbar puncture. In this scenario, circulating biomarkers would be of help for differential diagnosis and possibly for monitoring cognitive decline.
Promising candidates are small extracellular vesicles (EVs) that are extracellular membrane vesicles of endosomal origin with a diameter ranging from 30 to 150 nm. Initially referred to as "exosomes" by the international scientific community, they were later addressed by the International Society for Extracellular Vesicles (ISEV) as "small EVs" [3]. They are produced by all cell types and contain several molecular markers of their parental origin. They also include proteins, lipids and nucleic acids, named EV "cargo". The EV "cargo" varies depending on the physiological state of the cell [4]. Cells from the central nervous system (CNS), neurons, astrocytes, oligodendrocytes are also able to secrete small EVs into the extracellular compartment, both in physiological and pathological conditions [5]. They could thus reflect the state of their neural cell of origin, an aspect that may contribute to the diagnosis of neurodegenerative diseases [6]. In this regard, Fiandaca et al. were able to isolate small neural-derived extracellular vesicles (NDEVs) from plasma using a combination of chemical and immunochemical approaches based on specific antibody against neural L1 cell adhesion molecule (L1CAM) and to evaluate their protein content [7]. This challenging approach for the isolation of a specific subpopulation of vesicles has generated increasing interest in using small NDEVs as a source for biomarkers in neurodegenerative diseases including AD [8][9][10]. The ability to analyze the content of plasma small NDEVs has been demonstrated on several fronts to be relevant to AD pathogenesis and also highlights the role of small NDEVs as promising biomarkers. This hypothesis has also been supported by increasing evidence including levels of amyloid-β and tau proteins, as well as the synaptic proteins [11] involved in AD pathogenesis.
As mentioned above, the EVs' "cargo" also comprises nucleic acids such as RNA. This aspect also generated an interest in the field of neurodegenerative disorders after the discovery of EVs as mediators delivering microRNAs (miRNAs) in intercellular communication [12] or the source of miRNAs as candidates for biomarkers of disease [13,14].
MiRNAs are endogenous small noncoding RNAs of 21-23nt in length that are capable of controlling gene expression through post-transcriptional regulation. MiRNAs exert their regulatory effect by suppressing translation of mRNA through the binding to the 3 -untranslated region (UTR) of target mRNA or by degrading target mRNA. The same miRNA can target different mRNAs contemporarily, whereas a single mRNA can be regulated by different miRNAs.
MiRNAs have been identified in many biological fluids, such as plasma, serum, CSF, urine [15], highlighting their potential role as peripheral noninvasive biomarkers of several pathological conditions, including cardiovascular diseases, cancer and neurodegenerative diseases [16,17]. A growing body of evidence demonstrated that they are intimately involved in synaptic function and in specific signals during memory development [15]. Moreover, in vivo experiments showed that Aβ and Tau pathology drove the deregulation of some neuronal miRNAs (miR-142-5p, miR-146a-5p, miR-155-5p), alterations confirmed also in AD patients [18]. MiRNAs could also have a predominant role in driving the pathogenic process of the disease as it was shown that most altered miRNAs also target AD relevant pathogenic proteins [19]. Several miRNAs have been robustly identified as deregulated in brain tissue Cells 2020, 9,1443 3 of 20 from AD patients as recently reviewed in Herrera-Espejo et al. [20], whereas others were proposed as circulating peripheral biomarkers of disease, although none of them had the same regulation status in all studies [20].
Extracellular miRNAs in serum and plasma are found in different fractions [21]. Usually, they are encapsulated in membrane vesicles or are released from apoptotic bodies [22]. Most circulating miRNAs are, however, bound to proteins such as Argonaute2 [23].
The high heterogeneity of the results on circulating miRNAs levels in AD casts a shadow on their real diagnostic potential. Moreover, the origin of circulating miRNAs is heterogeneous itself and likely could not reflect the specific pathological status. Lastly, independent of the localization that could be protein-or vesicle-bound, miRNAs in serum or plasma could hardly trace back their cellular origin.
In this scenario, small NDEVs could be considered a promising source of miRNAs that could directly reflect the physiological condition of the nervous system without introducing confounding factors.
It was already proven that EVs represented an enriched source of noncoding RNAs of different types, such as miRNAs, that, in this way, result protected from RNase degradation. This peculiar aspect represents the solid foundation for their clinical application as diagnostic biomarkers. Moreover, the process of packaging of miRNAs into small EVs in cytoplasm is a finely regulated event that includes multiple steps, supporting the active functional role of miRNAs in these vesicles.
MiRNAs released from EVs could modulate the expression and function of amyloid precursor protein and tau proteins. EV-carried miRNAs could drive, via Toll-like receptors, inflammatory processes in AD and may also regulate neuroplasticity to relieve neurological damage [24].
Moreover, Lugli et al. found a consistent dysregulation of miRNA levels in plasma-derived EVs from AD patients pointing out a specific signature of seven miRNAs that were able to predict the group identity [12].
Given these premises, herein we carried out a comprehensive characterization of small NDEVs and a further analysis of their miRNA content for the detection of possible dysregulated miRNAs in patients with AD. We evaluated simultaneously miRNAs content from total plasma small EVs and small NDEVs to be certain of specifically isolating those of neuronal derivation from the others deriving from the total small EVs population.
Our final goal was to detect a specific small NDEVs miRNA signature in plasma from patients with AD. Assuming that pathogenic alterations in such NDEVs may reflect the brain environment, this research could lead to the identification of potential reliable peripheral biomarkers for early diagnosis of the disease.
Study Design and Sample Collection
Forty patients with AD were recruited at the Alzheimer Center of the University of Milan, Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico, between 2015 and 2017. All patients underwent a clinical interview, neurological and neuropsychological examination, routine blood tests, brain MRI and lumbar puncture (LP) for quantification of the CSF biomarkers Aβ, total tau and tau phosphorylated at position 181 (Ptau). Cut-off thresholds of normality were: Aβ ≥ 600 pg/mL; tau ≤ 500 pg/mL for individuals older than 70 years and ≤450 pg/mL for individuals aged between 50 and 70 years; Ptau ≤ 61 pg/mL [16]. The clinical diagnosis of AD was supported by CSF signature, consisting of decreased Aβ and increased tau and Ptau levels, according to the criteria of the International Working Group [2]. Details of the biomarker levels of all participants are provided in Supplementary Table S1 controls (CTRLs) were also collected. All individuals underwent LP in suspicion of a CNS disease and were discharged with no evidence of neurological diseases and cognitive impairment. None of them developed cognitive decline over time and, at inclusion, MMSE was ≥28. Whole blood samples, collected at the time of diagnosis, were allowed to sit at room temperature for a minimum of 30 min and a max of 2 h, after collection. Separation of the clot was done by centrifugation at 1000-1300× g at room temperature for 15-20 min. Plasma was removed and dispensed in aliquots of 550 µL into cryo-tubes and stored at −80 • C until use. Informed consent to participate in this study was given by all subjects or their caregivers. The study was approved by the local ethics committees (Parere 66/2016 Ethics Committee IRCCS Fatebenefratelli and Parere 532_2019bis del 13-6-2019 -Comitato Etico Milano Area 2).
Isolation of Total EVs from Plasma
Aliquots of 500 µL of plasma were centrifuged at 3000× g for 15 min to remove cells and debris The supernatant was transferred to a sterile vessel and incubated with 5 µL of purified thrombin ([500 U/mL], System Biosciences) to a final concentration of 5 U/mL for 5 min at room temperature. This step is required to remove the large amount of fibrin present in the plasma. After incubation, samples were centrifuged at 10,000 rpm for 5 min. Supernatants were incubated with 126 µL of ExoQuick precipitation solution (EXOQ; System Biosciences) and refrigerated for 30 min at 4 • C. The resulting suspension was centrifuged at 1500× g for 30 min at room temperature. The pellet was then re-suspended in 500 µL of 0.2 µm filtered 1X PBS and conserved at −80 • C until use.
Selective Capture of CD81 and L1CAM-Positive Small Neuronal-Derived EVs (NDEVs) from Plasma with Magnetic Beads and Sorting of EVs Based on Flow Cytometric (FACS) Analysis
The Exo-Flow Exosome Capture kit (System Biosciences) for the selective capture and flow sorting of NDEVs purification based on a particular surface marker was used according to the instruction of the manufacturer. The procedure enables selective capture for immunopurification and flow sorting to separate the distinct subpopulations of small EVs, based on the L1CAM or CD81 surface marker. Previously isolated small EVs were resuspended and bound to the magnetic beads for selective capture and subsequent FACS analysis and sorting. The specific beads involved have 9.1 µm diameter, a characteristic that enables great vesicle capture.
Supernatants containing the eluted vesicles were carefully removed and transferred to fresh tubes for further analyses. Supernatants were subjected to sorting by cell sorting (FACSAria SORP and FACSDiva software, Becton Dickinson, San Josè, CA) and L1CAM NDEVs were selected. In particular, small NDEVs were identified based on a gating strategy, including a first region on physical properties and a second one on FITC fluorescence intensity. Specifically, the protocol involves, for each sample and the negative control, the addition of the bead slurry (40 µL) to 1.5 mL tubes and the further incubation on a magnetic stand for 2 min. Then they were washed twice. The tubes with bead slurry were removed from the magnetic stand and 10 µL of mouse anti-human CD171 (L1CAM, neural adhesion protein) biotinylated capture antibody (clone 5G3, eBioscience) and anti-human CD81 (System Biosciences), both at a concentration of 100 ng/µL in 1X PBS, were added. After mixing, the tubes were incubated on ice for 2 h with gentle mixing by flicking every 30 min. The capture antibody-beads were suspended with 400 µL of wash buffer. The plasma isolated EVs (or PBS for negative control) were added to bead samples to a final total volume of 500 µL and placed on a rotating shelf at 4 • C overnight for capture. A portion of 240 µL of exosome stain buffer and 10 µL exo-fluorescein isothiocyanate (FITC) exosome staining solution were added to the beads and incubated on ice for 2 h with gently flicking every 30 min to mix. Samples were washed 3 times in place on the magnetic stand, followed by the addition of 300 µL wash buffer for FACS analysis, without vortexing. Flow-sort stained EV/bead complexes were incubated with 300 µL of exosome elution buffer for 40 min at room temperature on a rotating rack. Finally, the supernatants containing L1CAM positive EVs were obtained.
Transmission Electron Microscopy (TEM)
A total of 10 µL of EV-enriched solution was placed on a copper mesh and incubated for 10 min at room temperature. The grids were then dried by placing them sideways on filter paper and the EV-enriched fraction was contrasted by using a solution of saturated uranyl acetate in water for 5-10 min. Samples were subsequently dried. The copper mesh was then observed, and pictures captured with a transmission electron microscope (Leo912ab 80 kv).
Characterization of Small NDEVs by Nanoparticle Tracking Analysis (NTA)
Suspensions containing total small EVs and small NDEVs from patients and controls were analyzed using the Nano-Sight NS300 instrument (Malvern, Worcestershire, UK), a laser-based, light-scattering system which provides a reproducible platform for nanoparticle characterization. Prior to analysis, samples were diluted in PBS to obtain a range of 20-200 particles in the field of view (particles per frame, PF). For each sample, 5 videos of 60 s duration were taken. Data were processed using NanoSight NTA Software 3.2 (Malvern, Worcestershire, UK). The NTA post acquisition settings were optimized and kept constant between samples. Data obtained were particle size distribution (D-values; D10, D50 and D90: a particle size value indicating that, respectively, 10%, 50% and 90% of the distribution is below this value) and concentration (particles/mL).
Total Plasma EVs Protein Quantification
The protein concentration of total plasma EVs, extracted from two samples, was quantified by Micro BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer's specifications and using BSA (ThermoFisher Scientific, Waltham, MA, USA) as a standard. Values were extrapolated from this curve, using a third-order polynomial equation, with r 2 > 0.98 for each assay.
RNA Extraction from Total and Neural Derived Plasma EVs
Total RNA was extracted by using Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) that uses Acid-Phenol/Chloroform extraction to provide a robust front-end RNA purification step, followed by a final RNA purification over a glass-fiber filter. Ethanol was added to samples that were passed through a Filter Cartridge containing a glass-fiber filter which immobilizes the RNA. The filter was washed, and the RNA eluted with 30 µL low ionic-strength solution according to the manufacturer's instructions.
The RNA from total small EVs was analyzed using Agilent 2100 Bioanalyzer and RNA 6000 Nano kit (Agilent Technologies, CA, USA) for the determination of RNA concentration, purity, and integrity.
Expression Analysis of microRNA by TaqMan OpenArray Human Advanced MicroRNA Panel
Total RNA was reverse transcribed using the TaqMan Advanced miRNA cDNA Synthesis Kit according to the instructions of the manufacturer (Thermo Fischer Scientific, Waltham, MA, USA). Given the low RNA amounts obtained from NDEVs, cDNA was preamplified prior to the final real-time PCR step. The preamplification step was performed using the TaqMan PreAmp Master Mix and pooled gene-specific primers. The preamplified cDNA (diluted 1:20) was mixed with 2X TaqMan OpenArray Real-Time Master Mix to perform real-time PCR on the OpenArray plate. A 5 µL sample of PCR reactions were distributed into each well of 384-well plate and then samples with the master mix were loaded from the 384-well sample plate onto the OpenArray plate using the OpenArrayAccuFill System. Finally, PCR was run on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA, USA). For single-tube expression analysis, specific miRNA Advanced TaqMan probes were used (478532_mir, 477983_mir, 478619_mir, 478358_mir, 478293_mir) according to the instructions of the manufacturer.
RNase Treatment and Protection Assay
Small EVs isolated from 4 samples (total plasma and NDEVs) were used for miRNA RNase protection assay. Three conditions for the same sample were used prior to RNA extraction. The first condition was EVs treated with 20 mU/mL RNase I in order to eliminate all free circulating RNAs; the second condition consisted of EVs treated with 20 mU/mL RNase I and 1% TritonX-100 to disrupt membranes. The third consisted of EVs without treatments used as controls. All samples were incubated at room temperature for 20 min on a rocking wheel [25,26]. After incubation the RNA was extracted and the miRNA gene expression assay was carried out as previously described [16].
Target Gene Prediction and Bioinformatics Analysis
For miRNAs confirmed in the validation phase, target prediction was performed using TargetScan v 7.1 [27]. DIANA-mirPath was used to perform target prediction and pathway analysis based on two algorithms, microT-CDS and miRTarBase. The software performs an enrichment analysis of multiple miRNA target genes to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The graphical output of the program provides an overview of the parts of the pathway modulated by selected miRNAs. The statistical significance value associated with the identified biological pathways was calculated by mirPath [28]. Next, the tissue location of enriched expression for targets was determinate using FunRich v.3.0 (www.funrich.org) which combines multiple established databases including UniProt, Human Protein Atlas, Human Proteome Browser, Human Proteome Map, ProteomicsDB and Human Proteinpedia to infer regional and cell-type enrichment of target mRNAs for a given list of miRNAs [29].
Normalization and Statistical Analyses
Exploratory data analysis was performed using ClustVis (https://biit.cs.ut.ee/clustvis/). Normalized delta Cq-values (dCq) were used for clustering analysis based on Euclidean distance and complete linkage. Unit variance row scaling was applied for hierarchical clustering and principal component analysis (PCA) [30,31].
Results from the real time PCR will be calculated as Fold-Change [2ˆ(-∆∆Ct)], which is the normalized gene expression [2ˆ(-∆Ct)] in the AD group divided by the normalized gene expression [2ˆ(-∆Ct)] in the control group.
GraphPad Prism 6.0 software (San Diego, CA, USA) was used for statistical analysis. Fisher's exact test and the student's t-test were used to compare gender and age distribution. Test of normality was performed by using the Shapiro−Wilk's test. Subsequently, comparisons of ncRNAs levels, expressed by relative quantification (RQ), between patients and controls were performed by using the nonparametric Mann−Whitney test and the correction for multiple comparisons was performed using the post hoc Dunn's test. The selection of miRNAs for normalization was performed using the algorithm NormFinder. From this, the spike-in cel-miR-39 as well as miR-20b-5p and miR-21-5p were used to normalize across all Ct values using a combined geometric mean of all three Ct values [32].
Availability of Data and Materials
All data relevant to the study are included in the article or as supplementary information. Upon reasonable request, additional information (e.g., protocols) will be shared by the corresponding author.
Ethics Approval and Consent to Participate
All subjects gave written consent and agreed to be in the study, and biological specimens were obtained after consent with approval of local CE: Parere 66/2016 Ethics Committee IRCCS Fatebenefratelli and Parere 532_2019bis del 13-6-2019 -Comitato Etico Milano Area 2.
Characterization of Total and L1CAM Positive EVs from Plasma
A population consisting of 40 AD patients and 40 healthy controls (CTRLs) was investigated. The study was structured as follows: (1) discovery phase involving 20 AD patients and 20 CTRLs tested for 754 candidate miRNAs and (2) validation phase, involving the remaining 20 AD samples and 20 controls, for the validation of previously determined best hits miRNAs.
Participants' demographic and clinical information are summarized in Table 1. Small EVs fractions were characterized for the presence of specific surface markers, size, morphology with three different experiments as suggested by the ISEV [3]. Initially, the size of the plasma derived EVs, including the neural-derived fraction, was evaluated with NTA, then different vesicle markers were tested by Western blotting. Additionally, TEM analysis for total and small neural-derived EVs was performed. NTA confirmed that the total EV suspensions were enriched in small EVs. In particular, NTA revealed the following size distribution (mean ± SD): D10: 70 ± 1.3 nm; D50: 97 ± 1.9 nm; D90: 196 ± 9.9 nm with a particle concentration of 6.7 ± 0.8 × 10 11 particles/mL); the negative control was undetectable, i.e., below the detection limit of the instrument, thus not interfering with the sample analysis ( Figure 1). The protein concentration of the total plasma EVs was also evaluated in two samples, and an EV-associated protein concentration of 189.67 ± 98.2 µg/mL was reported. The ratio of particle counts and protein concentration as described by Webber and Clayton, 2013, was calculated, in order to test the quality of the purification process [33]. A ratio of 3.5 × 10 9 p/µg was obtained suggesting good purity of the total plasma EV preparations [33]. A substantial dimensional heterogeneity was detected by isolation with Exoquick, attributable to different types of vesicles, including exosomes, classified as under 100-150 nm, and larger microvesicles, usually above 100-150 nm. Due to the difference in the detected vesicle size and according to the "minimal information for studies of EVs (MISEV)" guidelines it was chosen to refer to them as small EVs [3].
NTA analysis on small NDEVs was not feasible, since the negative control had a signal intensity overlapping with the one from NDEVs suspension (Supplementary Figure S1) possibly due to antibody aggregates' cross-reactivity.
The neural-derived enriched fraction was isolated from the plasma of all the AD patients and all the healthy controls by using the neuronal marker L1CAM after sorting by flow cytometry (Figure 2a, b). The same approach was used to isolate total EVs present in plasma that were selected for the general marker CD81. In order to confirm the presence and the origin of the isolated vesicles, NDEVs and total EVs underwent further steps of characterization. A substantial dimensional heterogeneity was detected by isolation with Exoquick, attributable to different types of vesicles, including exosomes, classified as under 100-150 nm, and larger microvesicles, usually above 100-150 nm. Due to the difference in the detected vesicle size and according to the "minimal information for studies of EVs (MISEV)" guidelines it was chosen to refer to them as small EVs [3].
NTA analysis on small NDEVs was not feasible, since the negative control had a signal intensity overlapping with the one from NDEVs suspension (Supplementary Figure S1) possibly due to antibody aggregates' cross-reactivity.
The neural-derived enriched fraction was isolated from the plasma of all the AD patients and all the healthy controls by using the neuronal marker L1CAM after sorting by flow cytometry (Figure 2a,b). The same approach was used to isolate total EVs present in plasma that were selected for the general marker CD81. In order to confirm the presence and the origin of the isolated vesicles, NDEVs and total EVs underwent further steps of characterization. TEM revealed a homogenous population of morphologically distinctive particles, mainly ovalshaped, approximately 150 nm in diameter of EVs labeled with anti-CD81, known as general small vesicle marker, and L1CAM marker (Figure 2c,d). Furthermore, we did not find substantial differences in the morphology of EVs isolated from fresh samples (e) compared with frozen ones (f). Western blot analysis confirmed the presence of the neural marker L1CAM in the extracted vesicle subpopulation. As shown in Figure 2g, we found NDEVs immunoreactivity for both L1CAM and the common vesicle markers CD9, Tsg101 and the protein retromer Vps35.
MiRNAs Expression Profile in Total Plasma EVs and NDEVs in AD Patients and Healthy Subjects
RNA from total plasma EVs and NDEVs was initially isolated from 20 AD samples and 20 controls and the quality and size of extracted RNA were verified by bioanalyzer (Supplementary TEM revealed a homogenous population of morphologically distinctive particles, mainly oval-shaped, approximately 150 nm in diameter of EVs labeled with anti-CD81, known as general small vesicle marker, and L1CAM marker (Figure 2c,d). Furthermore, we did not find substantial differences in the morphology of EVs isolated from fresh samples (e) compared with frozen ones (f). Western blot analysis confirmed the presence of the neural marker L1CAM in the extracted vesicle subpopulation. As shown in Figure 2g, we found NDEVs immunoreactivity for both L1CAM and the common vesicle markers CD9, Tsg101 and the protein retromer Vps35.
MiRNAs Expression Profile in Total Plasma EVs and NDEVs in AD Patients and Healthy Subjects
RNA from total plasma EVs and NDEVs was initially isolated from 20 AD samples and 20 controls and the quality and size of extracted RNA were verified by bioanalyzer (Supplementary Figure S2). Thereafter, miRNAs were profiled by RT-qPCR using the TaqMan Open Array miRNA panel, which enables the testing of 754 human miRNAs. Raw data underwent quality control measures.
Applying a Crt cutoff < 28 and the amp score > 1 (measures of good amplification quality), a total of 57 miRNAs were detected across all 40 samples (Supplementary Table S2). For the normalization, Crt values of spike-in cel-miR-39 and miR-20b-5p along with the endogenous miR-21-5p were combined to generate the geometric mean using the available protocol of Andersen [32]. A specific distribution of miRNA expression was observed for both the EV populations analyzed, although there was an overlap for some miRNA expression between the fractions. The most variable miRNAs in the total EVs ( Figure 3) and NDEV (Figure 4) enriched fraction were selected based on the coefficient of variation and used for principal component analysis (PCA) and hierarchical clustering. These analyses of all identified miRNAs in both EV fractions revealed that the primary miRNA expression data were influenced by the disease. In particular, PCA showed a separation of samples based on disease status.
In order to confirm the specific vesicle-packed origin of these miRNAs, an RNase protection assay was performed prior to RNA extraction on the specific miRNAs, miR-223-3p and miR-190a-5p, miR-23a-3p, previously detected. Cells 2020, 9, x FOR PEER REVIEW 10 of 21 Figure S2). Thereafter, miRNAs were profiled by RT-qPCR using the TaqMan Open Array miRNA panel, which enables the testing of 754 human miRNAs. Raw data underwent quality control measures. Applying a Crt cutoff < 28 and the amp score > 1 (measures of good amplification quality), a total of 57 miRNAs were detected across all 40 samples (Supplementary Table S2). For the normalization, Crt values of spike-in cel-miR-39 and miR-20b-5p along with the endogenous miR-21-5p were combined to generate the geometric mean using the available protocol of Andersen [32]. A specific distribution of miRNA expression was observed for both the EV populations analyzed, although there was an overlap for some miRNA expression between the fractions. The most variable miRNAs in the total EVs ( Figure 3) and NDEV ( Figure 4) enriched fraction were selected based on the coefficient of variation and used for principal component analysis (PCA) and hierarchical clustering. These analyses of all identified miRNAs in both EV fractions revealed that the primary miRNA expression data were influenced by the disease. In particular, PCA showed a separation of samples based on disease status. As shown in Figure 5a, 59% of miRNAs expressed was found in total plasma EVs, but not in NDEVs, 15% overlapped between the two subpopulations and 26% was found only in the NDEVs (Supplementary Table S2). Specifically, considering the total EVs fraction, increased relative expression levels (greater than 2.5 fold) of miR-146b-5p, miR-181a-3p, miR-24-3p, miR-125a-5p, let-7b-5p and miR-27a-5p, miR-185-3p were observed (Figure 3b) in patients versus CTRLs, whereas decreased relative expression levels (lower than 2.5 fold) of miR-16-5p, miR-15b-5p, miR-30a-5p and miR-204-5p were detected, although the statistical significance was not reached. Considering miRNAs detected in NDEVs, a specific signature was observed: miR-1260a, miR-1-3p, miR-448, miR-628-3p, miR-653-5p, miR-452-3p, miR-502-3p and miR-190a-5p were in fact specifically detected in the neuronal-derived EVs fraction and an overall upregulation (greater than 2.5 fold) was observed in patients compared to CTRLs. Moreover, a Mann−Whitney test uncovered a statistically significant upregulation of miR-23a-3p, miR-223-3p, miR-190-5p (1.16 ± 0.49 versus 7.54 ± 2.5, p = 0.026; 9.32 ± 2.27 versus 0.66 ± 0.18, p < 0.0001; 2.9 ± 1.2 versus 1.93 ± 0.9, p < 0.05, respectively) Results showed that the miRNAs expression level was slightly decreased by treatment with RNase I, but it was mostly decreased when detergent was added to disrupt the membranes. These results suggested that miRNAs were all well protected by the EV membrane in total plasma as well as the neuronal-derived ones ( Figure 6). These data revealed that a significant amount of miR-223-3p, miR-190a-5p and miR-23a-3p existed in a vesicle associated manner. Figure 6. RNase protection assay. The miR-223-3p, miR-190-5p and miR-23a-3p were quantified by q-PCR in total and neural-derived plasma EVs. EVs were treated with or without RNase I and/or Triton X-100 (n = 4).
To deepen the effective robustness of statistically significant NDEVs' miRNA expression results (shown in Figure 5b), a further gene expression analysis of miR-23a-3p, miR-223-3p, miR-190-5p and miR-100-3p was done on NDEVs extracted from the validation cohort samples consisting of 20 AD patients and 20 CTRLs.
Results showed that the miRNAs expression level was slightly decreased by treatment with RNase I, but it was mostly decreased when detergent was added to disrupt the membranes. These results suggested that miRNAs were all well protected by the EV membrane in total plasma as well as the neuronal-derived ones ( Figure 6). These data revealed that a significant amount of miR-223-3p, miR-190a-5p and miR-23a-3p existed in a vesicle associated manner.
To deepen the effective robustness of statistically significant NDEVs' miRNA expression results (shown in Figure 5b), a further gene expression analysis of miR-23a-3p, miR-223-3p, miR-190-5p and miR-100-3p was done on NDEVs extracted from the validation cohort samples consisting of 20 AD patients and 20 CTRLs.
miRNA Targets and Pathways Prediction
The differentially deregulated microRNAs in patients were screened for their possible target genes and biological pathways using the TargetScan v7.1 algorithm. According to the analysis, a total of 1332 mRNA targets for miR-23a-3p, 412 for miR-223-3p, 3168 for miR-100-3p and 224 mRNA targets for miR-190a-5p were identified. Overlaps between mRNA targets is evident between the combination of two miRNAs, but no targets are shared among all of them (Figure 8a).
miRNA Targets and Pathways Prediction
The differentially deregulated microRNAs in patients were screened for their possible target genes and biological pathways using the TargetScan v7.1 algorithm. According to the analysis, a total of 1332 mRNA targets for miR-23a-3p, 412 for miR-223-3p, 3168 for miR-100-3p and 224 mRNA targets for miR-190a-5p were identified. Overlaps between mRNA targets is evident between the combination of two miRNAs, but no targets are shared among all of them (Figure 8a). Analysis with DIANA-miRpath (v.3.0) showed that combinations of at least two miRNAs overlapped in pathways such as steroid biosynthesis, proteoglycan in cancers, mTOR signaling and prion disease pathway (Figure 8b).
For miRNAs specifically detected in NDEVs, the analysis with DIANA-miRpath identified overlapping specific CNS pathways considering at least three miRNAs, such as the glioma, axon guidance, long term depression and calcium signaling ones (Figure 9a). To assess brain region and cell-type specific localization of NDEVs miRNAs, the FunRich v.3.0 online available tool was used [29]. Results revealed that mRNAs targets were depleted in peripheral blood cells, macrophages and red blood cells, as expected. Notably, the markers of the brain, hippocampus, cortex and cerebellum were instead enriched (Figure 9b) Analysis with DIANA-miRpath (v.3.0) showed that combinations of at least two miRNAs overlapped in pathways such as steroid biosynthesis, proteoglycan in cancers, mTOR signaling and prion disease pathway (Figure 8b).
For miRNAs specifically detected in NDEVs, the analysis with DIANA-miRpath identified overlapping specific CNS pathways considering at least three miRNAs, such as the glioma, axon guidance, long term depression and calcium signaling ones (Figure 9a). To assess brain region and cell-type specific localization of NDEVs miRNAs, the FunRich v.3.0 online available tool was used [29]. Results revealed that mRNAs targets were depleted in peripheral blood cells, macrophages and red blood cells, as expected. Notably, the markers of the brain, hippocampus, cortex and cerebellum were instead enriched (Figure 9b The percentage of targets showing regional enrichment (blue bar) and the significance of regional enrichment (red bar). Top x-axis is -log10 (p-value) of regional enrichment; bottom x-axis is the percentage of targets overlapping with the region.
Discussion
Herein, we performed for the first time a comprehensive characterization of small NDEVs, and identified a specific AD signature, consisting of significantly increased levels of miR-23a-3p, miR-223-3p, miR-190a-5p and decreased levels of miR-100-3p.
The need for peripheral biomarkers derives from the invasiveness of the procedure for CSF collection. In this scenario, NDEVs are candidates for harboring biomarkers reflecting CNS pathogenic AD alterations.
Recent studies showed that the NDEV enriched fraction represents a powerful reservoir of biomarkers for the investigation of key molecules involved in the pathogenesis of AD and related dementias [7][8][9].
The methodology developed by Fiandaca et al. [7] for the isolation of small NDEVs represents a modification of the original one published by Mitsuhashi, et al. [34], pioneers in the detection of exosomes in plasma from specific cellular derivation. The protocol basically involves an immunochemical enrichment of exosomes from a neural source using cocktails of biotinylated antibodies and subsequent steps of incubation with streptavidin agarose resin. Despite the published protocol being successfully applied for the determination of different proteins in neuronal-derived exosomes, there is still a lack of standardization of the method. Conversely, the methodology herein described could be easily approached for the harmonization of a shared protocol among laboratories, . The percentage of targets showing regional enrichment (blue bar) and the significance of regional enrichment (red bar). Top x-axis is -log10 (p-value) of regional enrichment; bottom x-axis is the percentage of targets overlapping with the region.
Discussion
Herein, we performed for the first time a comprehensive characterization of small NDEVs, and identified a specific AD signature, consisting of significantly increased levels of miR-23a-3p, miR-223-3p, miR-190a-5p and decreased levels of miR-100-3p.
The need for peripheral biomarkers derives from the invasiveness of the procedure for CSF collection. In this scenario, NDEVs are candidates for harboring biomarkers reflecting CNS pathogenic AD alterations.
Recent studies showed that the NDEV enriched fraction represents a powerful reservoir of biomarkers for the investigation of key molecules involved in the pathogenesis of AD and related dementias [7][8][9].
The methodology developed by Fiandaca et al. [7] for the isolation of small NDEVs represents a modification of the original one published by Mitsuhashi, et al. [34], pioneers in the detection of exosomes in plasma from specific cellular derivation. The protocol basically involves an immunochemical enrichment of exosomes from a neural source using cocktails of biotinylated antibodies and subsequent steps of incubation with streptavidin agarose resin. Despite the published protocol being successfully applied for the determination of different proteins in neuronal-derived exosomes, there is still a lack of standardization of the method. Conversely, the methodology herein described could be easily approached for the harmonization of a shared protocol among laboratories, as it is based on the use of commercially available products already proven to isolate exosomes with a good reliability and to be adaptable to the needs of specific enrichment for a neuronal population of extracellular vesicles [35]. To provide a complete and objective assessment of this methodology, it is important to mention that this isolation protocol has an important impact on the quality of isolated EVs [36], and on the amount of miRNAs obtained from the EVs' preparation [37].
However, since NTA analysis revealed a significant dimensional heterogeneity among the detected vesicles, we chose to address them as small EVs as suggested by the MISEV guidelines [3].
Regarding the current data available in literature on specific miRNAs as biomarkers of neurodegenerative diseases, there is limited evidence of an altered expression of plasma miRNAs in EVs from AD patients [12,24].
Recently, Chat et al. found miR-212 and miR-132 significantly downregulated in small NDEVs from AD patients using a relatively small sized SYBR green array based analysis testing 372 miRNA candidates [38].
However, a comprehensive high throughput miRNA investigation considering either small NDEVs and the plasma total ones has not been performed so far.
In the current study, for the first time, miRNA content associated to the specific NDEV population was analyzed, and we showed that miRNA NDEVs' cargo is different from that deriving from the overall EV population. Therefore, we identified a specific neuronal signature in peripheral circulation, likely associated to brain miRNA pathological dysregulations. As expected, due to the low amount of the specific neural-derived extracellular population and the subsequent relative RNA, only 26% of miRNAs investigated was reliably detected. All the miRNAs detected were already found in the extracellular vesicles from human biological samples [39,40]. In particular, miRNAs specifically found in NDEVs were already reported to be expressed in exosomes isolated from CSF [41].
Despite the finding of several dysregulations, both considering the total amount of vesicles and the neural-derived fraction, few miRNAs reached the statistical significance threshold. Among those, miR-23a-3p, miR-223-3p, miR-100-3p and miR-190-5p showed instead a significant dysregulation in NDEVs from AD patients compared with controls.
Analysis of pathway predictions using bioinformatics tools failed to reveal pathways robustly disease-correlated common to the four miRNAs, although they were demonstrated to be singularly involved in specific processes of the CNS such as axon guidance and long-term depression as well as in neurodegenerative diseases such as AD. A deeper analysis of available literature revealed a long history of correlation among the investigated miRNAs with dementia. MiR-223 for instance was already proposed as one of the candidate biomarkers for AD as free circulating as well as contained in serum exosomes [12,14,42]. In particular, we previously found a significant downregulation of free-circulating miR-223 and miR-23a levels in serum from AD patients [16]; on the other hand, in the present study, increased levels of NDEVs miR-223-3p and miR-23a-3p were found in patients compared with controls. An explanation of these apparently controversial findings could reside in the suggested function of exosomes as mediators of an active intercellular crosstalk. It is known that exosomes are able to load miRNAs selectively by a specific process, thus the EVs-derived miRNA profile is prespecified and not random, and this is related to the fact that EVs induce changes that are preprogrammed in the host cells [43][44][45]. In this case, increased levels of miR-223-3p in NDEVs could represent an effort by neurons to cope with inflammatory events occurring in AD since miR-223-3p is actively involved in immune response as a negative regulator of inflammation [14].
The same significant trend was found for miR-23a-3p levels in NDEVs; in this case results are concordant with its upregulated levels found in brain tissue from AD patients, although a reduction was found, such as free-circulating in serum or in CSF [14,41]. MiR-100-3p levels were found to be downregulated in NDEVs in patients. Evidence in a murine AD model suggests that the role of miR-100 in Aβ related AD pathogenesis may be related to the "ER stress-miRNAs-mTOR" axis [46]. On the contrary, no association was reported so far between miR-190a-5p and dementia. However, it was already detected in the CSF as free-circulating and, regarding its function, a possible modulation of adult neurogenesis and in vivo, contextual memory was proposed [47]. Interestingly, miR-190 was involved in neuroinflammation as its upregulation inhibited the expression of inducible nitric oxide synthase and other proinflammatory cytokines as IL-6, while it increased the expression of anti-inflammatory cytokines such as IL-10 in a Parkinson's disease mouse model. Furthermore, miR-190-5p reduced neuronal damage and favored the reduction of neuroinflammation in a PD model suggesting an active role in the disease [48].
As neuroinflammation is a common feature of neurodegenerative disease, miR-190 could act as a common dementia marker so its exclusive role in AD as biomarker needs to be further clarified. MiRNAs expression analysis performed with a human miRNA tissue atlas and Funrich v.3.0 revealed some specific miRNA enrichment in specific brain regions such as the hippocampus, cortex and cerebellum and depleted in peripheral blood cells, macrophages and red blood cells, highlighting the relevant role of these miRNAs in brain functions. This miRNA brain-specificity has been nicely reviewed recently by Herrera-Espejo et al. [20] who highlighted specific deregulation in brain tissue of seven miRNAs in AD patients. Interestingly, these miRNAs are involved in the regulation of key pathways, such as axon guidance, longevity, insulin and the MAPK signaling pathway.
Similarly, also miR-223-3p dysregulation seems to be involved in AD pathogenesis by modulating many other targets involved in differentiation/proliferation (i.e., NFI-A;/EBPbeta, Mef2c) and NF-kB pathways (i.e., STAT3, IKK). Moreover, it was found to contribute to other CNS diseases such as Multiple Sclerosis and PD [49,50].
Conversely, miR-23a-3p was found to be synaptically located in adult rat in vivo studies and is a negative regulator of transcription [51].
Three out of four significantly dysregulated miRNAs had already been detected in the EVs plasma population, but did not reach the statistical threshold, whereas miR-190a-5p was only detected in the neural-derived fraction, thus suggesting that miRNAs from plasma EVs and miRNAs from plasma NDEVs could be considered two different biomarker entities.
In conclusion, the four significant dysregulated miRNAs found in plasma NDEVs were already detected in the CSF and have some functions or may act in pathways related to the CNS, supporting the hypothesis of the usefulness to consider these EVs obtained from blood as a source of biomarkers for CNS pathologies. Given implications with neuroinflammation, it would be worth studying EVs from microglial origin. Nevertheless, at present there are no reliable extracellular vesicle markers for microglia [11].
Despite these promising results, some methodological limitations need to be acknowledged. In particular, the detection of NDEVs is hampered by the small numbers of brain-derived EVs that are secreted by potentially disease-relevant cells and transported into peripheral blood. These difficulties are derived from the complexity and technical limitations of current EV isolation methods that could influence the yield of RNA, also demonstrated by the low numbers of miRNAs detected.
The above mentioned technical limitations could explain the discrepancies between our results and the ones shown in the recent paper from Cha et al., [38]. The methodologic procedures, although considering the same L1CAM marker for selecting small NDEVs, were quite different as well as the criteria for patient selection itself that could represent a limitation as underlined by the author. Moreover, the authors highlighted the impossibility of completely ruling out the presence of some plasma RNA outside the vesicles as they did not perform any RNAse protection assay.
All these aspects underline the importance of a standardized procedure and the need for consensus guidelines for the analysis of RNA derived from specific populations of EVs.
Regarding the population considered for this study, there was a statistically different mean age at blood sampling between groups. Nevertheless, this cohort derives from a thorough analysis of all cases who underwent LP in suspicion of neurological disorders and were discharged without any disease. They were followed up over time (mean 3 years) and no cognitive impairment had occurred by follow-up, thus likely excluding the development of memory dysfunctions. Given all these considerations, a further replication step in a larger independent population is required to confirm this approach as a specific "window" on the brain. After that, maybe, other miRNAs or non-coding RNAs molecules could emerge and finally provide a molecular signature with a concrete diagnostic potential.
Conclusions
This is the first study that carries out the comparison between a total plasma EVs population and NDEVs in patients with AD, demonstrating the presence of a specific NDEV miRNA signature. In particular, significantly increased levels of miR-23a-3p, miR-223-3p, miR-190a-5p and significantly decreased miR-100-3p levels were observed in NDEVs isolated from AD patients, suggesting a possible "new peripheral window", possibly able to reflect pathogenic alterations occurring in the brain. | v3-fos-license |
2019-04-03T13:08:27.512Z | 2018-01-01T00:00:00.000 | 92787793 | {
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} | pes2o/s2orc | Changes in different fecal parameters with administration of bromelain and inulin in a rat model
ABSTRACT The present study was to investigate the effects of administrating bromelain, inulin, or a mixture of these ingredients on different fecal parameters in a rat model. Our results showed that taking bromelain (120 CDU/kg body weight) apparently increased fecal moisture by 18% and declined fecal mucinase activity (−36.3%). The incorporation of inulin (260 mg/kg body weight) could also result in some desirable changes including increased fecal moisture by 19%, declined fecal mucinase activity (−43.9%). The feeding of same amounts of bromelain and inulin could lead to significant increases in the presumptive counts of Bifidobacterium and Lactobacilli as well as the concentrations of various fecal short chain fatty acids (by 54–95%). This study suggested that the consumption of bromelain and inulin together might exert favorable effects in improving certain fecal parameters, and provide more hints for the development of functional food formulations.
Introduction
Human gut does not merely retain the nutrients from digested foods (Cencic & Chingwaru, 2010), but also provides a place for an enormous microbial growth. Various studies have revealed a relationship between the intestinal health and some intestinal lumen and feces-related parameters. These parameters included intestinal transit time, defecation frequency, intestinal lining integrity, fecal short chain fatty acids (SCFAs), fecal pH, and fecal moisture (Meyer & Stasse-Wolthuis, 2009;Olivares et al., 2006). The measurement of these parameters could be a useful clue to reflect the changes in the intestinal health and integrity.
Pineapple (Ananas comosus) is one of the most known nutritional fruits around the world. In addition to its flavorful taste, it has been used as both the traditional medicinal and food purposes (Chau & Wu, 2006). Pineapple possesses a proteolytic enzyme, namely, bromelain. It is categorized as one of the GRAS (Generally Recognized As Safe) enzymes. It could be found in both pineapple fruits and stems, but it exists in a larger quantity in stems than in fruits. Bromelain could be absorbed into blood circulation system (Bhattacharyya, 2008), and the typical daily dosage for different therapeutic applications (e.g. digestive disorders) ranged roughly from 4 to 40 CDU/kg body weight for a 60 kg adult (Roxas, 2008). It might benefit patients with postoperative ileus and prevent the enterotoxigenic Escherichia coli induced diarrhea by proteolytically reducing the binding ability of enteral mucosa (Mynott, Luke, & Chandler, 1996;Wen et al., 2006).
Inulin with a degree of polymerization (DP) > 3 is regarded as a non-digestible soluble fiber (Dai & Chau, 2017). It stays undigested in the small intestine and is gradually fermented in the large intestine (Schaafsma & Slavin, 2015). According to Meyer and Stasse-Wolthuis (2009), adults, unlike infants who only require 2 g of inulin per day, require at least 4-5 g of inulin daily to improve intestinal health and to trigger a bifidogenic effect. It contributed to multiple intestinal health functions such as promoted growth of intestinal microflora, micronutrient absorption, fermentation byproducts (i.e. SCFA) production, and relief of constipation (Meyer & Stasse-Wolthuis, 2009;Roberfroid, 2005).
In accordance to the fact that bromelain could be beneficial to gut health, our preliminary study has shown that the benefits from bromelain and inulin were different, for instance, with no apparent growth of lactic acid bacteria being observed with bromelain. This study tended to fill the literature gap to see if more beneficial effect could be achieved by feeding bromelain and inulin together.
Therefore, the aim of this study was to evaluate the effects of the bromelain and inulin consumption on different fecal parameters including fecal bacterial growth, fecal bacterial enzyme, and SCFA profiles.
Sample
In this study, bromelain (cat. no. Well3LE) was obtained from Top One Biotech Co. Ltd., Taiwan. The enzyme activity expressed in casein digestion unit (CDU) was about 1400 CDU/g. Inulin, soluble dietary fiber from chicory root (cat. no. AF-01), was obtained from CNI Venture (M) Sdn Bhd, Malaysia.
Diets and experimental design
The study protocol was approved by the Animal Care and Use Committee of National Chung Hsing University. Forty male Sprague Dawley (SD) rats aged seven weeks were purchased from BioLASCO Company, Taiwan. The SD rats weighing from 217.1 to 326.5 g were placed in animal room at 22 ± 1°C, 60 ± 5% humidity, and with 12-h light/dark cycle lightning. All animals were caged individually in a stainless steel cage and were fed chow diet (Laboratory Rodent Diet 5001, PMI Nutrition International/Purina Mills LLC, St. Louis, MO).
After an acclimation for 1 week, animals were divided into eight weight classes of five each. The rats in each weight class were randomly assigned to one of the five diet groups, including one control (feeding chow diet only) and four experimental groups, namely '2B', '2I', 'B+ I', and '2B+ 2I' groups. Specifically, the animals in group '2B' were given bromelain at a single dose of 120 CDU/kg bw. Group '2I' was given inulin (260 mg/kg bw). Group 'B+ I' was given a mix of bromelain (60 CDU/kg bw) and inulin (130 mg/kg bw) while the group '2B+ 2I' was provided with a mix of bromelain (120 CDU/kg bw) and inulin (260 mg/kg bw).
Throughout the experiment, water and feed were provided ad libitum. The feeding experiment was carried out for 28 days. Food intakes and body weights were recorded daily. Feces were collected, weighed, and analyzed for routine measurements. Some of the fecal samples left unused were stored at -20°C for further use.
Determination of fecal pH and moisture
According to the methods as described by Chau, Huang, and Chang (2005), fecal samples without urine and feed waste contamination were collected and analyzed for pH and moisture content. Fecal moisture content was determined by drying the fecal sample on aluminum foil trays to a constant weight in a 105°C air-oven. Fecal pH values were measured by homogenizing the fresh feces with deionized H 2 O in a 1:4 (w/v) ratio, followed by centrifugation at 1,006g for 10 min.
Determination of fecal mucinase activities
Fecal mucinase activity was determined by using the method of Shiau and Chang (1983). Fresh fecal samples were homogenized in 0.01 M phosphate buffer (pH 7.2, 1:50 w/v) for 30 min. After a centrifugation at 1,006g for 10 min, the supernatant was analyzed for mucinase activity. Protein in the supernatant was also determined by a protein assay kit (Cat No 500-0006, Bio-Rad). Mucinase activity, which was expressed as μmol, of reducing sugar released per min per mg of fecal protein was estimated by measuring the amount of reducing sugar released from porcine gastric mucin (M1778, Sigma).
Presumptive enumerations of Bifidobacterium, Lactobacilli and Escherichia coli
Fresh fecal samples were analyzed within 20 min immediately after the collection. The samples were immersed and mixed well in a sterile and anaerobic diluting solution (1:10 w/v). Serial ten-fold dilutions were prepared to acquire desired concentrations for analysis (Shieh, Shang, Liao, Zhu, & Chien, 2011). Presumptive counts of Bifidobacterium and Lactobacilli in the solutions were analyzed using Bifidobacterium iodoacetate medium 25 (BIM-25) and Rogosa agar, respectively, in an anaerobic incubator at 37°C for 72 h (Muñoa & Pares, 1988). For the enumeration of presumptive E. coli count, LEMB agar (Merck KGaA, Darmstadt, Germany) was used as a selective and differential medium, and cultured in an anaerobic chamber at 37°C for 48 h.
Determination of fecal short-chain fatty acids (SCFAs)
According to the methods as described by Huang, Chu, Dai, Yu, and Chau (2012) with slight modifications, the SCFA concentrations in the fecal samples were determined. Fresh fecal sample was homogenized with cold saline (0.9% w/v) at a ratio of 1:10 (w/v), followed by centrifugation at 1,006g for 10 min. Two millilitres of the supernatant was then mixed with 10 μL of isocaporic acid (internal standard) and 20 μL of 50% (w/v) sulfuric acid. After the SCFA extraction using diethyl ether, 1 μL of the ether layer was analyzed by a column (Agilent J & W HP-INNO Wax GC Column, 30 m, 0.25 mm. 0.25 µm) using a gas chromatograph (Agilent Technologies 7890A, California, USA) fitted with a flame ionization detector. The conditions were as follows: oven temperature, initially held at 80°C for 1 min and raised to 140°C at a rate of 20°C/min, then held at 140°C for another 1 min and raised again to 220°C at a rate of 20°C/min, followed by holding at 220°C for 2 more min; injector temperature, 140°C; detector temperature, 250°C; gas flow rate, 7 mL/min (carrier gas, helium).
Statistical analysis
All determinations expressed in mean ± standard deviation (SD) were analyzed by one-way ANOVA using the software of Statistical Product and Service Solutions (SPSS) (IBM Corp, version 20.0, Armonk, NY, USA). Values of P < 0.05 were considered statistically significant.
Results and discussion
All animals remained healthy and active over the whole experimental period. Table 1 summarizes the body weight gain, daily food intake, and daily water intake of rats among the five dietary groups. After 28 days of feeding, no apparent differences in the body weight gain (5.8-6.2 g), daily food intake (28.2-29.1 g/day), and daily water intake (42.7-43.9 g/ day) were noted among the five groups. Table 1 displays the comparison of fecal pH, fecal moisture and fecal weight in animals fed different diet groups. A significant reduction in fecal pH was observed in the 2B+ 2I group over the control while no apparent differences in fecal pH were noted among the control, 2B, 2I, and B + I groups. As compared with the control, fecal moisture contents were significantly (P < 0.05) increased by the inclusion of bromelain in 2B and 2B+ 2I groups (118-119%). A similar trend was also observed in fecal weight in the 2B and 2B+ 2I groups (123-128%). Since some studies have demonstrated that inulin could increase fecal water content and weight (Drabińska, Zieliński, & Krupa-Kozak, 2016;Slavin, 2013), our results revealed that the inclusion of inulin in diets at a dose of 260 mg/kg bw were not high enough to trigger an apparent change in these fecal indexes. However, Table 1 indicates that the feeding of bromelain at a relatively higher dosage (i.e. 2B and 2B+ 2I) was able to increase the moisture content and weight in fecal output. Wen et al. (2006) have also reported that bromelain was capable of increasing fecal moisture content for postoperative disorders and ameliorating constipation problem. More specifically, bromelain might improve defecation by increasing fecal moisture, fecal wet weight, and number of fecal pellets in postoperative rats, at least in part, by inhibiting colonic iNOS overexpression via NF-kappaB pathway. Table 2 shows the fecal presumptive counts of Bifidobacterium and Lactobacilli. When comparing with the control group (6.68 log CFU/g), the presumptive Bifidobacterium counts of the 2B (6.71 log CFU/g) and 2I (6.93 log CFU/g) groups did not demonstrate an apparent difference. A slight increase in the bacterial count of the B + I group (7.23 log CFU/g) over the control group was observed, but not as significant (P < 0.05) as the one shown in the 2B + 2I group (7.95 log CFU/g). As for the presumptive Lactobacilli count, the trend was somehow similar as the one seen in presumptive Bifidobacterium count. There was a major rise (P < 0.05) of the presumptive Lactobacillus count from 7.72 log CFU/g (control group) to 8.64 log CFU/g (2B + 2I group). It could be induced that consumption of the mixture of bromelain and inulin at a higher dose (2B+ 2I) effectively (P < 0.05) enhanced the growth of Bifidobacterium and Lactobacilli.
As shown in Table 2, a dosage of 260 mg/kg bw of inulin (2I group) did not result in any apparent growth in both the Bifidobacterium and Lactobacilli only until bromelain was added in the 2B+ 2I group. Consistent with these findings, animals fed with 260 mg/kg bw of inulin alone in this experiment did not demonstrate any growth promoting effect. Intriguingly, a supplement of bromelain (120 CDU/kg bw) together with inulin (260 mg/kg bw) was found to enhance the growth of these two bacteria to a further extent. With a stimulation of the presumptive counts of Bifidobacterium and Lactobacilli, proteolytic bacteria growth in the colon would Table 1. Effect of different diets on body weight gain, daily food intake, daily water intake, fecal pH, fecal moisture, and fecal weight of rats.
On the intestinal mucosa, mucin serves as a defense barrier layer to protect against bacterial invasion, enzymatic degradation, and toxic substances (Satchithanandam, Klurfeld, Calvert, & Cassidy, 1996). Mucinase in the hindgut and feces might catalyze a broad of metabolic transformations and the formation of toxic and carcinogenic substances. It also further hydrolyzes the protective mucin layer to exposes the intestinal cells to harmful substances (Shiau & Chang, 1983). Figure 1 illustrates that the basal mucinase activity in the control group was 2.9 units. No significant changes in the fecal mucinase activity were observed in the groups 2I and B + I (2.3-2.8 units) as compared with that of the control. Feeding the rats with bromelain at a higher dosage in both the 2B and 2B+ 2I groups could significantly (P < 0.05) decrease the mucinase activity by −36.3% and −43.9%, respectively, against the control.
Based on the above findings, major differences were observed among the control, 2B, and 2B+ 2I groups, while the results of the other groups (including 2I, and B + I) did not differ significantly from the control. Accordingly, the SCFA profiles analyses were solely conducted among the control, 2B, and 2B+ 2I groups (Figure 2). The changes in fecal SCFA profiles among these three groups were similar to those observed with the elevation in the presumptive counts of Bifidobacterium and Lactobacilli (Table 2). In general, SCFA concentrations in hindgut were associated with the consumption level of fermentable carbohydrate and the extent of microbial fermentation (Högberg & Lindberg, 2004). The SCFAs (i.e. acetate, propionate, and butyrate) produced in the colon could play an indispensable role in maintaining intestinal lining integrity and suppressing the pathogen growth (Meyer & Stasse-Wolthuis, 2009;Slavin, 2013).
The results depicted that the concentrations of acetic acid, propionic acid, butyric acid, and total SCFA in the fecal samples between the control and 2B groups were comparable to each other, and were found to be 106. 5-112.3, 73.1-80.0, 34.7-37.1, and 214.4-228.4 μmol/g, respectively. On the other hand, the fecal SCFA profiles of the 2B+ 2I group showed a consistently higher amounts of acetic acid, propionic acid, and butyric acid, which were up to 195%, 164%, 154%, and 178%, respectively, over the control. Some studies have showed that each of the SCFAs would perform a unique role. For instances, acetate could enhance the mucin secretion, propionate had the capability to decrease the de novo synthesis of fatty acid, and butyrate might suppress the neoplastic alterations in cancer cells (Barcelo et al., 2000;Nishina & Freedland, 1990;Pryde, Duncan, Hold, Stewart, & Flint, 2002).
An elevation in the concentration of SCFAs would stimulate mucosal cells to trigger a peristaltic reflex and hence to shorten gastrointestinal tract time (Grider & Piland, 2007). It is speculated that the increased gut motility might reduce the time for water reabsorption and therefore led to a significantly higher fecal moisture in 2B+ 2I group (Table 1). The elevated moisture retention in feces might, in turn, support a better growth of intestinal microflora, such as Bifidobacterium and Lactobacilli. A relative higher level of fermentation metabolites (i.e. lactic acid) was produced and led to a greater decline in fecal pH (Meyer & Stasse-Wolthuis, 2009). a-b Bars (mean ± SD, n = 8) among different groups with different letters are significantly different (P < 0.05). c Control: given chow diet only; 2B: given bromelain at a dose of 120 CDU/kg bw; 2I: given inulin at a dose of 260 mg/kg bw; B + I: given a mix of bromelain (60 CDU/kg bw) and inulin (130 mg/kg bw); 2B+ 2I: given a mix of bromelain (120 CDU/kg bw) and inulin (260 mg/kg bw). Figura 1. Efectos de distintas dietas en la actividad bacteriana en la mucinasa fecal. a-b Las barras (media ± DE, n = 8) con diferentes letras entre los distintos grupos son significativamente diferentes (P < 0.05). c Control: recibieron solo una dieta de alimentos; 2B: recibieron bromelina a una dosis de 120 CDU/kg bw; 2I: recibieron inulina a una dosis de 260 mg/kg bw; B + I: recibieron una mezcla de bromelina (60 CDU/kg bw) e inulina (130 mg/kg bw); 2B+ 2I: recibieron una mezcla de bromelina de (120 CDU/kg bw) e inulina de (260 mg/kg bw). bw = peso corporal
Conclusion
Based on the above findings in this study, as compared to taking bromelain alone, the administration of inulin together with bromelain could result in some further desirable changes including increased fecal moisture (by 19%), declined fecal mucinase activity (−43.9%), promoted growth of Bifidobacterium and Lactobacilli, and elevated concentrations of various fecal SCFAs (by 54-95%). Our results revealed that the consumption of bromelain and inulin together might exert favorable effects in improving certain fecal parameters. Future investigations should be directed to understanding the possible interactions between bromelain with inulin or some other prebiotic dietary fiber on potentiating the improvement of different fecal parameters.
Disclosure statement
No potential conflict of interest was reported by the authors. a Bars (mean ± SD, n = 8) of each fatty acid denoted with * differ from its corresponding control significantly (P < 0.05). b Control: given chow diet only; 2B: given bromelain at a dose of 120 CDU/kg bw; 2B+ 2I: given a mix of bromelain (120 CDU/kg bw) and inulin (260 mg/kg bw). | v3-fos-license |
2018-01-26T02:15:52.534Z | 2016-12-15T00:00:00.000 | 6069527 | {
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} | pes2o/s2orc | Catalytic activity of catalase–silica nanoparticle hybrids: from ensemble to individual entity activity† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04921d Click here for additional data file.
We demonstrate the electrochemical detection and characterization of individual nanoparticle–enzyme hybrids.
SiNP preparation and modification
For all measurements (UV-Vis and electrochemistry), the unmodified SiNPs were used as received and diluted to the desired concentration with ultrapure water from Millipore featuring a resistivity of not less than 18.2 MΩ.cm. The following procedure was used for modifying the SiNP with catalase: 79.5 L of SiNPs were added to 119.7 L of water. Next, 20 L of bovine catalase from stock solution (Sigma, 45 mg/ml) were added. Last, 54.8 L of 50 mM citrate phosphate buffer (pH=5.4) were added as well. The solution was left overnight for incubation at room temperature. Next, the solution was centrifuged (9000 RPM, Eppendorf 5430-R) for 30 min, the supernatant was removed and the pellet was washed with 219 L of water and 55 L of the citrate buffer. The centrifugation and washing process was repeated three times to insure that there is no residual catalase left in solution.
UV-Vis spectroscopy
UV-vis spectroscopy experiments were conducted in citrate-phosphate buffer solution (pH=5.4) using a Shimadzu spectrometer UV-1800 and quartz cells with a 1 cm optical path.
TEM
Silica dioxide nanoparticle characterization was performed using a transmission electron microscope (TEM) JEOL JEM-3000F equipped with an EDX spectrometer with an accelerating voltage of 300 kV. Sample preparation involved drop casting nanoparticle suspensions on holey carbon grids (Agar Scientific) and allowing the samples to dry. A size distribution histogram was plotted from the TEM image analysis of 233 NP (Fig. 1b), using ImageJ software. The mean size and standard deviation of the nanoparticles was estimated using a Gaussian fit (Origin 2015).
NTA
A NanoSight LM10 (NanoSight Limited, Amesbury, UK) was used to carry out nanoparticle tracking analysis. A 500 µl sample of SiNP was syringed into the viewing unit of the NanoSight and a red (638 nm) laser was used to illuminate the particles so they could be tracked. Measurements were recorded at 20 0C. NanoSight's NTA software was used to analyse the size distribution and concentration of the NPs. . For the chronoamperometric measurements a homemade potentiostat was used together with a carbon microelectrode as a working electrode (r = 3.5 m). Before all experiments the electrode was polished using micropolish alumina (Buehler) in the size sequence of 3.0 µm, 1.0 µm and 0.1 µm to a mirror-like finish. Data was recorded with a 4 kHz preamplifier filtered with a built-in passive 100 Hz filter. The properties of the homemade potentiostat were described previously. [1] Impact spikes were analysed using SignalCounter software developed by Dario Omanovic (Centre for Marine and Environmental Research, Ruder Boskovic Institute, Croatia). [2] Fig. S1 (a) NTA of the SiNP-Catalase (b) TEM of bare SiNP and (c) zeta potential of bare and catalase modified SiNP.
Surface coverage of catalase on a SiNP:
The absorption maximum of SiNP/Catalase hybrid ds in solution was at 405 nm and a value of 0.062 was recorded. Using the Beer-Lambert law we can calculate the concentration of bound catalase in solution: Since the concentration of the SiNP in solution was pre-determined to be 0.5 nM, we can estimate the number of catalase enzymes per SiNP to be: The radius of a single SiNP was 59 nm. The radius of catalase is estimated to be 5.12 nm. [3,4] Hence, the maximum number of enzymes that can be loaded on a SiNP can be approximated:
Theoretical calculation of SiNP impact frequency:
The steady-state current at a microdisk electrode of radius r, assuming a simple n electron reduction, is given by where n is the number of electrons transferred, F is the Faraday constant (C mol −1 ), C is bulk concentration (mol cm −3 ), D is diffusion coefficient (cm 2 s −1 ) and f(τ) is a function of time, t (s). A convenient single expression for f(τ) has been obtained from simulation by Shoup and Szabo and shown to correctly predict the current over the entire time domain with a maximum error of less than 0.6%. The Shoup and Szabo expression is: [5] f(τ) = 0.7854 + 0.8863τ −1/2 + 0.2146exp(0.7823τ −1/2 ) where τ = 4Dt/r 2 . Multiplication of this by the Avogadro constant, N A , converts the equation to a form referring to the number of particles. To determine the number of particle impacts expected within a given time, the Shoup-Szabo equation needs to be integrated and this has previously been performed by series expansion. [6] For a 100 pM particles in solution with a radius of 59 nm, the estimated upper value for the average impact frequency is ~ 50 impacts / 10 sec. The theoretical value is about an order of magnitude higher than the experimentally observed impact frequency and can be explained by an irreversible absorption process of the NP hybrids to the insulating glass surrounding the active microelectrode. [7] Theoretical calculation of irreversible two electron reduction of H 2 O 2 : The relation of the peak current (Ip) with the scan rate (υ) can be expected to follow the Randles-Ševčík equation for a two electron fully irreversible process: where I p is the peak current, α=0.3 is the electron transfer coefficient of the rate determining step, n=2 is the number of electrons transferred and assuming 1 st electron transfer is not the rate limiting step, F is the Faraday constant, 2 2 is the diffusion coefficient and equals to 1.71 × 10 −9 2 s -1 for hydrogen peroxide. [8] A is the area of the electrode (r=1.5 mm), [ ] is the hydrogen peroxide concentration, T is the absolute temperature, R is the gas constant and ν is the scan rate. | v3-fos-license |
2021-07-26T00:05:56.340Z | 2021-06-10T00:00:00.000 | 236300125 | {
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} | pes2o/s2orc | Extracellular membrane vesicles and nanotubes in Archaea
ABSTRACT Membrane-bound extracellular vesicles (EVs) are secreted by cells from all three domains of life and their implication in various biological processes is increasingly recognized. In this review, we summarize the current knowledge on archaeal EVs and nanotubes, and emphasize their biological significance. In archaea, the EVs and nanotubes have been largely studied in representative species from the phyla Crenarchaeota and Euryarchaeota. The archaeal EVs have been linked to several physiological processes such as detoxification, biomineralization and transport of biological molecules, including chromosomal, viral or plasmid DNA, thereby taking part in genome evolution and adaptation through horizontal gene transfer. The biological significance of archaeal nanotubes is yet to be demonstrated, although they could participate in EV biogenesis or exchange of cellular contents. We also discuss the biological mechanisms leading to EV/nanotube biogenesis in Archaea. It has been recently demonstrated that, similar to eukaryotes, EV budding in crenarchaea depends on the ESCRT machinery, whereas the mechanism of EV budding in euryarchaeal lineages, which lack the ESCRT-III homologues, remains unknown.
INTRODUCTION
Archaea have been recognized as a separate domain of life, besides Bacteria and Eukarya, only in 1977 by Carl Woese and colleagues who compared the ribosomal RNA (rRNA) gene sequences from diverse organisms (Woese and Fox 1977). The name Archaea, instead of archaebacteria, was proposed later on by Carl Woese to emphasize the fact that Archaea and Bacteria form two distinct lineages in the universal tree of life (Woese, Kandler and Wheelis 1990). Although archaeal cells are of the prokaryotic type, their informational systems (DNA replication, transcription, translation), as well as several membrane-associated machineries, such as the ATP synthase complex, the Sec secretion system and the signal recognition particles, are much more similar to those of eukaryotes. Similar to bacteria and eukaryotes, archaeal cells commonly secrete extracellular vesicles (EVs) and some produce tubular structures resembling bacterial nanopods and/or nanotubes (Gill, Catchpole and Forterre 2019).
The production of various types of EVs (apoptotic bodies, exosomes, microvesicles, etc.) and nanotube-like structures (tunnelling nanotubes, etc.) has been extensively studied in Eukarya (for reviews, see Yanez-Mo et al. 2015;Gill, Catchpole and Forterre 2019;Cordero Cervantes and Zurzolo 2021). In Bacteria, EVs were observed for the first time by electron microscopy in E. coli in 1966 (Knox, Vesk and Work 1966), but their biological importance was first dismissed and, hence, microbial EVs have been considered as artefacts of cell growth or lysis for many years (Coelho and Casadevall 2019). However, bacterial EVs are increasingly recognized to play important roles in many processes from pathogenesis, bacterial communication and biofilm formation, to horizontal gene transfer and protection against viral infections (Brown et al. 2015;Jan 2017;Gill, Catchpole and Forterre 2019). Nanotubes have been described in Bacteria more recently (Baidya et al. 2018;Gill, Catchpole and Forterre 2019), but their physiological role remains controversial (Baidya, Rosenshine and Ben-Yehuda 2020;Pospíšil et al. 2020). In Archaea, EVs have been first described over two decades ago when they were found to carry protein toxins (Prangishvili et al. 2000). Although until recently, archaeal EVs received relatively little attention, perhaps due to the fact that their discovery was from the very beginning linked to a defined function (i.e. toxin transfer), archaeal EVs were not dismissed by the archaeal community as cellular 'junk,' as in some other branches of microbiology (Coelho and Casadevall 2019). The research on archaeal EVs has primarily focused on two orders of hyperthermophilic species, Sulfolobales and Thermococcales, whereas nanotubes have been primarily described in Thermococcales and Haloarchaea.
A major difference between Archaea and Bacteria that probably influences the respective mechanisms of EV and nanotube production is the structure of their cell envelopes. Similar to most eukaryotic cells, most Archaea are monoderms, i.e. their envelope consists of a single membrane (Ellen et al. 2010;Albers and Meyer 2011). Important exceptions are Ignicoccus hospitalis and members of the order Methanomassilicoccales that are diderm archaea with an outer membrane and a periplasmic space (Dridi et al. 2012;Klingl 2014;Heimerl et al. 2017). Most archaea lack rigid cell wall and this may facilitate the production of EVs and nanotubes. The exceptions are methanogenic archaea of the orders Methanobacteriales and Methanopyrales in which the membrane is surrounded by a peptidoglycan-like polymer, referred to as pseudomurein layer (Steenbakkers et al. 2006;Albers and Meyer 2011;Klingl 2014), and halophilic archaea of the genus Halococcus that are surrounded by a complex and rigid heteroglycan cell wall (Steber and Schleifer 1975). The cytoplasmic membrane is surrounded by a paracrystalline protein surface (S-) layer usually composed of a single main glycoprotein (40-200 kDa) that is capable of self-assembly into highly ordered structures (Rodrigues-Oliveira et al. 2017). As in eukaryotes, the outer surfaces of archaea are also often covered with abundant glycoproteins and the protein N-glycosylation pathways exhibit important similarities between Archaea and Eukarya, suggesting a common origin (Nikolayev, Cohen-Rosenzweig and Eichler 2020).
For a long time, Archaea have been divided into two major phyla based on rRNA sequence comparisons: Crenarchaeota and Euryarchaeota (Woese, Kandler and Wheelis 1990). Crenarchaeota includes thermophilic or hyperthermophilic species, whereas members of Euryarchaeota are phenotypically very diverse, including (hyper)thermophiles, mesophiles, methanogens and halophiles. Most cultivated species belong to these two phyla and all experimental studies on archaeal EVs have been performed either on Crenarchaeota (order Sulfolobales) or Euryarchaeota (order Thermococcales and class Halobacteria). In 2008, a third major archaeal phylum was proposed, the Thaumarchaeota (Brochier-Armanet et al. 2008). Many thaumarchaeal species, either mesophilic or thermophilic, have now been cultivated, but they all turned out to be very fastidious and the production of EVs has not yet been studied in this phylum.
Although many eukaryotic features are common to all archaea, others are specific to only some lineages, phyla or superphyla. This is the case for the endosomal sorting complexes required for transport (ESCRT) machinery proteins that are responsible not only for cell division but also for EV biogenesis in eukaryotes (Vietri, Radulovic and Stenmark 2020). Both eukaryotic-like proteins of the ESCRT machinery, ESCRT-III and Vps4 ATPase, are conserved in Crenarchaeota (except for Thermoproteales). Members of the Thaumarchaeota also encode the ESCRT proteins that are very similar to those of Crenarchaeota (Caspi and Dekker 2018;Lu et al. 2020). However, despite producing EVs, members of the order Euryarchaeota, such as Thermococcales and Halobacteriales, do not encode the ESCRT machinery but only Vps4 homologues. Instead, the euryarchaeal lineage relies on the bacterial-like FtsZ-based system for cell division. This suggests that EV biogenesis in different archaeal lineages occurs by very different mechanisms. Thus, mechanistic comparisons of EV budding in archaea and eukaryotes might provide insights into the evolution and diversification of this important process in different cellular lineages.
In recent years, a wealth of new archaeal phyla have been described from the reconstructions of metagenome-assembled genomes, vastly expanding the known diversity of Archaea and the range of their ecological distribution (Adam et al. 2017;Spang, Caceres and Ettema 2017). Several new phyla corresponding to small archaea have been grouped in the DPANN superphylum (referring to the first described constituent lineages, Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota) (Rinke et al. 2013). ESCRT machinery proteins and many other eukaryotic-like proteins missing in Euryarchaeota are also absent in DPANN archaea. These miniature archaea have reduced genomes and most of them lack essential metabolic pathways, such as lipid biosynthesis, suggestive of parasitic lifestyles. Consequently, most DPANN members are probably ectosymbionts of larger archaeal hosts (Dombrowski et al. 2019). The best characterized of these symbiotic interactions is the association of Nanoarchaeum equitans (a member of the Nanoarchaeota) with its host, the diderm crenarchaeon Ignicoccus hospitalis. Remarkably, membrane vesicles that might be implicated in the interactions between Nanoarchaeum and Ignicoccus have been observed budding from the internal cytoplasmic membrane of I. hospitalis (see later) (Junglas et al. 2008).
Another major superphylum, the Asgardarchaeota, has attracted much attention because some phylogenetic analyses of universal protein concatenation have suggested that eukaryotes emerged from within this phylum (Spang et al. 2015;Zaremba-Niedzwiedzka et al. 2017;Williams et al. 2020). This hypothesis is still debated because Archaea are monophyletic, with Asgard archaea branching between euryarchaea and crenarchaea in other universal protein phylogenies (Da Cunha et al. , 2018Jay et al. 2018). Interestingly, the genomes of Asgardarchaeota encode more eukaryotic-like proteins than most other archaeal phyla (Zaremba-Niedzwiedzka et al. 2017), including actin, tubulin and additional components of the ESCRT system (Caspi and Dekker 2018;Lu et al. 2020). Moreover, these proteins are also more closely related to their eukaryotic homologues than those from other Archaea. Asgard archaea should therefore serve as valuable models to study the role of these proteins in the biogenesis of archaeal EVs and/or nanotubes. However, only one Asgard archaeon, Candidatus Prometheoarchaeum syntrophicum, has been successfully cultivated yet, and only in symbiosis with other microorganisms (Imachi et al. 2020). Interestingly, electron microscopy analyses have suggested that this organism produces both EVs and nanotubes (see later).
PRODUCTION OF EVS BY ARCHAEA OF THE PHYLUM CRENARCHAEOTA
Nearly all studies dealing with the production of EVs in crenarchaea have been performed in organisms of the order Sulfolobales. These archaea are thermoacidophiles thriving in acidic (pH 2-3) terrestrial hot springs that have been studied as model organisms by biochemists and molecular biologists from the very beginning of archaeal research, mainly because they are aerobes and are easy to cultivate (Schocke, Brasen and Siebers 2019). Moreover, many genetic tools are now available for several Sulfolobus species (Peng et al. 2017). The earliest reports about archaeal EVs came from studies carried out on Sulfolobus islandicus (Prangishvili et al. 2000). These EVs, 90-230 nm in diameter and coated with the S-layer, were shown to be associated with an antimicrobial protein, termed 'sulfolobicin,' that inhibits the growth of related Sulfolobus species. Subsequently, EV-associated toxins were identified in some other Sulfolobus species and were shown to be encoded by a two-gene operon (Ellen et al. 2011). Disruption of these genes has shown that the two sulfolobicin proteins, dubbed SulA and SulB, are required for the antimicrobial activity (Ellen et al. 2011). Notably, their association with EVs was not necessary for the antimicrobial activity since purified sulfolobicins were still active once extracted from EVs by alkaline carbonate treatment.
Characterization of EVs produced by Sulfolobus acidocaldarius, Saccharolobus solfataricus and Sulfurisphaera tokodaii showed that lipid and protein profiles of the parental cells were different from those of the corresponding EVs (Ellen et al. 2009). Interestingly, the protein content analyses have shown that EVs from all three species carry some components of the ESCRT machinery, namely, ESCRT-III-1 and ESCRT-III-2 and the Vps4 ATPase (also called CdvC) (Ellen et al. 2009). All three proteins play a key role in the Sulfolobus cell division (Lindås et al. 2008;Samson et al. 2008;Liu et al. 2017;Tarrason Risa et al. 2020). Given that in eukaryotes the ESCRT machinery is responsible not only for the cytokinesis but also among other functions, for EV budding (Vietri, Radulovic and Stenmark 2020), it has been hypothesized that production of Sulfolobus EVs also depends on the ESCRT machinery.
Direct evidence that budding of Sulfolobus EVs depends on the ESCRT machinery has been recently provided by an in-depth characterization of the EVs from S. islandicus (Liu et al. 2021b). Proteomic analysis has shown that highly purified S. islandicus EVs (Sis-EVs; Fig. 1A and B) carry 413 proteins, including all six components of the Sulfolobus ESCRT machinery, with ESCRT-III-2 and ESCRT-III-1 being in the top-10 of the most abundant EV proteins. Western blot analysis confirmed that both proteins were present and strongly enriched in the Sis-EVs. Using a CRISPR-based knockdown system, it was demonstrated that the four archaeal ESCRT-III homologues and the AAA+ ATPase Vps4 were all required for EV production, whereas the archaeaspecific component CdvA appeared to be dispensable (Liu et al. 2021b). Importantly, using synchronized S. islandicus cultures, it was shown that EV production is linked to cell division (Fig. 1C) and coincides with the natural, cell cycle-linked changes in the expression of ESCRT-III homologues, in particular, ESCRT-III-1 and ESCRT-III-2. Consistently, overexpression of ESCRT-III-1 and ESCRT-III-2 from a plasmid resulted in 200-250% increase in vesiculation, while overexpression of other ESCRT machinery components had little effect on EV production (Liu et al. 2021b). Based on these findings, it has been suggested that ESCRTmediated EV biogenesis has deep evolutionary roots and predates the divergence of eukaryotes and archaea. Interestingly, it has been recently shown that in virus-infected S. islandicus cells, ESCRT machinery mediates asymmetric cell division, whereby normal-sized cells are budding from the giant virus-infected cells (Liu et al. 2021a), topologically resembling the EV budding.
A hypervesiculation phenotype was obtained in cells overexpressing the CdvA protein (Liu et al. 2021b). The latter protein does not appear to participate in normal EV budding, because CRISPR-mediated depletion of the cdvA transcripts had little effect on the EV production. However, when the CdvA was overexpressed from a plasmid, the EV production was boosted by several folds. It was suggested that the hypervesiculation is the result of excessive binding of CdvA to the membrane (Liu et al. 2021b). Regardless of the exact mechanism, it appears that EVs can be produced by different mechanisms even in the same organism. The normal EV production is evidently linked to cell division in Sulfolobus but it is possible that under different physiological conditions and upon exposure to different stressors EVs could be produced through different pathways.
Besides ESCRT machinery components, Sis-EVs carry a diverse subset of the S. islandicus proteome, including diverse proteases and nucleases (Liu et al. 2021b). However, highly purified Sis-EVs showed no toxicity against other Sulfolobus, Saccharolobus and Sulfurisphaera species tested, suggesting that EVmediated transfer of sulfolobicins might not be a general phenomenon. Notably, proteins carried by Sis-EVs were not randomly included from the S. islandicus proteome. Indeed, comparison of the Sis-EV and S. islandicus proteomes showed that Sis-EV protein fraction is strongly enriched in membrane proteins as well as proteins from particular functional categories, including the cell division (as discussed earlier), cell motility, posttranslational modification, protein turnover and secretion as well as energy production and conversion, and inorganic ion transport and metabolism (Liu et al. 2021b).
Sis-EVs cargo includes not only diverse proteins but also chromosomal and plasmid DNA. Importantly, Sis-EVs protect the cargo DNA from nucleases as well as the harsh physicochemical conditions of the extracellular milieu and can transfer it to recipient cells (Liu et al. 2021b). The possibility to transfer DNA via EVs was previously observed with archaea of the order Thermococcales (see later) and also in bacteria (Domingues and Nielsen 2017). The term 'vesiduction' has been proposed for DNA transfer mediated by EVs as a fourth way of DNA transfer, besides transformation, transfection and conjugation (Soler and Forterre 2020).
Moreover, Sis-EVs can also support the heterotrophic growth of S. islandicus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Thus, it is becoming clear that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments. Indeed, S-layer-covered EVs have been detected directly in an environmental sample collected from a terrestrial hot spring (Baquero et al. 2020;Liu et al. 2021b), providing evidence that EVs are not a laboratory artefact.
It has been suggested that Sulfolobus EVs also promote biomineralization (Kish et al. 2016). Whilst S-layers have long been implicated in mineral formation, the underlying mechanisms remained unresolved. A study using Sulfolobus acidocaldarius, a hyperthermophilic archaeon isolated from metal-enriched environments, demonstrated a passive process of iron phosphate nucleation and growth within the S-layer of cells and cellfree S-layer 'ghosts' during incubation in a Fe-rich medium. In addition, EVs of ∼175 nm in diameter were formed and released in response to S-layer encrustation by minerals. These EVs were fully encrusted by minerals, even when cells were only partially encrusted (Kish et al. 2016). The authors proposed that these EVs are produced in an attempt to remove sections of damaged Slayer.
Besides Sulfolobales, the production of EV vesicles in Crenarchaeota was reported in I. hospitalis, a member of the order Desulfurococcales. Ignicoccus hospitalis is a diderm archaeon with an inner cytoplasmic membrane and an outer membrane separated by a fairly large periplasmic space (20-1000 nm in width). Vesicles are produced by budding from the inner membrane and numerous vesicles can accumulate in the periplasm and fuse with the outer membrane (Näther and Rachel 2004). Ignicoccus hospitalis often hosts cells of the tiny archaeon N. equitans attached to its surface (Huber et al. 2002;Küper et al. 2010). Nanoarchaeum equitans has the smallest known genome for an archaeon (0.49 Mb) and cannot synthesize many essential components, including lipids (Waters et al. 2003). It is assumed that these components could be delivered from the cytoplasm of I. hospitalis to N. equitans via vesicles that reach the outer membrane at the position of the symbiont attachment (Junglas et al. 2008). Unfortunately, there are presently no genetic tools available to address the mechanism of vesicle production in this fascinating system.
PRODUCTION OF EVS BY ARCHAEA OF THE PHYLUM EURYARCHAEOTA
Most studies dealing with the production of EVs in Euryarchaeota have been performed with archaea of the genus Thermococcus (order Thermococcales). Members of the Thermococcales are strictly anaerobic, hyperthermophilic sulfur reducers, which are placed at the base of the Euryarchaeota in most archaeal phylogenies (Adam et al. 2017;Da Cunha et al. 2017). They are rather easy to cultivate under laboratory conditions, requiring typical equipment for the cultivation of anaerobes, and are typically abundantly present in environmental samples from hydrothermal vents, both terrestrial and marine. Massive production of EVs was first observed in the course of screening for viruses a collection of Thermococcales isolated from hydrothermal deep-sea vents (Soler et al. 2008). Subsequently, EVs were also observed in several reference strains widely used as model organisms for the study of hyperthermophiles, such as Thermococcus kodakarensis Marguet et al. 2013). These EVs (50-150 nm) are covered with the S-layer and are apparently produced by budding ( Fig. 2A-C).
Since the genomes of Thermococcales and of other Euryarchaeota do not encode for ESCRT-III homologues (Makarova et al. 2010), the mechanism of EV production in this phylum is likely to be different from that postulated for Sulfolobales (Liu et al. 2021b). Indeed, the genomes of Thermococcales encode three putative homologues of the Vps4 ATPase, but disruption of any of their genes in Thermococcus kodakarensis did not affect EV production (Gill, Catchpole and Forterre, unpublished result). In contrast with the result obtained with Sulfolobales, the biochemical characterization of purified EVs from three species of Thermococcales (T. kodakarensis, T. gammatolerans and Thermococcus sp. 5-4) revealed that protein and lipid profiles of EVs and cell membranes from the same species have a similar composition ). However, the major protein present in both cell membranes and EVs of Thermococcus species, the oligopeptide binding protein OppA, was also found in Sulfolobus EVs (Ellen et al. 2009;Liu et al. 2021b).
In addition to the typical EVs, some members of the Thermococcales, such as T. prieurii or T. kodakarensis, produce numerous intracellular dark vesicles that bud from the host cells (Gorlas et al. 2015) (Fig. 2E and F). Energy-dispersive X-ray spectroscopy analyses revealed that these dark vesicles are filled with sulfur, and hence they have been termed 'sulfur vesicles' (SVs). The presence of SVs was exclusively observed when elemental sulfur S)0) is added into the growth medium, suggesting that these SVs could be produced to prevent the toxic intracellular accumulation of S(0) and/or polysulfides, thus playing a key role in sulfur detoxification. Surprisingly, the SVs are not produced by all species of Thermococales, suggesting significant differences in the sulfur metabolic pathways (Gorlas et al. 2015). More recently, it has been observed that Thermococcales SVs and EVs are actively involved in the production of iron-sulfide biominerals (Gorlas et al. 2018), suggesting a defensive function of EVs that might allow Thermococcales to survive in a broad range of extreme environments characterized by the high iron and sulfide contents (Gorlas et al. 2018) (Fig. 2F and G).
The EVs produced by members of the Thermococcales were shown to be often associated with either chromosomal or plasmid/viral DNA (Soler et al. 2008(Soler et al. , 2011Gaudin et al. 2013Gaudin et al. , 2014Choi et al. 2015). As recently observed with Sulfolobus EVs, DNA enclosed within EVs produced by Thermococcales is more resistant to thermodenaturation than free DNA, suggesting a protective role of the EVs (Soler et al. 2008), which is likely to be vital for horizontal gene transfer in extreme geothermal environments. The EVs of Thermococcales can indeed transfer DNA between cells. It was demonstrated that EVs of T. kodakarensis can be used to transfer plasmid DNA into plasmid-free cells .
Thermococcus onnurineus cells produce heterogeneous populations of EVs, which differ in terms of size and DNA content (Choi et al. 2015). EVs always encapsidate ∼14-kb-long DNA fragments. However, sequencing of the packaged DNA revealed that all regions of the T. onnurineus genome are represented in EVs, except for a 9.4-kb region. The authors speculated that this region might participate in DNA packaging and/or EV production (Choi et al. 2015). Interestingly, a T. onnurineus mutant in which the 9.4-kb region has been deleted still produces EVs but without associated DNA, supporting the original hypothesis (Kim, pers. comm.). This 9.4-kb region encodes various enzymes involved in sulfur metabolism and/or hydrogen production, with the possible roles of these enzymes in DNA packaging being unclear.
Remarkably, EVs produced by T. nautili, which contains three plasmids, selectively incorporate only two of these plasmids, pTN1 and pTN3, but not pTN2 (Soler et al. 2011;Gaudin et al. 2014). The reason for this specificity is unknown and purified EVs did not contain proteins encoded by pTN1 or pTN3. Notably, pTN3 is a defective virus belonging to the viral realm Varidnaviria (formerly the PRD1-Adenovirus lineage). This observation reinforces the idea that EVs of Thermococcales can serve as vehicles for the intercellular transport of extrachromosomal DNA (Soler et al. 2011;Gaudin et al. 2014). EVs containing viral DNA have also been detected in Bacteria and Eukarya (Gill, Catchpole and Forterre 2019) and the term 'viral vesicle' has been proposed for these biological entities. Interestingly, in silico analysis of the DNA associated with bacterial EVs isolated in diverse marine environments has revealed the presence of many viral genes, suggesting that such 'viral vesicles' are abundant in nature, alongside true virions and EVs containing cellular DNA (Soler et al. 2015).
Some EVs have been recently detected in cultures of Methanocaldococcus fervens, a methanogenic hyperthermophile belonging to the order Methanococcales (Thiroux et al. 2021). The natural isolate of M. fervens is a lysogen producing a headtailed virus MFTV1 (Krupovic, Forterre and Bamford 2010;Thiroux et al. 2021). Methanocaldococcus fervens cultures exposed to copper displayed greater production of EVs, but lower virus production, suggesting an interplay between EV production and virus life cycle (Thiroux et al. 2021). Notably, Methanocaldococcus and Thermococcus species inhabit the same deep-sea hydrothermal vent ecosystems and were shown to share several groups of non-conjugative plasmids, some of which could have been exchanged horizontally through EVs (Krupovic et al. 2013). EVs and structures resembling nanotubes were observed in cultures of Aciduliprofundum boonei (Reysenbach et al. 2006;Reysenbach and Flores 2008), a thermoacidophilic euryarchaeon distantly related to Thermococcales and Methanococcales.
Haloarchaea appear to represent a very promising model for the study of EVs in Archaea. Haloarchaea are halophilic and aerobic microorganisms that thrive in up to 5.5 M NaCl, i.e. salt concentrations approaching saturation, and are responsible for the pink colour of many hypersaline seas and lakes around the globe due to the specific carotenoid pigments that they produce. A seminal study describing EVs produced by Halorubrum has uncovered entities blurring the canonical frontiers between plasmids and viruses (Erdmann et al. 2017). Indeed, in contrast to plasmid vesicles produced by T. nautili, the membranes of EVs from Halorubrum carrying the plasmid pR1SE contain mostly proteins encoded by this plasmid, resembling the packaging of viral genomes by capsid proteins. Many of these plasmidencoded proteins were found in EVs by mass spectrometry analysis. These peculiar 'plasmid vesicles' were proposed to be the prototype of a new type of biological entities called 'plasmidions,' standing for membrane vesicles mimicking virions (Forterre, Da Cunha and Catchpole 2017). Similar to EVs of Sulfolobus (Liu et al. 2021b) and Thermococcus (Soler et al. 2008;Gaudin et al. 2014), Halorubrum EVs can mediate the transfer of plasmid DNA (pR1SE and derivatives). Notably, the plasmids carried by Halorubrum EVs can integrate into haloarchaeal replicons (note that Haloarchaea harbour several circular replicons), and subsequent excision from these replicons generates plasmid derivatives with different segments of the host chromosome, possibly leading to the horizontal transfer of the host genes by vesiduction (Erdmann et al. 2017).
NANOTUBES IN ARCHAEA
It has been known for a long time that eukaryotic cells can produce long tubular structures, often known as tunnelling nanotubes or sometimes microvillus (Lou et al. 2012;Rustom 2016;Nawaz and Fatima 2017;Cordero Cervantes and Zurzolo 2021). These nanotubes, formed by extrusion from the cytoplasmic membrane, can have very different lengths and thickness and can contain actin and/or tubulin filaments (for review, see Gill, Catchpole and Forterre 2019). They can connect different cells across substantial distances and have different physiological roles. Nanotubes (also called nanopods) were reported more recently in Bacteria (Baidya et al. 2018). It has been proposed that they could play a role in transferring nutrients, electrons or genetic material between cells. Bacterial nanotubes have been extensively studied in Bacillus subtilis (Baidya, Rosenshine and Ben-Yehuda 2020 and references therein). More recently, it has been suggested that these structures are not physiologically relevant since 'they are exclusively extruded from dying cells as a result of biophysical forces' (Pospíšil et al. 2020). The production of nanotubes by bacteria, especially Gram-positive, indeed raises questions because extrusion of nanotubes cannot take place without formation of apertures in the thick peptidoglycan layer. Considering the high number of studies that have emphasized important roles for nanotubes in bacteria during these last few years, the question of their physiological relevance should become a hot topic.
In their first studies on the Thermococcales EVs, Forterre and colleagues noticed the presence of strings of EVs enclosed within an elongated membrane structure covered by the S-layer (Soler et al. 2008), resembling the nanopods or nanotubes later observed in bacteria (Fig. 3). It remains to be definitively demonstrated that such structures are normally produced by living cells in natural habitats, as in the case of EVs from Sulfolobales. However, nanotubes from Thermococcales are sometimes produced in abundance (Soler et al. 2008) and throughout different growth phases (Gauliard, pers. comm.), suggesting that they could be physiologically relevant. Some species of Thermococcales are able to produce giant nanotubes that can reach several micrometers in length and are often filled with EVs (Fig. 3). EVs present within these nanotubes are usually smaller than free EVs ( Fig. 3A and B) and larger vesicle-like structure are sometimes located in the extremities of the nanotubes (Fig. 3C), suggesting that these structures could be involved in the transport and/or formation of EVs. Nanotubes often connect cells of Thermococcales together (Fig. 3D) and it was suggested that they could be involved in transfer of materials (nucleic acids and proteins) between cells (Marguet et al. 2013).
During the 80s, the Mevarech group has described an original mechanism of gene transfer between species of the genus Haloferax (order Halobacteriales) (Mevarech and Werczberger 1985;Rosenshine, Tchelet and Mevarech 1989). These transfers can be interspecific and were shown to be bidirectional, leading to genetic hybrids through DNA recombination between the parental genomes (Naor et al. 2012). They do not seem to involve EVs but instead cell-cell bridges were recently observed by electron cryo-tomography (Sivabalasarma et al. 2020) (Fig. 4). The Slayer-covered nanotube-like protrusions are 100 nm in diameter and connect the cells that are up to 2 μm apart (Rosenshine, Tchelet and Mevarech 1989;Sivabalasarma et al. 2020) (Fig. 4C). These structures were shown to allow the diffusion of cellular materials, such as ribosomes (Fig. 4D).
A hyperthermophilic crenarchaeon of the genus Pyrodictium was found to form extracellular tubules with an outer diameter of around 25 nm, which interconnected the cells and led to the formation of extensive networks (Horn et al. 1999;Nickell et al. 2003). These extracellular tubes formed by Pyrodictium are much thinner than those produced by Thermococcus spp. (Soler et al. 2008;Marguet et al. 2013), and it is not known whether their biogenesis is mechanistically related to the EV production.
Finally, very long tubular structures similar to those observed in Thermococcales have been occasionally observed to be associated with cells of the first cultivated Asgard archaeon, Prometheoarchaeum synthrophicum (Imachi et al. 2020). However, these nanotubes do not connect cells together, suggesting divergence in physiological role. Imachi and co-workers have suggested that similar structures present in the Asgard ancestor of eukaryotes have facilitated the engulfment of an aerobic bacterial symbiont at the onset of eukaryogenesis (Imachi et al. 2020).
PERSPECTIVES
Although the research on archaeal EVs and nanotubes is still in its infancy, it is already clear that these structures, especially EVs, play a profound role in archaeal physiology and environmental adaptation. Archaeal EVs were shown to contain cellular, plasmid or viral DNA. Vesiduction has now been demonstrated for several archaeal phyla (Thermococcales, Haloarchaea and Sulfolobales). As a result, archaeal EVs probably play an important role in the evolution and plasticity of cellular archaeal genomes and their mobilome, thus enabling archaeal adaptation in very diverse and often extreme environments. Future investigations on archaeal EVs should now more systematically test their ability to facilitate horizontal gene transfers by vesiduction in order to have a more complete overview of their importance in archaeal evolution. Moreover, the molecular mechanism of DNA recruitment into EVs is yet to be understood.
The existence of diverse mechanisms of EV production in Archaea is exemplified by the abundant production of EVs in Crenarchaeota and Euryarchaeota that encode and lack the ESCRT system, respectively. The discovery of proteins involved in EV production in Thermococcales and Haloarchaea is a major challenge right now. Hopefully, the availability of powerful genetic tools for these organisms will help to identify such proteins in the near future. One possibility is that some proteins involved in cell division in Euryarchaeota are also involved in EV production, as is the case for ESCRT proteins in Crenarchaeota. Another interesting possibility is that EV formation depends on proteins involved in polar lipids biosynthesis, especially those involved in the modification of the polar head groups. It was suggested that EV production in Bacteria and Eukarya is controlled, at least partly, by the regulation of enzymes involved in the incorporation of polar lipids in order to create asymmetry between the outer and inner membrane leaflets (Gill, Catchpole and Forterre 2019). However, this hypothesis cannot be directly transposed to Archaea that often have monolayer membranes formed by long tetraether lipids. In that case, the classical models of membrane fission and fusion that involve the transient opening of the bilayer cannot be applied (Relini et al. 1996). Notably, monolayer membranes are especially preva-lent in Sulfolobales and Thermococcales, which have been more thoroughly studied for EV production. Indeed, in many Sulfolobales species, 95-100% of lipids in the membrane are membranespanning C 40 glycerol dibiphytanyl glycerol tetraethers (Quemin et al. 2015;Kasson et al. 2017;Liu et al. 2018;Wang et al. 2019). One possibility is that membrane curvature in organisms with monolayer membranes is induced by the accumulation of larger polar head groups in the outer surface of the monolayer. Alternatively, patches of membrane could adopt bilayer structure by favouring the local clustering of diether lipids or that of tetraether lipids adopting the horseshoe conformation, as recently observed in the membranes of some archaeal viruses (Kasson et al. 2017). In the future, a better knowledge of the archaeal lipid biosynthetic pathways, especially that of the formation of polar head group, would enable the genetic manipulations necessary to test different hypotheses.
The production of nanotubes observed in some Archaea is another aspect worthy of further studies. Curiously, although nanotubes have not yet been observed in Sulfolobales, some species of Thermococcales produce abundant nanotubes that could be linked to EV production. Studying the still mysterious mechanisms for EV production in these archaea thus should also bring information on nanotubes, their relevance and physiological roles. Interestingly, it was shown that some nanotubes in eukaryotes are formed from EVs (Rustom 2016). This suggests that nanotubes could be also relevant structure in Archaea and studying their connection to EVs is likely to become a hot topic of research in the near future. It will be especially interesting to see whether, besides Lokiarchaeota, other Asgard archaea produce nanotubes and whether the eukaryotic-like actins encoded by these archaea are involved in the formation of these structures. A possible evolutionary connection between the pathway for nanotube formation in Archaea and Eukaryotes will be worth exploring in Asgard archaea or Bathyarchaea, which encode actin and/or tubulin homologues. | v3-fos-license |
2020-01-11T14:03:42.506Z | 2020-01-09T00:00:00.000 | 210134071 | {
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} | pes2o/s2orc | Enantio‐ and Diastereoselective Synthesis of Homopropargyl Amines by Copper‐Catalyzed Coupling of Imines, 1,3‐Enynes, and Diborons
Abstract An efficient, enantio‐ and diastereoselective, copper‐catalyzed coupling of imines, 1,3‐enynes, and diborons is reported. The process shows broad substrate scope and delivers complex, chiral homopropargyl amines; useful building blocks on the way to biologically‐relevant compounds. In particular, functionalized homopropargyl amines bearing up to three contiguous stereocenters can be prepared in a single step.
Chiral homopropargyl amines are used in the synthesis of many natural products, and biologically and medicinally important molecules. [1][2][3] Most methods for homopropargyl amine synthesis involve the union of imines and propargylic or allenic substrates. These methods deliver racemic homopropargylic amines [4] and asymmetric variants selectively generate products with a stereocenter adjacent to the amino group (Scheme 1 A). In general, these methods use a transition metal catalyst and chiral ligand, or imines bearing a chiral auxiliary. [5] Constructing homopropargyl amines with more than one stereocenter, particularly if the stereocenters are adjacent, is a more challenging process (Scheme 1 B), few procedures address this goal and these require difficult-toaccess reagents and/or chiral auxiliaries. [6] Thus, a general preparation of chiral homopropargylic amines, bearing multiple stereocenters, from readily-accessible substrates, remains an important challenge.
Copper-catalyzed borylative transformations are a powerful method for uniting unsaturated hydrocarbons and electrophiles. [7] Importantly, these methods produce densely functionalized, chiral molecules from simple, achiral substrates, and use cheap and non-toxic transition metal catalysts. We and others have described efficient routes to amines through the multicomponent coupling of imines with hydrocarbon pro-nucleophiles and boron reagents. [8][9][10] Krische pioneered the use of enynes as hydrocarbon pro-nucleophiles in transition metal-catalyzed transformations, [11][12][13] however, in both reductive and borylative coupling, the asymmetric union of imines and enynes remains an unmet challenge. [14] We envisaged a new approach to homopropargyl amines involving the copper-catalyzed enantio-and diastereoselective multicomponent coupling of imines, enynes, and diboron reagents (Scheme 1 C). Furthermore, through routine oxidation of the carbon-boron bond, biologically relevant 1,3amino alcohols would be accessible. [15] Herein, we disclose an efficient method for obtaining functionalized chiral homopropargyl amines, bearing up to three stereocenters and various synthetic handles (amino, boron, alkynyl), using an inexpensive, non-toxic, and readily-available copper catalyst, and a commercial phosphine ligand.
We explored the copper-catalyzed coupling of imine 1 a, 1,3-enyne 2 a and bis(pinacolato)diboron (B 2 pin 2 ). Using CuCl and (S,S)-Ph-BPE (L1), the desired product 3 a' (PG = PMP) was obtained in 70 % yield and the major diastereoisomer was found to have an ee of 53 % (Table 1, entry 1). After screening reaction conditions with imine 1 a, we turned our attention to N-phosphinoylimine 1 b. With this imine, the enantioselectivity and diastereoselectivity of the reaction increased (89 % ee, > 95:5 dr), however, only 37 % yield of the desired product was obtained (entry 2). By screening the copper salt, base, and solvent, we found that the use of CuOAc, KOMe, and THF was optimal; 3 a was obtained in high yield, with excellent diastereoselectivity and enantioselectivity (entry 3). [16] X-ray crystallographic analysis of 3 d revealed the relative and absolute stereochemistry of the Scheme 1. Enantioselective transition metal-catalyzed nucleophilic addition to imines for the synthesis of homopropargyl amines. PG = protecting group; X = PG or chiral auxiliary; Pin = pinacolato.
The reaction tolerated electron-donating and electronwithdrawing substituents on the aryl ring of the aldimine; the desired products were obtained in high yield and with excellent enantio-and diastereoselectivity (Scheme 2). For example, electron-rich aldimines were well tolerated in the reaction and only a slight decrease in enantioselectivity was observed when an ortho-methoxy substituent was used (3 b). Similarly, imines bearing electron-withdrawing groups at the ortho-, meta-, and para-positions (3 e-3 j), including halogen (3 e-3 g, 3 i), ester (3 h), and trifluoromethyl (3 j) substituents, also performed well. The reaction also proceeded efficiently when heteroaryl-aldimines were used (3 l-3 o). The reaction could be executed on a gram scale without significant detriment to the yield or selectivity (3 a). Attempts to use an aliphatic aldimine in the process were unsuccessful (See Supporting Information).
Aryl-substituted 1,3-enynes bearing electron-donating groups delivered the corresponding products in good to excellent yield and with high enantioselectivities (Scheme 3, 4a-4d). Mixed results were obtained when using electrondeficient enynes; for example, whereas the bromo-substituted product 4e was prepared in good yield, with high selectivity, an ester substituent severely affected the efficiency of the coupling (4 f). The use of an alkyl substituted enyne gave 4h in low yield but with high enantiocontrol. Substitution at the terminal position of the alkene was investigated: E-enynes gave products 6b-6d in good to high yield, with good diastereoselectivity and excellent enantioselectivity. The structure of 6b was confirmed by X-ray crystallography. [16] The use of Z-enyne 5a-Z gave alternative diastereoisomeric product 6a. Thus, the process delivers amino alcohols bearing three contiguous stereocenters with essentially complete enantiocontrol. Amine 3 a was readily hydrogenated, to give the branched chain alkane 7 a, and the b-amino acid derivative 7 b was accessed by oxidation of 7 a (Scheme 4). Biologically-and medicinally-relevant N-containing heterocycles were also prepared, for example, azetidine 7 c, or 2,3-dihydropyrrole 7 d through p-activation of the alkyne bond using a Au-Ag catalyst system. [17] The phosphinoyl group could be removed to reveal the free amine 7 e, [9a] which was subjected to urethanation to give oxazinone 7 f.
Regioselective borocupration provides intermediate A (1), [12a, 13a] which is proposed to undergo propargyl-to-allenyl isomerization to B (2) (Scheme 5 A). [12d] We propose that intermediate B is the major allenyl-copper isomer in the reaction. [12d] Coupling of the allenyl-copper intermediate B with imine 1 b (C re , 3) gives chiral homopropargylic amine D and closes the catalytic cycle (4). [12b-d] Scheme 5 B provides an explanation for the anti-diastereoselectivity observed in the reaction. Coupling (3) between allenyl intermediate B and imine 1 b can occur from attack at either the re face (C re ) or the si face (C si ) of the imine. However, reaction at the si face (C si ) incurs unfavorable interactions between the N-phosphinoyl group and the -CH 2 Bpin group and is disfavored.
In conclusion, a highly enantio-and diastereoselective coupling of imines, 1,3-enynes, and diborons using an inexpensive copper catalyst and a commercial ligand, delivers chiral homopropargyl amines with up to three contiguous stereocenters. The products provide access to important targets, including b-amino acids and N-heterocycles. | v3-fos-license |
2018-11-22T17:06:29.514Z | 2018-11-22T00:00:00.000 | 53744413 | {
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} | pes2o/s2orc | Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum
Background Metabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis. Results Engineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone. Conclusion Overexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production. Electronic supplementary material The online version of this article (10.1186/s13068-018-1317-3) contains supplementary material, which is available to authorized users.
Background
Phaeodactylum tricornutum is a unicellular model pennate diatom that can accumulate lipids particularly when it is subjected to environmental stresses [1][2][3][4][5]. Recently, P. tricornutum has emerged as a model candidate for lipid enhancement by metabolic engineering, owing to its high lipid content and the availability of a sequenced genome and genetic tool kit. However, engineered strains accumulate lipids to less than their theoretical maximum [6,7] and contain a wide range of fatty acid moieties that hinder their commercial exploitation [8,9]. Furthermore, the triacylglycerol (TAG) biosynthetic pathway is complex and is regulated by various metabolic nodes. Therefore, there is a pressing need to identify the key metabolic nodes and unravel their regulatory mechanisms so that the full potential of microalgal fuel production potential can be realized.
Triacylglycerols in microalgae are primarily stored in lipid droplets (LDs) and serve as energy reservoirs [10]. In microalgae, TAG can be synthesized via the de novo synthetic pathway, the fatty acyl-CoA-dependent Kennedy pathway, and/or the acyl-CoA independent PDAT pathway. The Kennedy pathway comprises three sequential acylations of a glycerol backbone that are catalyzed by glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) [11]. The GPAT, LPAT, and DGAT transfer the acyl moiety to the sn-1, sn-2, and sn-3 positions of a glycerol backbone for TAG assembly, respectively. This leads to the generation of lysophosphatidic acid (LPA), phosphatidic acid (PA), diacylglycerol (DAG), and the end product TAG. In higher plants, TAG is assembled in the endoplasmic reticulum (ER) from its precursor DAG and stored in cytosolic LDs. However, in Chlamydomonas reinhardtii, Fan et al. [12] found that TAG is produced from DAG in the chloroplast and accumulated in both the chloroplasts and cytosol, thus implying the existence of prokaryotic plastid and eukaryotic ER-localized TAG biosynthetic pathways.
GPAT and LPAT catalyze the initial committed steps. This leads to the formation of key substrates for TAG biosynthesis, and hence, they are considered to be the ratelimiting enzymes [13,14]. In higher plants, ten GPAT isoforms have been identified, of which two have been reported to be involved in the Kennedy pathway [15][16][17]. In the model diatom P. tricornutum, GPAT overexpression significantly enhances TAG content and alters the fatty acid profile [18]. Our previous study showed that overexpression of LPAT significantly increases TAG content and polyunsaturated fatty acid levels in P. tricornutum [19]. Plastidial LPAT has prokaryotic catalytic activity and prefers C16 fatty acids, whereas ER-localized LPAT prefers C18 fatty acids [20][21][22]. Interestingly, Chlamydomonas and related green algae have been shown to possess ER-LPAT, but they exhibit greater activity on C16:0 as plastidial LPATs [23]. Therefore, it is important to understand the acyl-specific catalytic mechanism underlying algal LPAT as well as its pivotal role in producing TAG.
We introduced both GPAT1 and LPAT1 genes into P. tricornutum cells to produce microalgal strains with high lipid yields. Molecular analyses revealed the expression of the two genes in the transgenic lines. We obtained transgenic strains that showed significantly elevated TAG contents without affecting algal biomass. This study also investigated the potential mechanisms underlying lipid accumulation triggered by GPAT1 and LPAT1 overexpression and elucidated the prokaryotic pathway for TAG assembly in diatoms. A presumptive pathway that transports TAG precursors from the chloroplast to the LDs is proposed for diatoms.
Algal strain and culture conditions
Phaeodactylum tricornutum (Strain CCMP-2561) was procured from the Provasoli-Guillard National Center for Marine Algae and Microbiota (East Boothbay, USA). It was maintained in f/2 medium at 20 ± 0.5 °C under a 12 h:12 h light/dark cycle with 200 μmol photons m −2 s −1 irradiance. Before preparation of the transgenic cells, the algal cells were acclimated in modified f/2 medium without Si. In the nitrogen (N) or phosphorus (P)-depleted experiments, the cells grown under N-replete conditions were harvested by centrifugation (4400×g for 10 min), washed with an N or P-free medium, and resuspended in N or P-free medium. In the NADPH inhibition experiment, different concentrations of sesamol (0-2 mM) were added to the medium, and the cells were harvested after 48 h treatment for further analysis. The analyses of the samples taken from the media containing different concentrations of sesamol were performed in triplicate. In addition, cells harvested on day 4 were used for biomass analysis while cells harvested on day 7 were used for the determination of lipid productivity.
Construction and transformation of the double overexpression system
Total RNA was isolated from algal cells harvested on day 7 using a Plant RNA Kit (Omega, USA) and transcribed into cDNA with a HiScript II Reverse Transcriptase Kit (Vazyme, China) according to the manufacturer's instructions. The full-length coding regions for GPAT1 (Accession No.: XP_002177014.1) and LPAT1 (Accession No.: XP_002176893.1) were amplified using the primers shown in Additional file 1: Table S1. The GPAT1 and LPAT1 gene fragments were purified using a gel extraction kit (Omega, USA) and cloned into the expression vectors pHY18 and pHY21, respectively, through the homologous recombination method, using a CloneExpress II One Step Kit (Vazyme, China). The recombinant plasmids (pHY18-GPAT1 and pHY21-LPAT1) were first linearized and then electroporated into algal cells using Gene Pulser Xcell equipment (Bio-Rad, USA) at a 1:1 ratio (w/w) according to Wang et al. [8]. The transformed cells were grown in f/2 liquid medium without antibiotics for at least 2 days, then selected on f/2 solid medium with chloramphenicol (250 mg L −1 ) and zeocin (100 mg L −1 ). Genomic PCR was performed to verify the integration of the expressions containing the GPAT1 and LPAT1 cassettes in the diatom. The PCR method has been previously described by Kang et al. [24]. Briefly, both WT and transgenic cells were harvested, and their genomic DNA was extracted. Then the genomic DNA was used as the template for the PCR. Taq PCR StarMix (GenStar, China) was also used to run the PCR. The antibiotic gene Shble and CAT in the expression cassette were amplified using primers Shble-f, Shble-r, CAT-f, and CAT-r, whereas the 18S rDNA gene was detected using primers 18s-f and 18s-r (Additional file 1: Table S1). The expected PCR product lengths of Shble, CAT , and the 18s rDNA were 349 bp, 500 bp, and 498 bp, respectively.
Quantitative real-time PCR, western blotting, and enzymatic activity assays
Total RNA was extracted for qRT-PCR using a Plant RNA Kit (Omega, USA) and its concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme, China). The qRT-PCR reactions were performed in eight-strip realtime PCR tubes containing 20 μL AceQ qPCR SYBR Green Master Mix (Vazyme, China). The relative transcript abundance was calculated by the 2 −ΔΔCt method after the expression had been normalized to that of the endogenous housekeeping gene β-actin. Three biological replicates were analyzed. The primers for the qRT-PCR are listed in Additional file 1: Table S1.
Western blot analysis was used to examine the expression of the target proteins. Total protein was extracted from algal cells with RIPA lysis buffer (Beyotime, China) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). The total protein concentration was determined with a BCA protein quantification kit (Beyotime, China). After quantification, the proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-transferred to a polyvinylidene difluoride membrane pre-activated by methanol. The membrane was incubated with primary anti c-Myc antibody (1:3000, Abcam, UK) or anti-flag antibody (1:2000, Sigma, USA) overnight at 4 °C after it had been blocked with skimmed milk for 1 h at 4 °C. This was followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, CST, USA) for 2 h at 4 °C. Then, the membrane was washed three times in pre-cooled PBST (Sangon, China) and developed with a chemiluminescence system (Millipore, USA). Endogenous β-actin was used as an internal control.
The activity levels of GPAT1, LPAT1 and malic enzyme (ME) were measured using a plant GPAT activity spectrophotometry assay kit (Bangyi, China), a plant LPAT activity spectrophotometry assay kit (Bangyi, China), and a ME colorimetric quantitative detection kit (Ke Ming Co., China), respectively, according to the manufacturer's instructions.
Measurement of photosynthetic parameters
The effective photochemical efficiency of photosystem II (Fv/Fm) and the electron transport rate (ETR) were measured using a PhytoPAM Phytoplankton Analyzer (Walz, Germany). Non-photochemical quenching (NPQ) was calculated using the formula: NPQ = (Fm − Fm′)/ Fm′ [25]. Chlorophyll a and c were quantified using an Agilent 1200 HPLC system (Agilent Technologies, USA) with a Symmetry C8 column as described previously [26]. Cell density was calculated daily through the direct count method, with a microscope and a bright-line Neubauer hemocytometer. Specific growth rate (μ) of culture in log phase was calculated following the equation reported by Nur et al. [27].
Lipid, protein, and carbohydrate analysis
The relative neutral lipid (NL) content was determined with the Nile-red staining method as described previously with minor modification [28]. Briefly, 50 μL Nile red solution (0.1 mg mL −1 in acetone) was added to 5 mL algal culture and incubated for 20 min in the dark at 37 °C. Then the mixture was transferred to a 96-well plate, and the relative fluorescence intensity was measured with a microplate reader (Bio-Tek, USA) at an excitation wavelength of 480 nm and an emission wavelength of 592 nm. Total lipids (TLs) were determined gravimetrically according to a previously described method [8]. Approximately, 50 mg lyophilized algal cells were ground to a powder in liquid nitrogen and transferred to 10 mL tubes. Then 3.8 mL mixed solvent (methanol/chloroform/water, 2:1:0.8, v/v/v) was added, and the mixture was sonicated for 15 min at 200 W. Then 2 mL of chloroform/water (1:1, v/v) was added, and the solution was mildly vortexed. The suspension was separated into two layers by centrifugation at 2000 rpm for 5 min. The upper phase was discarded, and the lower phase was collected in a pre-weighed tube. The extract was dried under a stream of N 2 and weighed.
The TAG was determined by extracting the total lipids from the algal cells as described as above and dissolving them in chloroform. The lipids were separated by thin layer chromatography on a silica plate, using the developing solvent hexane/diethyl ether/acetic acid (85:15:1, v/v/v). The separated TAG was visualized by iodine vapor, scraped off the plate, and dissolved in chloroform. The solution was centrifuged at 12,000×g for 10 min, and the supernatant was collected, dried by N 2 steam flow, and TAG was gravimetrically determined. Fatty acid composition was determined as fatty acid methyl esters with a gas chromatography-mass spectrophotometer equipped with an NIST 147 spectrum library, according to the method described by Balamurugan et al. [19]. The peak areas were normalized to the internal standard methyl nonadecylate (Aladdin, China).
The total carbohydrate content was obtained following the phenol-sulfuric acid method [29]. Briefly, cell pellets were resuspended in 1 mL ddH 2 O. Then 1 mL 5% phenol solution was added to the mix, followed by 5 mL sulfuric acid (95-98%, v/v). The solution was incubated in a water bath at 25 °C for 10 min, and the absorbance of the solution was read at a wavelength of 483 nm. Based on the curve of glucose standard, carbohydrate content per liter of culture was obtained. This value was divided by cell density to get carbohydrate content per cell. Total soluble protein was extracted with the method described above and determined using a BCA protein quantification kit (Beyotime, China). According to the standard curve of bovine serum albumin, total soluble protein per liter of culture was calculated.
Confocal microscopy and subcellular localization analysis
One milliliter of culture was stained with 10 μL Nile red solution (0.1 mg mL −1 in acetone) and incubated at 37 °C for 30 min in the dark. The stained cells were observed under an LSM880 laser-scanning confocal microscope (Zeiss, Germany) with an excitation wavelength of 514 nm, an emission wavelength of 596 nm, and a detection wavelength range of 539-652 nm.
The subcellular localization of GPAT1 and LPAT1 was determined by fusing EGFP with the 3′-terminus of the target gene in pPhAP1-EGFP following the previous method [30]. The recombinant plasmids containing the target genes were electroporated into algal cells. The cells were observed under an LSM880 confocal laser scanning microscope (Zeiss, Germany), and the EGFP fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength range of 510-555 nm. Chlorophyll auto-fluorescence was detected at an excitation wavelength of 488 nm and an emission wavelength range of 625-720 nm.
Statistical analysis
All experiments were carried out in triplicate, and the data are expressed as mean ± SD. All statistical tests were performed using the SPSS statistical package 19.0. Paired t-tests were used to compare two groups. The results were considered to be significantly different at p < 0.05 (*) or p < 0.01 (**).
Sequence analysis and construction of the expression system
The sequence structure of GPAT1 and LPAT1 predicted using SMART (http://smart .embl-heide lberg .de/) [31] and ChloroP (http://www.cbs.dtu.dk/servi ces/Chlor oP/) [32] showed that GPAT1 contained a chloroplastic signal peptide, a LPLAT superfamily and two transmembrane regions, whereas LPAT1 had a plastidial signal peptide, a transmembrane region, and a PlsC domain (Additional file 1: Figure S1), thus suggesting that they are both membrane proteins localized to the chloroplast. However, the subcellular localization of GPAT1 and LPAT1 predicted using HECTAR [33] revealed that LPAT1 localized to the chloroplasts, whereas GPAT1 localized to the mitochondria (Additional file 1: Table S2). The results indicated that bioinformatic prediction is inaccurate. To further validate the localization of GPAT1 and LPAT1, we used pPhAP1 with EGFP to express the fusion protein for GPAT1 or LPAT1 (Additional file 1: Figure S2B and D). The confocal microscopy images showed that both GPAT1 and LPAT1 localized to the chloroplasts (Additional file 1: Figure S3), thus verifying the previous immunoelectron microscopy results [18,19].
The full-length coding sequences for GPAT1 and LPAT1 were PCR amplified and inserted into the vectors pHY-18 and pHY-21, respectively, for co-overexpression (Additional file 1: Figure S2A and C).
Verification of transgenic strains using molecular approaches
Putative transformants were screened using solid medium supplemented with chloramphenicol and zeocin. More than ten independent microalgal colonies were selected and inoculated into a liquid medium with chloramphenicol and zeocin for further screening. Then the cells were transferred to fresh medium without antibiotics for further experimental analysis. Genomic PCR was performed to detect the integration of the expression cassette in the transgenics. As expected, two amplicons (349 bp and 500 bp) were amplified in the two overexpression (OE) lines, OE1 and OE2, whereas no such bands were detected in the wild-type (WT) and negative control (B) (Additional file 1: Figure S4). This result demonstrated that the two expression cassettes harboring GPAT1 and LPAT1 were successfully introduced into P. tricornutum cells. Subsequently, qRT-PCR was performed to examine the GPAT1 and LPAT1 transcript levels in P. tricornutum. Figure 1a, b shows that the relative transcript levels of GPAT1 and LPAT1 were significantly higher in the OE lines than in the WT cultures over the 13 day culture period. They reached a maximum at day 7 and were upregulated by 5.11-6.64-fold in the transgenic lines compared with the WT. Interestingly, the expression of both the GPAT1 and LPAT1 transcripts gradually decreased after day 7, possibly because of nutrient depletion. In the two vectors, a c-Myc tag and a 2 × flag tag were ligated to the 3′-terminus of GPAT1 and LPAT1, respectively, so that their protein expression could be detected. Western blot analysis showed that two specific cross-reactive protein bands, which corresponded to the expected protein molecular weights, were present in the OE lines, whereas no such protein bands were detected in the WT cultures (Fig. 1c). These results indicated that the introduced GPAT1 and LPAT1 were successfully expressed in the transgenic cells. In addition, the enzymatic activities of GPAT1 and LPAT1 were assayed. The activities of the two enzymes were considerably higher in the OE lines than the WT, and this increase in activity occurred over the entire growth phase and reached a maximum on day 7 (Fig. 1d, e). These results confirmed that GPAT1 and LPAT1 were functional in the OE lines.
Effect of GPAT1 and LPAT1 overexpression on photosynthesis
Assessment of cellular physiological characteristics is crucial if P. tricornutum is to be commercially exploited. In this study, we measured a number of important photosynthetic parameters. Figure 2 shows that chlorophyll a or c content, the ETR, and Fv/Fm were significantly higher in the OE lines at day 4 than the WT cultures, whereas NPQ was lower. These results indicated that photosynthetic efficiency was elevated in the OE lines, in accordance with the enhanced growth rate of the OE lines during the mid-log phase (green dotted frame in Fig. 2f, Table 1). We also determined the transcript abundance of two key genes, phosphatidic acid phosphohydrolase (PAP) and monogalactosyldiacylglycerol synthase (MGD), which directly participate in diacylglycerol Fig. 1 Molecular characteristics of GPAT1 and LPAT1 co-overexpressed lines (OE1 and OE2) and wild-type (WT). a Relative expression abundance of GPAT1 during the whole growth period (13 days), β-actin was served as an internal reference gene; b relative expression abundance of LPAT1 during the whole growth period, β-actin was served as an internal reference gene; c Western blot analysis for detection of c-Myc and flag. Cells were collected on day 7. Lane 1 and 2 represent OE1 and OE2 lines, respectively. β-actin was served as a housekeeping protein; d changes of GPAT1 enzymatic activity during the whole growth phase; e changes of LPAT1 enzymatic activity in microalgae during the whole growth phase. A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level. Each value represents the mean ± SD (n = 3) Fig. 2 Physiological characterization of GPAT1 and LPAT1 dual overexpression lines (OE1 and OE2) and wild-type (WT) during the whole growth period (13 days). a Chlorophyll α content; b chlorophyll c content; c non-photochemical quenching (NPQ); d electron transport rate (ETR); e effective photochemical efficiency of photosystem II (Fv/Fm); f growth curve. A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level. Each value represents the mean ± SD (n = 3)
Table 1 Specific growth rate, biomass and lipid productivity of overexpression lines (OE-1 and OE-2) and wild-type (WT)
a Determined in the log phase. Specific growth rate and biomass were measured on day 4 while total lipid productivity and TAG productivity were determined on day 7. Each value represents the mean ± SD (n = 3). A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level (DAG) and monogalactosyldiacylglycerol (MGDG) biosynthesis. Interestingly, the PAP and MGD transcription levels were significantly higher in the OE lines than the WT cultures after day 4 (Fig. 5i, p), which suggested that the DAG and MGDG content in the OE lines had also increased. In algae, DAG is the precursor of thylakoid membrane lipids [34,35], which are crucial for high-efficiency photosynthesis [36][37][38][39]. It is likely that the overproduction of DAG and membrane lipids, triggered by GPAT1 and LPAT1 coordinated overexpression, might have led to the higher photosynthetic efficiency in OE cells. It has been reported that in A. thaliana, increased levels of thylakoid membrane lipids elevate photosynthetic efficiency [40]. In addition, to further support the notion, we determined the content of glycolipids and found that cellular glycolipid content was significantly higher in the OE lines at day 4 than the WT cultures (Additional file 1: Figure S5). Glycolipids are main components in thylakoid membrane lipids and have been shown to play a crucial role in maintaining photosynthetic performance [41][42][43]. Thus, the elevated photosynthetic efficiency in the OE lines may be related to the increase in glycolipids.
Co-overexpression altered the contents of cellular components
The total carbohydrate and soluble protein contents of the OE lines and WT cultures were analyzed at days 4 and 7. Figure 3a shows that the total carbohydrate content was significantly higher in the OE lines than the WT cultures on day 4, but decreased by day 7. There was no significant difference in soluble protein between the WT cultures and the OE lines on day 4, but soluble protein in the OE lines suddenly decreased on day 7 (Fig. 3b). We hypothesize that during the mid-log cultivation phase, the elevated photosynthesis in the OE lines might have required an increased CO 2 supply and, therefore, more of the carbon flux was directed towards carbohydrate synthesis. However, in the late-log phase, carbon precursors and energy sources might have been redirected to lipogenesis in the OE lines. In cyanobacteria, sucrose permease (encoded by CscB), a proton symporter, is involved in the transportation of sucrose into the cell across the cell membrane through proton symport [44].
CscB overexpression led to significant increases in biomass, photosystem II activity, and chlorophyll content, thus indicating that elevated photosynthesis redirects carbon flux to carbohydrate biosynthesis in cyanobacteria [44]. The results also confirmed that the biomass of the OE lines also increased, in accordance with the elevated photosynthesis levels (Table 1). Cellular lipids were also analyzed. The relative NL, TL, and TAG content was considerably higher in the OE lines after day 7 than in the WT culture (Figs. 3c, d and 4). This result suggests that overexpression of GPAT1 and LPAT1 effectively promoted the accumulation of cellular lipids, especially TAG. The dual overexpression lines had a higher total lipid content (50.4% of dry biomass) than the single overexpression GPAT1 or LPAT1 cell lines used in previous studies (lipid levels 42.6% or 42.9% of dry biomass, respectively) [18,19]. Laser scanning confocal microscopic analysis of Nile-red stained cells indicated that the volume of the LDs was significantly higher in the OE lines than the WT cultures (Additional file 1: Figure S6). This result was similar to an observation by Talebi et al. [45] in which the carbon flux from starch was found to be channeled to fatty acid synthesis when AccD and ME were overexpressed in Dunaliella salina. There were no significant changes in NL, TL, and TAG under the -N or -P conditions (Fig. 4). Therefore, the double overexpression OE lines might potentially reach their maximum lipid accumulation under normal nutrient conditions. The results from this study are in accordance with our previous observation that cells cease to accumulate lipids after the lipid content reaches a maximum [46,47]. Additionally, the P. tricornutum lipid productivity was calculated to assess whether P. tricornutum could potentially be used as a feedstock for biofuels. Table 1 shows that total lipid productivity was 1.023 ± 0.071 mg day −1 10 9 cells −1 for WT, whereas it reached 2.737 ± 0.195 m g day −1 10 9 cells −1 and 2.779 ± 0.209 mg day −1 10 9 cells − 1 , respectively, for the two OE lines. There was also a significant TAG productivity increase in the OE lines. Altogether, the data demonstrated that double overexpression strains may potentially increase lipid production without altering cellular biomass.
Effect of collective GPAT1 and LPAT1 overexpression on key genes involved in photosynthesis and TAG synthesis
Co-overexpression has a crucial role in controlling photosynthetic efficiency and lipid content. Therefore, we attempted to elucidate the effect of double overexpression on the expression of other key genes involved in photosynthesis and lipogenesis. Figure 5a-d shows that the photosynthetic related genes AtpC, PsbO, PsbM, and PetJ were significantly higher in OE lines on day 4 than in the WT cultures, but after mid-log phase, their expression declined. Furthermore, genes involved in TAG biosynthesis, including GPAT2, LPAT2, PAP, DGAT2A, DGAT2B, and DGAT2D, were detected during the stationary phase (Fig. 5), and their transcript abundance was significantly higher in the OE lines than the WT cultures. The data further suggested that photosynthesis was higher during the growth phase and that the resultant photosynthetic carbon was potentially used for lipogenesis during the stationary phase. Interestingly, the expression of GPAT3 and LPAT3, which were predicted to be localized to the ER, was not significantly different between the WT cultures and OE lines, which suggested that the ER-localized TAG biosynthetic pathway was not affected by the double overexpression of these genes. The results also indicated that the plastidial TAG biosynthetic pathway has a promising role in lipid accumulation. Malic enzyme and glucose-6-phosphate dehydrogenase play crucial roles in providing lipogenic NADPH to microalgae [48][49][50]. Therefore, this study examined whether double overexpression might also control the expression of NADPH-associated genes in OE lines. Interestingly, the transcript expression of G6PD and particularly ME was higher in the OE lines than the WT cultures after day 4 (Fig. 5n, o). However, the mechanism underlying such regulation remains unclear. Sesamol, a potent inhibitor of ME activity and NADPH supply, was used to determine the relationships among NADPH, lipid content, and ME expression in OE lines [51,52]. The results showed that 1.5 mM sesamol significantly reduced the increase in ME expression levels, ME enzyme activity and NADPH content in the OE lines, which in turn led to a decrease in lipid accumulation in the OE lines (Additional file 1: Figure S7). These analyses revealed that double overexpression elevated NADPH content and showed that ME has a significant role in providing lipogenic NADPH.
TAG synthesis mediated by GPAT1 and LPAT1
In microalgae, the fatty acyls for TAG assembly can be both de novo synthesized and recycled from membrane polar lipids. Our results showed that there was a significant increase in TAG content when the fatty acid content increased, which suggested that de novo synthesized fatty acids have an important role in TAG assembly (Fig. 6a). This study and previous studies [18,19] showed that GPAT1 and LPAT1 are localized in the diatom chloroplasts. However, the confocal microscopy images revealed that LDs were present in the cytoplasm and that most of them surrounded the chloroplast (Additional file 1: Figure S6). Furthermore, it is unclear how overexpression of GPAT1 and LPAT1 led to enlarged LDs in the cytoplasm. Fan et al. [12] suggested that C. reinhardtii can utilize DAG from chloroplasts to synthesize TAG, and that the TAG formed in this way is deposited in both the chloroplasts and the cytoplasm LDs. However, it has been recently shown that these chloroplast LDs in C. reinhardtii are actually LDs implanted within the chloroplast invaginations, which are part of the outer membrane of the chloroplast [53]. Therefore, we inferred that, like C. reinhardtii, diatoms may use a pathway that transports TAG precursors, such as DAG, from the chloroplasts to the LDs through a close association with the outer membrane of the chloroplast. In vascular plants, glycerolipids are assembled by prokaryotic or eukaryotic pathways. In the prokaryotic pathway, a 16-carbon acyl group is incorporated into the sn-2 position of glycerolipids in plastids by the substratespecific activity of plastidial LPAT, whereas in the eukaryotic pathway, an 18-carbon acyl group is added to the sn-2 position of glycerolipids in the ER [54]. The results from this study demonstrated that C16:0 dominated the TAG sn-2 position (Fig. 6b, d), which suggested that the sn-2 fatty acids in diatom TAGs are mainly assembled by the prokaryotic pathway. Similar observations have been found in green algae [12,[55][56][57]. In addition, the relative content of C16:0 and C16:1 at the TAG sn-2 position was significantly higher in the OE lines than the WT cultures (Fig. 6d), which suggested that LPAT1 may contribute more to prokaryotic TAG biosynthesis in diatoms. The phylogenetic analyses revealed that LPAT1 in P. tricornutum, CrLPAAT1 in C. reinhardtii, and LPAT1 in A. thaliana belonged to the same clade and were divergent from the ER-located CrLPAAT2 in C. reinhardtii (Additional file 1: Figure S8). In C. reinhardtii, the plastidlocated CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor [22]. These results suggest that the plastidial LPAT1 in P. tricornutum may have a similar function to the CrLPAAT1 in C. reinhardtii. C18:0 and C18:1 were significantly higher at the TAG sn-1/sn-3 position in the OE lines than the WT cultures, whereas C16:0 and C16:1 were significantly lower (Fig. 6c, e). The TAG positional Relative expression levels of genes related to photosynthesis, lipid and NADPH biosynthesis in overexpression lines (OE1 and OE2) and wild-type (WT). a-d Photosynthetic related genes including ATPase gamma subunit (AtpC), oxygen-evolving enhancer protein (PsbO), photosystem II subunit (PsbM) and cytochrome c6 (PetJ); e-m TAG biosynthetic related genes including glycerol-3-phosphate acyltransferase 2 (GPAT2), glycerol-3-phosphate acyltransferase 3 (GPAT3), lysophosphatidyl acyltransferase 2 (LPAT2), lysophosphatidyl acyltransferase 3 (LPAT3), phosphatidic acid phosphatase (PAP), diacylglycerol acyltransferase 1 (DGAT1), diacylglycerol acyltransferase 2A (DGAT2A), diacylglycerol acyltransferase 2B (DGAT2B) and diacylglycerol acyltransferase 2D (DGAT2D); n, o NADPH biosynthetic related genes including malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD); p MGD, a monogalactosyldiacylglycerol synthase gene. Each value represents the mean ± SD (n = 3). The gene information and primers are shown in Additional file 1: Table S1. Each value represents the mean ± SD (n = 3) analysis demonstrated that double overexpression resulted in differential changes in the fatty acid profiles of the TAG sn-2 and sn-1/sn-3 positions. In C. reinhardtii, the C18 fatty acids were more abundant than the C16 acyl chains at the sn-1/sn-3 position in TAG, which suggested that the availability of fatty acids or substrate specificity of acyltransferases may play an important role in cells [12]. The results from this study indicated that the C18 fatty acid percentage content was higher than the C16 percentage content in the OE lines, but they were very similar in the WT cultures. It is probable that more C18 fatty acids were available or preferred in the double overexpression system. Based on the aforementioned discussion, a schematic pathway for TAG assembly and LD formation in GPAT1 and LPAT1 overexpression cells is proposed (Fig. 7).
Conclusion
In this study, we report the functional importance of the TAG biosynthetic machinery in the overproduction of engineered fatty acids after the double overexpression of GPAT1 and LPAT1 in oleaginous P. tricornutum. The TAG content and maximum lipid content was considerably elevated in the OE lines, but cellular biomass did not decrease. GPAT1 and LPAT1 localized to plastids. Their increased expression elevated photosynthetic efficiency during the early growth phase and triggered the expression of lipogenic genes. LPAT1 shows prokaryotic activity, which prefers the transfer of the 16-carbon acyl group to the sn-2 position on TAG. These results suggest that the plastidial TAG pathway is involved in enhancing cellular lipid content. Collectively, the results provide valuable insights that may be used to genetically improve lipid production by manipulating suitable metabolic targets. c relative abundance of total C16 and C18 fatty acids in sn-1/3 position of TAG in cells harvested on day 7; e relative abundance of main fatty acids in sn-1/3 position of TAG in cells harvested on day 7. C16 represents the sum of C16:0 and C16:1 while C18 represents the sum of C18:0, C18:1, C18:2 and C18:3. Each value represents the mean ± SD (n = 3). A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level. Each value represents the mean ± SD (n = 3)
Additional file
Additional file 1: Figure S1. Protein structure of GPAT1 and LPAT1 obtained using SMART (http://smart .embl-heide lberg .de) and ChloroP (http://www.cbs.dtu.dk/servi ces/Chlor oP/). TM, transmembrane; SP, signal peptide; LPLAT and PlsC represented the conserved domains in proteins. Figure S2. Schematic representation of the expression vectors employed in this study. GPAT1 and LPAT1 genes were cloned into the expression vectors pHY18 (A) and pHY21 (C), respectively under the control of promoter PfcpC. An omega leader sequence and "ACC" nucleotides were inserted between the promoter and the target gene for boosting protein translation. For subcellular localization, GPAT1-EGFP (B) and LPAT1-EGFP (D) were employed under the control of promoter ProPtAP. Figure S3. Subcellular localization of GPAT1 and LPAT1 in P. tricornutum cells. A, Microscopy images of a representative wild-type cell; B, Microscopy images of a representative transgenic line with co-overexpression of GPAT1 and EGFP; C, Microscopy images of a representative transgenic line with co-overexpression of LPAT1 and EGFP. From left to right, fluorescence of EGFP, autofluorescence of chloroplasts, differential interference contrast (DIC), fluorescence images overlaid on DIC image. Scale bars represent 5 μm. Figure S4. PCR validation by agarose gel electrophoresis to verify the antibiotic gene Shble (349 bp), CAT (500 bp) and endogenous gene 18s rDNA (498 bp) PCR product. V1, pHY 21 vector (containing Shble gene); V2, pHY 18 vector (containing CAT gene); OE1 and OE2, individually overexpressed transformants; WT, wild type; B, negative control; M, marker. Figure S5. Glycolipid content in overexpression lines (OE1 and OE2) and wild-type (WT) harvested at day 4 and 7. A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level. Each value represents the mean ± SD (n = 3). Figure S6. Confocal microscopy images for detecting lipid droplet morphology in cells harvested on day 7. A, wild-type cells; B and C, transgenic lines. Left to right, fluorescence of Nile-red stained lipid droplets; autofluorescence of chloroplasts; fluorescence overlay; differential interference contrast (DIC). Scale bars represent 5 μm. Figure S7. Responses of wild-type (WT) and transgenic lines to sesamol treatment. Cells were harvested on day 7 and subjected to Fig. 7 Proposed pathway for TAG assembly in GPAT1 and LPAT1 overexpression lines of P. tricornutum. Regular font represents substrate or product; bold font represents enzyme; red bold font represents upregulated enzyme; purple bold font represents overexpressed enzyme. ACCase acetyl CoA carboxylase, MCAT malonyl-CoA:ACP transacylase, KAS 3-ketoacyl-ACP synthase, KAR 3-ketoacyl-ACP reductase, HD 3-hydroxyacyl-ACP dehydratase, ENR enoyl-ACP reductase, FAT fatty acyl-ACP thioesterase, LACS long-chain acyl-CoA synthetase, MGDG monogalactosyldiacylglycerol, MGD MGDG synthases, GPAT glycerol-3-phosphate acyltransferase, LPAT lysophosphatidate acyltransferase, PAP phosphatidic acid phosphatase, DGAT diacylglycerol acyltransferase, AtpC ATPase gamma subunit, PsbO oxygen-evolving enhancer protein, PsbM photosystem II subunit, PetJ cytochrome c6, ME malic enzyme, G6PD glucose-6-phosphate dehydrogenase, G-3-P glycerol-3-phosphate, LPA lysophosphatidic acid, PA phosphatidic acid, DAG diacylglycerol, TAG triacylglycerol, LD lipid droplet sesamol treatment for 48 h. A, Relative NADPH content in WT cells treated with different concentration of sesamol; B, Relative NADPH content in transgenic lines and WT treated with 1.5 mM sesamol; C, Relative neutral lipid content in transgenic lines and WT treated with 1.5 mM sesamol; D, ME expression level of transgenic lines and WT treated with 1.5 mM sesamol; E, ME enzyme activity of transgenic lines and WT treated with 1.5 mM sesamol. A significant difference between WT and OE lines is indicated at the p < 0.05 (*) or p < 0.01 (**) level. Each value represents the mean ± SD (n = 3). Figure S8. Phylogenetic tree showing relationship among LPATs from various organisms including plants (A. thaliana, B. napus, R. communis, O. sativa), microalgae (P. tricornutum, C. reinhardtii) and microbes (E. coli, S. cerevisiae). All the sequences were retrieved from NCBI and TAIR. The phylogenetic tree was established by MEGA7 using Maximum Likelihood method based on Poisson correction model. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are represented along the branches. Red frame indicates ER-located CrLPAAT2 from C. reinhardtii; blue frame indicates plastidial LPAT1 from P. tricornutum. Table S1. Primers used in this study. Table S2. Subcellular localization of GPAT1 and LPAT1 predicted using HECTAR. | v3-fos-license |
2018-03-24T13:10:31.313Z | 2018-03-23T00:00:00.000 | 4167107 | {
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} | pes2o/s2orc | Diatom frustules protect DNA from ultraviolet light
The evolutionary causes for generation of nano and microstructured silica by photosynthetic algae are not yet deciphered. Diatoms are single photosynthetic algal cells populating the oceans and waters around the globe. They generate a considerable fraction (20–30%) of all oxygen from photosynthesis, and 45% of total primary production of organic material in the sea. There are more than 100,000 species of diatoms, classified by the shape of the glass cage in which they live, and which they build during algal growth. These glass structures have accumulated for the last 100 million of years, and left rich deposits of nano/microstructured silicon oxide in the form of diatomaceous earth around the globe. Here we show that reflection of ultraviolet light by nanostructured silica can protect the deoxyribonucleic acid (DNA) in the algal cells, and that this may be an evolutionary cause for the formation of glass cages.
, Transmittance, reflectance and absorption spectra from NP monolayer (first row), CW and CR monolayers (second row).
Frustule preparation
The diatoms Coscinodiscus wailesii (CW), Coscinodiscus cf. radiatus (CR), Nitzschia sp. (N) and Navicula perminuta (NP) were cultivated separately in 0.2 µm filtered seawater (salinity 32), enriched with f/2 medium with silica. CW and CR were kept for 13 days at a temperature of ~15°C and an irradiance of ~100 µmol photons m -2 s -1 in a 16L:8D hours light cycle. NP and N were cultivated during 28 days at a temperature of ~4°C at an irradiance of ~70 µmol photons m -2 s -1 in a 22L:2D hours light cycle. Light was provided from fluorescent tubes (Osram Lumilux L36 W/865
Preparation of frustule monolayers
The frustule monolayer was created by spreading a dispersion of frustules in chloroform onto an aqueous surface. The formation of the monolayer was facilitated by a nonionic surfactant (Triton X-100, Sigma-Aldrich). The resulting monolayer was transferred to a cleaned glass slide.
SEM
Scanning electron microscopy (SEM) imaging was performed using a Zeiss Leo at 5kV acceleration voltage. Prior to imaging the samples were sputtered using a Leica EM SCD500 sputter coater with 10 Å Pt to increase contrast and enable high magnification imaging.
Numerical simulations
Finite-element method (FEM) simulations were performed using the COMSOL Multiphysics® software package solving the partial differential equations for the electromagnectic perturbation in the time domain field. In order to save computing resources, the complex structure of the frustules was reduced to a 2D representation based on the geometrical dimension extracted from the SEM images. In the case of frustules with radial symmetry, a cross section of the pores hierarchy was considered. Only two inner layers (foramen and cribrum) were included in the simulations. In order to consider the contribution of the neighboring structures, periodic conditions were applied to the boundaries along the x axis. In the case of frustules with bilateral symmetry (NP), the cross sections were taken along the apical and transapical axis. To avoid artifacts due to reflection in the boundaries perfect matching layers were applied where they were needed. Built-in refractive indexes were used for air (n=1) and SiO 2 (n=1.45).
In all cases, a plain wave incidence with a positive propagation vector parallel to the y axis and a wavelength of 260nm is considered. Based on the symmetry of the periodic arrays, the electric field source employed was polarized in the x direction and only considered the components in the x-y plane. In order to save computational resources, only a portion of the entire structure is represented. In the case of centric diatoms, only one hole of the inner plate (foramen) and this environment in the cribrum was considered. In addition, based on the hexagonal periodicity of centric diatoms, periodic boundaries along the x axis, were applied to include the contribution of the nearest neighbors. For the NP geometry, one arrangement of pores was used, considering two possible geometries; along the apical and transapical axes. In each case, the geometry used is outlined in black in Figure S2.
The electromagnetic field distribution and rate of energy flow in the near field (10µm) is shown in Figure S2a, c, e, g, after the interaction with CW, CR, NP (short axis) and NP (long axis) respectively. Figure S2b, d, f, h, correspond to the rate of energy flow for CW, CR, NP (short axis) and NP (long axis) respectively, where the white arrows represent the normalized flow direction.
The electric field and rate of energy flow in the near field (10µm) after CW ( Figure S2a Figure S2g and h). This effect can be related to the relation between pore size at the first interface (cribrum) and wavelength of the incident light (260nm) which is relevant for light scattering phenomena. Geometrical parameters were extracted from the SEM images (Fig. 1, Fig. S5), and the materials are SiO 2 and air for the surrounding medium.
Positive photoresist
The positive photoresist (Shipley MICROPOSIT S1813, sensitive to wavelengths between 350 to 450nm) was spin coated unto Si wafer at 4000 rpm (corresponding to roughly 1.5 µm thickness according to the manufacturer (http://www.microchem.com/PDFs_Dow/S1800.pdf)). The organic film was soft baked at 100°C for 5 minutes to remove solvents. A droplet of frustule containing solution was dried out for 5 minutes on the surface of the film. The film with frustules was put under a fluorescent microscope (Zeiss M200) equipped with optical filters. As illumination with red light did not affect the film it was possible to illuminate and focus on an area of interest. Switching to blue light, however, immediately started the light induced degradation process as visible in the microscope. After exposure the film for 1-5 seconds, the film was developed in 0.1 M NaOH for 1 minute where the exposed areas were Transmittance, reflectance and absorption spectra Figure S3, Transmittance, reflectance and absorption spectra from NP monolayer (first row), CW and CR monolayers (second row). Figure S4, Photo bleaching of a PEDOT:PSS layer after exposure to UVR (components between 250 and 360nm) during 24h. The light colour areas in each picture were covered during the exposure time with a-NP, b-CW and c-CR frustules. Scale bar represents 100µm.
Photobleaching of a PEDOT:PSS layer
As shown in Figure S4, when frustules are spread onto a PEDOT film, they effectively reduce the photobleaching of PEDOT. The regions covered with show clear diatom-shaped areas on the overall bleached film, which is directly related to the wave redistribution by frustule induced UVR scattering.
SEM imaging of frustule geometries | v3-fos-license |
2018-12-27T07:45:03.745Z | 2012-03-28T00:00:00.000 | 94736667 | {
"extfieldsofstudy": [
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} | pes2o/s2orc | Improving Cell Surface Functional Expression of F508 CFTR: A Quest for Therapeutic Targets
Cystic fibrosis (CF) is largely a protein misfolding disease. The deletion of a phenylalanine at residue 508 (F508) in the cystic fibrosis transmembrane conductance regulator (CFTR) accounts for 70% of all disease-causing alleles and is present in at least one copy in 90% of CF patients (Kerem et al., 1989). The F508 mutation impairs the conformational maturation of nascent CFTR (Lukacs et al., 1994), and arrests it in an early folding intermediate (Zhang et al., 1998). As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) (Cheng et al., 1990) in a chaperone-bound state (Yang et al., 1993), The ER-accumulated mutant CFTR fails to efficiently couple to the coatomer complex II (COPII) ER export machinery (Wang et al., 2004), and is degraded by the ubiquitin proteasome system through the ER-associated degradation (ERAD) pathway (Jensen et al., 1995; Ward et al., 1995), leading to loss of CFTR function at the cell surface. The folding defect of F508 CFTR appears kinetic in nature (Qu et al., 1997). A small fraction of F508 CFTR is able to exit the ER but the escaped mutant protein is not stable at the cell periphery and is rapidly cleared through lysosomal degradation (Lukacs et al., 1993). This second defect further reduces the cell surface localization of this mutant CFTR. Aside from localization defect, the F508 mutation also impairs the channel gating of CFTR, leading to reduced open probability (Dalemans et al., 1991). The threefold defect of F508 CFTR stems from its defective conformation, and impairs the CFTR functional expression at the cell surface, leading to severe clinical phenotype. Given the autosomal recessive inheritance of the disease, improving plasma membrane functional expression of F508 CFTR will benefit the vast majority of CF patients (Gelman & Kopito, 2002). Numerous research efforts have been made to improve F508 CFTR cell surface functional expression, including elevating its expression, reducing its degradation, enhancing the efficiency of its maturation, increasing its post-ER stability and improving its channel gating. Restoring F508 CFTR conformation will potentially improve its ER folding, its cell surface stability and its channel gating, leading to efficient F508 CFTR rescue. In this chapter, multiple approaches for F508 CFTR rescue will be reviewed, and their advantages as well as limitations will be discussed.
Introduction
Cystic fibrosis (CF) is largely a protein misfolding disease.The deletion of a phenylalanine at residue 508 (F508) in the cystic fibrosis transmembrane conductance regulator (CFTR) accounts for 70% of all disease-causing alleles and is present in at least one copy in 90% of CF patients (Kerem et al., 1989).The F508 mutation impairs the conformational maturation of nascent CFTR (Lukacs et al., 1994), and arrests it in an early folding intermediate (Zhang et al., 1998).As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) (Cheng et al., 1990) in a chaperone-bound state (Yang et al., 1993), The ER-accumulated mutant CFTR fails to efficiently couple to the coatomer complex II (COPII) ER export machinery (Wang et al., 2004), and is degraded by the ubiquitin proteasome system through the ER-associated degradation (ERAD) pathway (Jensen et al., 1995;Ward et al., 1995), leading to loss of CFTR function at the cell surface.
The folding defect of F508 CFTR appears kinetic in nature (Qu et al., 1997).A small fraction of F508 CFTR is able to exit the ER but the escaped mutant protein is not stable at the cell periphery and is rapidly cleared through lysosomal degradation (Lukacs et al., 1993).This second defect further reduces the cell surface localization of this mutant CFTR.Aside from localization defect, the F508 mutation also impairs the channel gating of CFTR, leading to reduced open probability (Dalemans et al., 1991).The threefold defect of F508 CFTR stems from its defective conformation, and impairs the CFTR functional expression at the cell surface, leading to severe clinical phenotype.Given the autosomal recessive inheritance of the disease, improving plasma membrane functional expression of F508 CFTR will benefit the vast majority of CF patients (Gelman & Kopito, 2002).
Numerous research efforts have been made to improve F508 CFTR cell surface functional expression, including elevating its expression, reducing its degradation, enhancing the efficiency of its maturation, increasing its post-ER stability and improving its channel gating.Restoring F508 CFTR conformation will potentially improve its ER folding, its cell surface stability and its channel gating, leading to efficient F508 CFTR rescue.In this chapter, multiple approaches for F508 CFTR rescue will be reviewed, and their advantages as well as limitations will be discussed.
originates in NBD1 but spread throughout the whole molecule through domain-domain interactions, leading to a global conformation defect (Du et al., 2005;Du & Lukacs, 2009;Roy et al., 2010).Restoring wild-type-like global conformation is required for F508 CFTR to pass the quality control and egress from the ER (Roy et al., 2010).Second site mutations in NBD1 have been identified that suppress the F508 processing defect (Teem et al., 1993), and at least some of such suppressing mutations can act co-translationally on the NBD1 misfolding (Hoelen et al., 2010).Therefore, the de novo folding of F508 CFTR at both cotranslational and post-translational levels can be targeted for its rescue.
CFTR quality control in the ER
Newly synthesized CFTR undergo quality control before it can exit the ER.ER quality control starts even before CFTR is fully translated (Fig. 2).A membrane-associated ubiquitin ligase complex containing the E3 RMA1, the E2 Ubc6e and Derlin-1 mediates CFTR co-translational quality control (Sun et al., 2006;Younger et al., 2006).BAP31, an integral membrane protein that associates with Derlin-1 as well as the amino terminus of CFTR, promotes F508 CFTR retrotranslocation from the ER and its subsequent degradation by the cytoplasmic 26S proteasome (B.Wang et al., 2008).P97/valosincontaining protein interacts with gp78/autocrine motility factor receptor in coupling CFTR ubiquitination to its retrotranslocation and proteasome degradation (Carlson et al., 2006;Vij et al., 2006).Interestingly, gp78 was found to cooperate with RMA1 in the ERAD of F508 CFTR (Morito et al., 2008).Moreover, ubiquitin C-terminal hydrolase-L1 (UCH-L1) protects CFTR from co-translational ERAD (Henderson et al., 2010).This cotranslational quality control of CFTR appears to be regulated by cytoplasmic Hsc70 as DNAJB12 was recently found to cooperate with Hsc70 and RMA1 in F508 CFTR degradation (Grove et al., 2011).Consistent with this, we found that Hsp105, a nucleotide exchange factor (NEF) for Hsc70, promotes co-translational ERAD of CFTR (Saxena et al., 2011a).
A second ER quality control step occurs largely post-translationally, which is mediated through Hsc70 and cochaperone CHIP (Meacham et al., 2001) (Fig. 2).CHIP functions as a scaffold for the formation of multi-subunit E3 ubiquitin ligase for the post-translational ERAD of CFTR, and such degradation activity is also dependent upon Hsc70, Hdj-2 and the E2 UbcH5a (Younger et al., 2004).Interestingly, HspBP1 and BAG-2, two other NEFs for Hsc70, inhibits the CHIP-mediated post-translational ERAD of CFTR (Alberti et al., 2004;Arndt et al., 2005), suggesting a dual role for Hsc70 in regulating co-translational and posttranslational ERAD of CFTR.
Nevertheless, inhibiting CFTR ERAD is not sufficient for F508 CFTR to efficiently exit the ER (Jensen et al., 1995;Pagant et al., 2007).Obviously, another quality control system is responsible for the retention of the foldable pool of F508 CFTR in the ER, the mechanism of which is less clear.We recently showed that the ER exit code and domain conformation both contribute significantly to the exportability of CFTR (Roy et al., 2010).Therefore, chaperone association and/or ER exit code presentation might be two important factors for this last checkpoint of the ER quality control of CFTR.A better understanding of its mechanism will lead to much greater improvement in F508 CFTR maturation.
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Improving Cell Surface Functional Expression of F508 CFTR: A Quest for Therapeutic Targets 337
ER-to-Golgi sorting signals within CFTR
Exit of proteins from the ER requires specific sorting signals on the cytoplasmic side of the ER membrane which is recognizable by the cargo selection complex (Sec23/24) of COPII (Aridor et al., 1998;Nishimura et al., 1999;Miller et al., 2002).A di-acidic ER exit motif (DAD) was identified in the NBD1 of CFTR (Fig. 1), and substitution of the second acidic residue (leading to DAA) abolishes CFTR association with Sec24 and dramatically reduces the export efficiency of CFTR (Wang et al., 2004).The F508 mutation also reduces CFTR association with Sec24 (Wang et al., 2004) but the underlying mechanism might be different from the DAA mutant.Using in situ limited proteolysis to probe the domain conformation of CFTR (Zhang et al., 1998), we showed that the DAA mutant has similar domain conformation as wild-type CFTR despite its inability to efficiently exit the ER (Roy et al., 2010).This is in stark contrast to F508 CFTR which displays global conformational defects including NBD1 (Du et al., 2005;Du & Lukacs, 2009;Roy et al., 2010).Furthermore, DAA CFTR displays lower chaperone association and higher post-ER stability when compared with F508 CFTR (Roy et al., 2010).Therefore, the conformational defects in F508 CFTR contribute significantly to its misprocessing.
ER retention/retrieval signals have been found in the cytoplasmic domains of multiple transmembrane cargo proteins (Nilsson et al., 1989;Zerangue et al., 1999).An RXR ER retention/retrieval signal serves as a quality control check point for the assembly of oligomeric cargo proteins in the ER (Zerangue et al., 1999;Margeta-Mitrovic et al., 2000).The RXR signals are exposed in individual subunits or in incompletely assembled oligomers but are concealed only when the proper assembly of the oligomer is achieved.This mechanism prevents the cell surface expression of improperly assembled cargo molecules.Multiple RXR motifs have been identified in the cytoplasmic domains of CFTR (Fig. 1), and the replacement of key arginine residues results in F508 CFTR rescue, suggesting that the RXR motifs might contribute to the ER retention/retrieval of F508 CFTR (Chang et al., 1999).It is proposed that such RXR motifs are shielded by domain-domain interactions in wild-type CFTR but become exposed when the F508 is deleted (Kim Chiaw et al., 2009).In fact, peptides designed to mimic such a sorting motif were found to functionally rescue F508 CFTR (Kim Chiaw et al., 2009).As the key RXR motif in NBD1 contributes significantly to F508 CFTR global conformation (Qu et al., 1997;Hegedus et al., 2006;Roy et al., 2010), it is unclear if the RXR-mimetics rescue F508 CFTR by influencing F508 CFTR conformation.Determining whether the RXR-mimetics are able to bind to the RXR sorting receptor or whether they block the retention/retrieval of other RXR-containing cargo molecules will provide a definitive answer.
Defining F508 conformational defects
F508 resides in NBD1 (Fig. 1), and therefore the deletion of this residue should naturally affect the conformation of NBD1.Early in vitro studies using purified NBD1 revealed a kinetic folding defect in F508 NBD1 (Qu & Thomas, 1996;Qu et al., 1997).However, the crystal structure of F508 NBD1 revealed no major conformational change from the wildtype domain (Lewis et al., 2005).In the mean time, it was found that F508 mutation causes major conformation changes in NBD2 (Du et al., 2005), highlighting the importance of domain-domain interactions in F508 misfolding.This notion was strengthened by the finding that F508 side chain contributes significantly to NBD1 folding in the context of fulllength CFTR (Thibodeau et al., 2005), and by the finding that F508 residue mediates the contact between NBD1 and cytoplasmic loop 4 (CL4) in MSD2 (Serohijos et al., 2008a).Therefore, deletion of F508 triggers a global conformational change in CFTR, leading to misprocessing (Du & Lukacs, 2009).
The apparent lack of a detectable NBD1 conformational change as a result of the F508 mutation remains an enigma as how can the F508 mutation trigger such a profound global conformational change without significantly impacting NBD1 conformation in the first place?The finding that some of the solubilization mutations included in F508 NBD1 for crystallography studies actually rescue the F508 processing defect in the context of fulllength CFTR reopened this question (Pissarra et al., 2008).Another twist in our understanding of the impact of F508 deletion on NBD1 conformation came from the finding that the removal of the regulatory insert (RI), a 32-residue segment within NBD1 that is unique to CFTR but not shared by the NBD1's of other ABC transporters, renders F508 NBD1 soluble, dimer-forming and displaying wild-type-like conformation (Atwell et al., 2010).Another study shows that, in the context of full-length CFTR protein, removal of the RI restores maturation, stability and function of F508 CFTR, suggesting that the RI contributes significantly to F508 misfolding in NBD1 (Aleksandrov et al., 2010).
Using in situ limited proteolysis, we identified a definite conformational change within NBD1 as a result of F508 mutation (Roy et al., 2010).The F508 NBD1 conformation, like the conformation of other domains of F508 CFTR, resembles the conformation of an earlier folding intermediate of wild-type CFTR (Zhang et al., 1998;Roy et al., 2010).Furthermore, rescue of F508 CFTR using low temperature or R555K substitution leads to NBD1 as well as global conformational reversion, suggesting that conformational correction is prerequisite for the rescue of the folding and export defects of F508 CFTR (Roy et al., 2010).Using crystallography and hydrogen/deuterium exchange mass spectrometry, Lewis, et al. showed that F508 mutation increases the exposure of the 509-511 loop and increases the dynamics in its vicinity (Lewis et al., 2010).Consistent with the above, a conformational change in F508 NBD1 was observed using a cysteine-labelling technique, and such conformational change is reversed by second site mutations in NBD1 (He et al., 2010).Interestingly, the second site mutations also restore the interactions between NBD1 and its contacting domains (He et al., 2010).Combination of G550E, R553M and R555K suppressor mutations in NBD1 produces a dramatic increase in F508 CFTR processing, and this is accompanied by the enhanced folding of F508 NBD1 both in isolation and in the context of full-length CFTR (Thibodeau et al., 2010).An interesting finding is that while NBD2 is not required for CFTR processing (Pollet et al., 2000), it contributes to F508 CFTR rescue by second site mutations as well as by low temperature (Du & Lukacs, 2009;Cheng et al., 2010).Furthermore, the rescue of F508 CFTR by suppressor mutations requires a continuous fulllength CFTR peptide (Cheng et al., 2010), suggesting a role for peptide backbone tension in F508 CFTR rescue (Thibodeau et al., 2005).
Taken together, F508 mutation causes increased exposure of the 509-511 loop in NBD1 and increases its dynamics.These changes not only alter the conformation of NBD1, but through NBD1's interface with CL4 and NBD2, alter the conformation of other domains, leading to global conformational change.Second site mutations within NBD1 can partially correct the F508 NBD1 conformational defect, which spread to other domains through domaindomain interactions, leading to partial restoration of global conformation as well as processing.Conformation repair is at the heart of F508 correction.
Elevating F508 CFTR expression
The severe reduction in F508 CFTR cell surface functional expression results from its defective export, reduced peripheral stability, and subnormal channel gating.Nevertheless, a small fraction of the mutant CFTR can leak from the ER and make its way to cell surface.One simple approach to enhance F508 CFTR cell surface localization is to increase its expression.This can be achieved in cells heterologously expressing F508 CFTR under the control of metallothionein promoter by treatment with sodium butyrate (Cheng et al., 1995).In CF airway epithelial cells, 4-phenylburyrate, a histone deacetylase (HDAC) inhibitor dramatically increases the expression of F508 CFTR at the protein level (Rubenstein et al., 1997).Recently, a group of other HDAC inhibitors including Trichostatin A, suberoylanilide hydroxamic acid (SAHA) and Scriptaid were found to potently increase F508 CFTR transcription in CFBE41o-cells (Hutt et al., 2010).
Interestingly, over-accumulation of F508 CFTR in the ER induces the unfolded protein response (UPR) (Gomes-Alves et al., 2010), and induction of UPR inhibits CFTR endogenous transcription (Rab et al., 2007).The UPR-induced CFTR transcriptional repression is mediated through the transcription factor ATF6, and both DNA methylation and histone deacetylation contribute to this process (Bartoszewski et al., 2008).Therefore, there is a limit to which the transcription of endogenous F508 CFTR can be increased but HDAC inhibitors may potentially alleviate the UPR-induced CFTR transcriptional repression (Fig. 2).
The expression of F508 CFTR can also be regulated at the post-transcriptional level.A recent study revealed that the synonymous codon change of I507 in the F508 allele can cause mRNA misfolding, leading to reduced rate of translation and/or impaired cotranslational folding of F508 CFTR (Bartoszewski et al., 2010).Therefore, codon-dependent mRNA folding represents a new mechanism by which F508 CFTR expression can be regulated.Although it is not realistic to change the nucleotide sequence of F508 CFTR in CF patients, identification of this novel mechanism opens up new opportunities for therapeutic intervention at the level of mRNA processing, folding, and stability.
Reducing F508 CFTR ERAD
The vast majority of F508 CFTR synthesized in the cells is degraded through the ERAD pathway (Jensen et al., 1995;Ward et al., 1995).Inhibition of ERAD will certainly increase the steady state level of F508 CFTR in the ER and subsequently increase its cell surface localization (Fig. 2).Significant advance in understanding the mechanism of ERAD of F508 CFTR has been achieved during the past 16 years.Hsc70 has been found to regulate both the co-translational and post-translational ERAD of F508 CFTR with two distinct sets of cochaperones (Meacham et al., 2001;Zhang et al., 2001;Alberti et al., 2004;Arndt et al., 2005;Grove et al., 2011;Saxena et al., 2011a).While the functional relationship between the two remains unclear, multiple cochaperones, such as CHIP (Meacham et al., 2001), HspBP1 (Alberti et al., 2004), BAG-2 (Arndt et al., 2005), Hdj-2 (Younger et al., 2004), DNAJB12 (Grove et al., 2011) and Hsp105 (Saxena et al., 2011a) may be targeted for increasing the steady state levels of F508 CFTR.Moreover, 4-phenylbutyrate, which rescues F508 CFTR (Rubenstein et al., 1997), was found to reduce the expression level of Hsc70, subsequently decreases its association with F508 CFTR, and therefore inhibits the ERAD of F508 CFTR (Rubenstein & Zeitlin, 2000).More recently, a soluble sulfogalactosyl ceramide mimic that inhibits the Hsp40-activated Hsc70 ATPase activity, promotes the rescue of F508 CFTR from ERAD (Park et al., 2009).In addition to Hsc70, small heat-shock proteins (sHsps) preferentially associate with F508 CFTR and promote its ERAD (Ahner et al., 2007).It is believed that small heat-shock proteins bind to misfolded F508 CFTR, prevent its aggregation and maintain its solubility during the ERAD (Ahner et al., 2007).
Enhancing F508 CFTR maturation
Despite its obvious importance in rescuing F508 CFTR, relatively little is known concerning how to improve the maturation of F508 CFTR in the ER.The major reason is that F508 CFTR hardly matures if at all at physiological temperature.However, at reduced temperature, F508 CFTR does achieve conformational maturation much more efficiently, leading to greatly enhanced functional expression at the cell surface (Denning et al., 1992).Interestingly, such a temperature-sensitive phenotype is cell-dependent, suggesting that cellular machinery plays an essential role in the process (X.Wang et al., 2008).We found that the increased conformational stability provided by low temperature combines with chaperone actions in facilitating F508 CFTR maturation at reduced temperature (Roy et al., 2010).Therefore, the temperature-dependent maturation of F508 CFTR serves as an excellent model system in understanding the role of the cellular chaperone machinery in the forward folding of F508 CFTR.
Mild heat shock greatly potentiates the temperature rescue of F508 CFTR, and this is dependent upon transcription, suggesting that the upregulation of heat inducible chaperones promotes F508 CFTR maturation (X.Wang et al., 2008).Using a series of chaperone-or cochaperone-deficient cell lines, we demonstrate that an Hsp70-Hsp90 chaperone network operates on the cytoplasmic face of the ER membrane facilitating the maturation of F508 CFTR at reduced temperature.Cochaperone Hop, which physically and functionally links Hsp70 and Hsp90 through its multiple tetratricopeptide repeat (TPR) domains, is essential for the temperature-dependent maturation of F508 CFTR, and Hsp105 is an integral player in the system (Saxena et al., 2011b).We also found that Hsc70, Hsp90, Hop, Hsp105 and Hdj-2 are functionally linked during the temperature rescue of F508 CFTR.Depletion of Hsp90, Hop or Hsp105 also reciprocally reduces some or all of other chaperone components (Saxena et al., 2011b).It is highly likely that these folding components, and perhaps other yet unidentified chaperones or cochaperones, form a functionally organized chaperone network on the cytoplasmic side of the ER membrane, facilitating the conformational maturation of F508 CFTR at reduced temperature.Given a clear role for Hsp90 in wild-type CFTR maturation at physiological temperature (Loo et al., 1998), we believe such a cytoplasmic chaperone network functions in the cell under physiological conditions.While its effect on F508 CFTR maturation is more pronounced at reduced temperature, it should also impact F508 CFTR maturation at the physiological temperature.Consistent with this prediction, overexpressing Hsp105 promotes F508 CFTR processing at both the reduced and physiological temperatures (Saxena et al., 2011a).An indepth analysis of this process will reveal novel molecular targets that promote the maturation of F508 CFTR (Fig. 2).
Another approach to enhance F508 CFTR maturation is through transcomplementation (Cormet-Boyaka et al., 2004).Such rescue requires co-expression of a sizeable segment of CFTR that contains wild-type sequence corresponding to the region where F508 is located.Such transcomplementation does not result in changes in Hsc70 association but is believed to improve F508 CFTR forward folding through intra-and/or inter-molecular domaindomain interactions.A related but distinct approach to promote F508 CFTR maturation is to co-express a fragment of F508 CFTR containing NBD1 and R domains (Sun et al., 2008).This mutant fragment of CFTR can actually sequester key chaperone components from the endogenous F508 CFTR and lead to its rescue.Moreover, co-expressing an N-terminal truncated CFTR mutant (264) can not only transcomplement F508 CFTR but also dramatically increases the protein expression levels of both wild-type and F508 CFTR (Cebotaru et al., 2008).As the 264 mutant CFTR associates with VCP and HDAC6, two components involved in retrotranslocation of proteins from the ER, and is more efficiently degraded by the proteasome than F508 CFTR, high level expression of this mutant may interfere with F508 CFTR ERAD and hence increase its steady state level.Taken together, co-expression of CFTR fragments might rescue F508 CFTR by improving its folding, helping it escape ER quality control and protecting it from ERAD.As these fragments have much lower molecular weight than full-length CFTR, they can be used as potential agents for CF gene therapy.
Increasing F508 CFTR peripheral stability
The F508 CFTR has reduced conformational stability in post-ER compartments and therefore turns over rapidly at the cell periphery (Lukacs et al., 1993;Sharma et al., 2001;Sharma et al., 2004).Increasing F508 CFTR half-life at cell periphery is an important strategy for effective rescue of F508 CFTR.CAL, a Golgi-associated, PDZ domaincontaining protein that binds to the C-terminus of CFTR, reduces the half-life of CFTR at the cell surface (Cheng et al., 2002).RNA interference of endogenous CAL in CF airway epithelial cells increases plasma membrane expression of F508 CFTR and enhances transepithelial chloride current (Wolde et al., 2007).The Na + /H + exchanger regulatory factor (NHERF), a subplasma membrane PDZ domain protein, competes with CAL in associating with CFTR and promotes its plasma membrane localization (Cheng et al., 2002).Knockdown of NHERF1 promotes the degradation of temperature-rescued F508 CFTR at the cell surface of human airway epithelial cells (Kwon et al., 2007).Expression of dominantnegative dynamin 2 mutant K44A increases CFTR cell surface expression, and counteracts the effect of CAL overexpression on CFTR cell surface stability (Cheng et al., 2004).SNARE protein syntaxin 6 binds to CAL and reduces CFTR cell surface stability in a CAL-dependent manner (Cheng et al., 2010).Therefore, CAL and its functional partners are viable molecular targets for increasing cell surface stability of F508 CFTR (Fig. 2).
Cytoplasmic chaperone Hsc70 was shown to mediate the uncoating of clathrin-coated vesicles (Schmid & Rothman, 1985;Chappell et al., 1986) and hence regulates the peripheral trafficking of membrane bound cargo proteins such as CFTR.Recently, a more direct role for cytoplasmic Hsp70-Hsp90 chaperone network in regulating F508 CFTR peripheral quality control was revealed, where Hsc70, Hsp90, Hop and other chaperone components collaborate with the ubiquitin system in promoting the cell surface degradation of this mutant CFTR (Okiyoneda et al., 2010).This finding uncovers a great number of new potential chaperone targets for regulating cell surface stability of F508 CFTR.However, as the cytoplasmic Hsp70-Hsp90 chaperone network also facilitates the maturation of CFTR in the ER (Loo et al., 1998;Meacham et al., 1999;Wang et al., 2006), a critical balance must be maintained between the two seemingly opposing effects of the Hsp70-Hsp90 chaperone network at the ER and the peripheral levels in order to effectively rescue F508 CFTR (Fig. 2).
Of particular interest, we found that Hsp105 is involved in both processes.At the ER level, Hsp105 facilitates the Hsp70-Hsp90-mediated maturation of F508 CFTR at reduced temperature (Saxena et al., 2011b).At the peripheral level, Hsp105 preferentially associates with the rescued F508 CFTR, and stabilizes it in post-ER compartments (Saxena et al., 2011a).It is currently unclear whether Hsp105 functionally relates to Hsc70, Hop and Hsp90 at the cell periphery or it act on its own.While Hsp105 acts in the same direction as the cytoplasmic Hsp70-Hsp90 network at the ER level, it acts in opposite direction to Hsc70, Hop and Hsp90 at the cell periphery.Understanding these aspects is critical to the effective enhancement of F508 CFTR cell surface functional expression by modulating cytoplasmic chaperone machinery.
Improving F508 CFTR channel gating: Potentiator or corrector?
Although the primary defect in F508 CFTR is impaired export (Cheng et al., 1990), it has aberrant channel gating as reflected in reduced channel open probability (Dalemans et al., 1991).Correcting such a defect will also improve the overall cell surface functional expression of F508 CFTR.The G551D substitution in CFTR, a mutation causing severe CF, does not impact its export to plasma membrane but primarily impairs its channel opening (Tsui, 1995;Li et al., 1996).VX-770, a small molecule potentiator (improving channel gating) developed for G551D CFTR by the Vertex Pharmaceuticals Inc., also increases the channel open probability of F508 CFTR (Van Goor et al., 2009).Interestingly, small molecule compound VRT-532 display both corrector (improving maturation) and potentiator activities for F508 CFTR by binding directly to the mutant protein (Wellhauser et al., 2009).Recently, a fragment of a phenylglycine-type potentiator was successfully linked to a fragment of a bithiazole corrector to form a "hybrid" potentiator-corrector molecule, the cleavage of which by intestinal enzymes is able to release separate potentiator and corrector for F508 rescue in vivo (Mills et al., 2010).Using high-throughput screen, multiple small molecules with independent potentiator and corrector activities for F508 CFTR were also identified (Phuan et al., 2011).Using the above approaches, more efficient rescue of F508 CFTR can be achieved.
Conformational repair: One stone and three birds
Given that the root cause of CF in the majority of patients lies in the conformational defects of F508 CFTR, repairing its conformational defects will potentially lead to improved export, stability (both in the ER and at the cell periphery) and channel gating.Effective development of novel approaches in conformational repair relies on a thorough understanding of the conformational defects of F508 CFTR and their correction.Given that F508 residue resides in NBD1, NBD1 is a central domain for the understanding of F508 conformational repair.In addition, as domain-domain interactions within CFTR play an important role in altering or maintaining CFTR global conformation (Du et al., 2005;Du & Lukacs, 2009), key interfaces between different domains are also important in CFTR conformational repair (Serohijos et al., 2008a).
An excellent attempt was made early on in screening for suppressor mutations in NBD1 which restores the export of F508 CFTR (Teem et al., 1993).This was made possible by swapping a portion of CFTR NBD1 into yeast STE6 gene encoding an ABC transporter that delivers -factor out of the cell which is necessary for mating.When F508 mutation is included into the STE6-CFTR chimera, the yeast fails to transport -factor.Using this system, second site mutations within the CFTR NBD1 portion were identified that rescue F508 CFTR (Teem et al., 1993;Teem et al., 1996).Interestingly, R555K, one of such F508 suppressor mutations, causes a global conformational reversion in F508 CFTR, leading to increased export and enhanced post-ER stability (Roy et al., 2010).R555K, when combined with other rescue subsitutions, improves F508 CFTR conformation and processing (Chang et al., 1999;Hegedus et al., 2006), and significantly increases the open probability of F508 CFTR (Roxo-Rosa et al., 2006).These data support the notion that conformational repair is a highly effective approach for enhancing F508 CFTR cell surface functional expression, ameliorating all three facets of F508 defect.
Another approach for designing NBD1 conformational repair employs molecular dynamics simulation.Molecular dynamics has the advantage over structural biology in that it reveals information on folding kinetics and dynamics.Using this approach, key differences in the distribution of meta-stable intermediates have been identified between wild-type and F508 NBD1, and additional rescue mutations can be designed (Serohijos et al., 2008b).These rescue mutations, if validated experimentally, will significantly advance our understanding of NBD1 folding both alone and in the context of full-length CFTR.
High resolution crystal structure of full-length CFTR is currently unavailable.However, the crystal structures of multiple ABC transporters including the p-glycoprotein have been solved (Locher et al., 2002;Dawson & Locher, 2006;Aller et al., 2009).Attempts to use these structures as bases for modeling full-length CFTR have provided new insights into the role of F508 residue in domain-domain interactions (Jordan et al., 2008;Loo et al., 2008;Serohijos et al., 2008a;Mornon et al., 2009).These studies, when backed up by biochemical analyses, are an excellent start point to probe F508 global conformational defects and their repair.
Large-scale target identification for the rescue of F508 CFTR
Aside from the above mechanism-based identification of therapeutic targets for F508 rescue, several large-scale target identification regimes have been quite successful.The functional follow-up of these studies has yielded and will yield many novel molecular targets.
The first such attempt was to use proteomics to identify CFTR-interacting proteins between wild-type and F508 CFTR (Wang et al., 2006), which revealed, among others, an ERassociated chaperone network facilitating CFTR biogenesis and quality control.In an attempt to gain information on the potential mechanism of F508 chemical rescue by 4phenylbutyrate, a pharmacoproteomic approach was used to identify changes in protein expression in CF airway epithelial cells in response to 4-phenylbutyrate treatment (Singh et al., 2006).This approach was then followed by a comparison of F508 CFTR-interacting proteins between the chemically rescued (by 4-phenylbutyrate) and genetically repaired (by introducing wild-type CFTR) CF airway epithelial cells (Singh et al., 2008).Protein targets involved in the ERAD, protein folding and inflammatory response have been identified, and proteins that were modulated in the ER as well as on the plasma membrane have been isolated (Singh et al., 2008).
Recently, a high-throughput functional screen was designed to identify proteins that promote the rescue of F508 CFTR (Trzcinska-Daneluti et al., 2009).In this study, 450 different proteins were fused to a chloride-senstive yellow fluorescent protein and were expressed in a F508 CFTR-expressing stable cell line.The cells were screened for their ability to rescue the F508 functional defect at the plasma membrane.Several proteins that are known to rescue F508 CFTR as well as novel target proteins have been identified.Further functional characterization will reveal their usefulness as potential therapeutic targets.
Another excellent approach worth noting is the use of functional small interfering RNA screen to identify proteins that are involved in peripheral quality control of F508 CFTR (Okiyoneda et al., 2010).This approach took advantage of a well developed cell surface ELISA assay measuring CFTR plasma membrane localization, where three HA-tags have been engineered in an extracellular domain of CFTR.The siRNAs targeting a great number of ubiquitin E3 ligases, ESCRT proteins, E2 enzymes and chaperone/cochaperones were introduced into the above cells, and the plasma membrane stability of the rescued F508 CFTR-3HA was quantified.This study led to the identification of an Hsp70-Hsp90 chaperone network facilitating the peripheral quality control of F508 CFTR.Functional followup of these chaperone proteins will not only reveal critical mechanistic information but also uncover yet unidentified molecular targets.
Small molecule modulators for F508 CFTR
One of the major strategies for developing effective therapeutics for CF is to identify small molecule compounds that can improve F508 CFTR cell surface functional expression.Using cell-based functional assay for CFTR-mediated chloride conductance combined with high-throughput screening of small molecule compound libraries, multiple CFTR modulators have been identified, affecting F508 CFTR trafficking and/or channel function (Van Goor et al., 2006;Verkman et al., 2006).Once promising scafolds have been identified, structural optimization can be performed to enhance their biological activities, pharmacokinetics, and safety.In fact several of the above compounds are currently in clinical trial for treating CF.
While the functional screening as mentioned above has the benefit of identifying small molecule compounds that improve the aggregate endpoint readout on F508 CFTR cell surface functional expression, the mechanisms by which these compounds do so are unknown.The compounds can either bind directly to CFTR to affect its folding and/or channel gating, or they can bind to other cellular proteins that regulate CFTR biogenesis, cell surface protein-protein interactions, or its degradation.Understanding these mechanistic aspects of a specific CFTR modulator will lead to the design and identification of additional molecular targets and CFTR modulators.This is especially important as only a limited number of efficacious CFTR modulators have been identified through the functional screen.In order to obtain an FDA-approved drug for CF, more of such compounds are desparately needed to feed into the CF drug discovery pipeline.
Recently, new screening strategies have been designed to improve the variety of workable lead compounds.These compounds might not have been identified during the functional screen because they do not provide the above-the-threshold functional readouts.However, if they have special properties that can enhance certain key aspects of F508 CFTR rescue, such compounds can be further engineered or optimized to produce a much greater efficacy in terms of functional rescue of F508 CFTR.A new strategry has been developed where small molecule compound libraries were screened by their ability to improve the plasma membrane localization of F508 CFTR (Carlile et al., 2007).
A conformation-based virtual screen for F508 CFTR modulators represents one step further as it aims at the core defect of F508 CFTR (i.e.abberrant conformation).Recently, one attempt was made by the EPIX Pharmaceuticals Ltd to identify small molecule correctors for F508 CFTR (Kalid et al., 2010).In this study, a total of three potential small molecule binding cavities were identified at a number of domain-domain interfaces of CFTR, and small molecule compounds were screened in silico for their ability to bind to these cavities.The initial hits derived from the virtual screen were then subjected to functional screen, which yielded a ten-fold increase in hit rate as compared to conventional screen regimes.
An alternative to the above high-throughput screening approach is to explore the possibility of using FDA-approved drugs for other conditions or other small molecule compounds that are safe for human use for rescuing F508 CFTR.Sodium 4-phenylbutyrate is approved for clinical use in patients with urea cycle disorders.4-Phenylbutyrate, like sodium butyrate, is also a transcriptional regulator that inhibits HDAC (Jung, 2001).4-Phenylbutyrate was shown to rescue F508 CFTR through a number of mechanisms including biosynthesis, folding and transport (Rubenstein et al., 1997;Rubenstein & Zeitlin, 2000;Choo-Kang & Zeitlin, 2001;Wright et al., 2004;Singh et al., 2006).More recently, SAHA (Vorinostat), an HDAC inhibitor approved by FDA for the treatment of cutaneous T cell lymphoma through epigenetic pathways (Monneret, 2007), was shown to restore cell surface functional expression of F508 CFTR to 28% of wild-type level (Hutt et al., 2010).Doxorubicin (Adriamycin), a cancer chemotherapy agent, increases cell surface functional expression of F508 CFTR through increasing its folding, promoting its chaperone dissociation and inhibiting its ubiquitination (Maitra et al., 2001;Maitra & Hamilton, 2007).Sildenafil (Viagra) was also shown to promote F508 CFTR apical trafficking by unknown mechanism (Dormer et al., 2005).S-Nitrosoglutathione (GSNO), an endogenous bronchodilator (Gaston et al., 1993), was found to increase the expression and maturation of F508 CFTR in airway epithelial cells (Zaman et al., 2006).Interestingly, GSNO was recently found to function at least in part through inhibiting Hop expression (Marozkina et al., 2010), suggesting that small molecules compound can promote F508 CFTR rescue through modulating chaperone machinery.
Chaperone environment: A critical but complex part of the equation
Cellular chaperone machinery plays an important role in the synthesis, maturation, quality control of CFTR (Fig. 2).Due to misfolding, F508 CFTR has more extensive association with molecular chaperones (Yang et al., 1993;Jiang et al., 1998;Meacham et al., 1999;Wang et al., 2006;Sun et al., 2008;Roy et al., 2010).Therefore, the impact of chaperone machinery on F508 CFTR is greater than on wild-type CFTR.This notion is further underscored by the recent finding that cytoplasmic Hsp70-Hsp90 chaperone network promotes the peripheral quality control of F508 CFTR (Okiyoneda et al., 2010).Modulating chaperone environment can not only impact the quality control of F508 CFTR at either the ER or the peripheral level but also can dramatically influence its maturation (Loo et al., 1998;Zhang et al., 2002;Saxena et al., 2007;Saxena et al., 2011b).
Heat shock response is a transcriptional program by which cells upregulate the expression of an array of genes including those encoding molecular chaperones to cope with the massive need for protein folding and degradation as a result of elevated temperature or toxic agents (Morimoto et al., 1990).Therefore, conditions or agents that induce heat shock response will up-regulate the cellular chaperone machinery to enhance folding and ERAD of F508 CFTR.Consistent with this finding, mild heat shock dramatically potentiates the temperature-rescue of F508 CFTR (X.Wang et al., 2008).Another cellular response that upregulate the cellular chaperone machinery is the unfolded protein response (UPR) (Sidrauski et al., 1998).This is particularly relevant to F508 CFTR as over accumulation of this mutant protein in the ER induces such a response, leading to downregulation of CFTR endogenous transcription (Rab et al., 2007;Bartoszewski et al., 2008).Aside from the above two, the inherent variation in the cellular chaperone machinery among different tissues or cell types will also significantly affect the cell surface functional expression of F508 CFTR (Varga et al., 2004;X. Wang et al., 2008;Rowe et al., 2010).Therefore, understanding the functional organization of the chaperone machinery in airway epithelial cells is highly relevant to the development of effective rescue strategies for F508 CFTR.
Certain chemicals such as celastrol can globally influence the cellular chaperone machinery through inducing the heat shock response (Westerheide et al., 2004).Other epigentic modulators can also influence the expression of mutliple molecular chaperones (Wright et al., 2004;Hutt et al., 2010).Interfering with ER lumenal chaperone activities by depleting the ER calcium stores promotes the escape of F508 CFTR from the ER quality control and enhances its cell surface expression (Egan et al., 2002).Certain small molecule compounds directly modulate the expression or activity of molecular chaperones (Jiang et al., 1998;Loo et al., 1998;Marozkina et al., 2010).Furthermore, small molecule compound can act as chemical chaperones to stabilize the conformation of F508 CFTR, enhancing its cell surface functional expression (Brown et al., 1996;Fischer et al., 2001).
Conclusion
The F508 mutation is present in over 90% of CF patients.This mutation impairs the conformational maturation of CFTR leading to defective export, reduced stability and abberrant channel gating.Improving the cell surface functional expression of this mutant CFTR will benefit the vast majority of CF patients.While many approaches can be taken toward this goal, conformational rescue is the most effective, postitively impacting all three molecular defects of F508 CFTR.The F508 CFTR molecule is the most important target for the development of therapeutics.A clear understanding of its biogenesis, quality control and conformation is fundamental.In the cell, the synthesis, folding, quality control, trafficking and degradation of CFTR is dependent upon its interactions with multiple cellular machineries (Fig. 2).Such interactions provide additional opportunites for therapeutic interventions.The cellular protein homeostasis as regulated by the chapeorone machinery provides an important chemical environment for F508 CFTR.Such an enironment is regulated by multiple cellular responses or epigenetic modulators.Understanding the relationship between such cellular environment and F508 CFTR cell surface functional expression will provide additional molecular targets for intervention.
Acknowledgment
We thank the Cystic Fibrosis Foundation, the American Heart Association and the University of Toledo Health Science Campus for support. | v3-fos-license |
2019-05-29T14:22:48.031Z | 2019-05-28T00:00:00.000 | 167220133 | {
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} | pes2o/s2orc | A hydrofluoric acid-free method to dissolve and quantify silica nanoparticles in aqueous and solid matrices
As the commercial use of synthetic amorphous silica nanomaterials (SiO2-NPs) increases, their effects on the environment and human health have still not been explored in detail. An often-insurmountable obstacle for SiO2-NP fate and hazard research is the challenging analytics of solid particulate silica species, which involves toxic and corrosive hydrofluoric acid (HF). We therefore developed and validated a set of simple hydrofluoric acid-free sample preparation methods for the quantification of amorphous SiO2 micro- and nanoparticles. To circumvent HF, we dissolved the SiO2-NPs by base-catalyzed hydrolysis at room temperature or under microwave irradiation using potassium hydroxide, replacing the stabilizing fluoride ions with OH−, and exploiting the stability of the orthosilicic acid monomer under a strongly basic pH. Inductively coupled plasma – optical emission spectroscopy (ICP-OES) or a colorimetric assay served to quantify silicon. The lowest KOH: SiO2 molar ratio to effectively dissolve and quantify SiO2-NPs was 1.2 for colloidal Stöber SiO2-NPs at a pH >12. Fumed SiO2-NPs (Aerosil®) or food grade SiO2 (E551) containing SiO2-NPs were degradable at higher KOH: SiO2 ratios >8000. Thus, hydrofluoric acid-free SiO2-NP digestion protocols based on KOH present an effective (recoveries of >84%), less hazardous, and easy to implement alternative to current methods.
Results and Discussion
Molar ratio of KOH: SiO 2 for complete SiO 2 dissolution-mechanism. The results of the method optimization using the High_SiO 2 digestion method outlined in the method section and Table 1 are presented in Fig. 2. The dissolution of colloidal SiO 2 into Si(OH) 4 species was less dependent on the concentration of KOH, and more on the ratio of KOH to SiO 2 , which optimally is >1.2, and the pH, which should be >12. A volume of 10.0 mL of 0.1 M KOH (final concentration 82 mM) solubilized up to 50 mg of colloidal SiO 2 -NPs in suspension (Fig. 2). This corresponds to a molar ratio of 1.2 KOH: SiO 2 , in line with the results from Yang et al. 19 . The same volume of 0.05 M KOH still dissolved up to 30 mg colloidal SiO 2 -NPs (molar ratio: 1.0 KOH: SiO 2 ), but did not dissolve 50 mg SiO 2 -NPs anymore (molar ratio: 0.6 KOH: SiO 2 ), apparent from the high particle counts per second detected by DLS in that particular sample (Fig. 2). A slightly elevated DLS signal was also observed for the molar ratio of 1.0 KOH: SiO 2 . These results demonstrate that at least an equimolar concentration of KOH and optimally an excess of >20% is needed to dissolve SiO 2 . The constant ratio suggests that KOH fulfills a two-fold purpose: (a) installing a pH of >12 for the base-catalyzed hydrolytic degradation of hydrated silica 31 , and (b) neutralizing the Si(OH) 4 liberated during this reaction to maintain the high pH. The threshold pH of >12 corresponds to the 14 mM KOH that are not neutralized by Si(OH) 4 in the sample digested with 1.2 KOH: SiO 2 (82 mM KOH, 68 mM SiO 2 ), and is in agreement with the pH of 9-12 reported by Croissant et al. to dissolve SiO 2 -NPs 31 . We therefore adapted 0.1 M KOH and 1.2 KOH: SiO 2 ratio as minimum values for further digestions for colloidal SiO 2 -NPs, and slightly more for fumed SiO 2 NPs based on our observations (discussion below).
For further investigation on the dissolution mechanism, the incompletely digested sample containing 50 mg colloidal SiO 2 -NPs in suspension with the insufficient 0.6 KOH: SiO 2 ratio (Fig. 2) was dialyzed after the digestion against water for 1 d to remove KOH and dissolved Si species, and then inspected by transmission electron microscopy (TEM, Fig. 2). The structure of these partially dissolved SiO 2 -NPs revealed electron-transparent www.nature.com/scientificreports www.nature.com/scientificreports/ nanopores and more surface roughness compared to the dense, non-porous, and smooth structure of freshly synthesized SiO 2 -NPs (Fig. 2), confirming results of Li et al., who, based gas adsorption analysis results, ascribed some nanoporosity to colloidal SiO 2 -NPs due to aggregation-based NP growth 32 . An alternative explanation for the porosity is that the dissolution does not progress from the surface towards the core, but is targeted on specific silanol bonds 31 throughout the molecular structure. These observations are in line with Park et al., who showed that SiO 2 -NPs undergo a shape evolution due to Si-O bond-breaking and bond-making caused by hydroxyl ions, leading to rearrangement of high-energy bonds in the core 33,34 . No remaining NPs were observable by TEM in the samples digested using ratios >0.6 KOH: SiO 2 (Fig. 2 Fig. 3 and their fitting parameters in Supplementary Table S2. As apparent from the high R 2 (0.9987-0.9998), both Si and Y were stable in the concentration range of the calibrations under all conditions. As expected, calibrations exclusively containing acids showed the most stable Si signal (R 2 = 0.9998), and the most complex calibration was slightly more unstable (R 2 = 0.9987). A similar trend was observed for the signal of the internal standard yttrium. Only subtle signal suppression of Si or Y due to the matrix were observable: the maximal relative difference between the sensitivity of the different calibrations was 7.7% for Si and 9.5% for Y. For Si, the highest sensitivity (652 ± 9.5 counts/(µg L −1 )) was observed for the . Key steps and reagents used to hydrolytically degrade SiO 2 nanoparticles under basic conditions using potassium hydroxide, and detect dissolved Si and Si(OH) 4 , respectively, under acidic conditions. The SiO 2 concentrations stated are those used for the method development. (A) HF-free procedure for detection by inductively coupled plasma -optical emission spectrometry (ICP-OES) method suitable for complex matrices and accurate detection of low Si concentrations. (B) Procedure for detection by the colorimetric method using a UV-vis spectrophotometer. This method involves in situ HF, was used to validate method A, and is suitable for simple sample matrices. www.nature.com/scientificreports www.nature.com/scientificreports/ matrix-matched + H 2 SO 4 + digested calibration, and the lowest for the BgS calibration (602 ± 3 counts/(µg L −1 )). The digestion and addition of 0.1 M KOH moderately stabilized free Si. However, Fig. 3 shows that neither the acids used (2.25 M H 2 SO 4 , 0.5% HCl, and 2.0% HNO 3 ), nor the 0.1 M KOH, nor the digestion in the microwave led to a change of the Si signal noticeable in the statistical scatter of the data. For Y, the trends in matrix effects were somewhat different than for Si, and the highest sensitivity (18204 ± 321 counts/(µg L −1 )) was observed for the BgS calibration, in agreement with HSAB theory (stabilizing effect of soft nitrate ligands on the soft Y metal ions which is less effective for hard Si ions) 35 . The variability of the Y intercept was somewhat increased due to an accidental systematic second addition of internal standard which had to be corrected in the data by subtraction. The Y calibrations suffered from slight sensitivity loss under the matrix-matched KOH conditions by ~2-7%. Nevertheless, the absolute sensitivity for Y was excellent throughout all experiments. We therefore used the matrix-matched + H 2 SO 4 + digested calibrations with the highest sensitivity for Si for all measurements shown in Fig. 4 and Table 2. www.nature.com/scientificreports www.nature.com/scientificreports/ Repeatability and recovery. The measured concentrations of the SiO 2 -NP suspensions (Table 1) digested via the method KOH0.1 are compared with their calculated concentrations of Si in Fig. 4. A total recovery of Si/SiO 2 of 85 ± 2% was achieved with an instrument limit of detection of 41 µg L −1 and an instrument limit of quantification of 80 µg L −1 SiO 2 ( Table 2). The method was linear in the investigated range of injected Si (373-1981 µg L −1 ) which corresponds to 1.88-8.53 mg L −1 SiO 2 during the digestion. The relative error of the Si sensitivity, which can be attributed to measurements being taken over the course of multiple days by different investigators with different calibration matrices, was 31%. Three outliers are present among the hundred-twenty repeated measurements in Fig. 4. We attribute them to human pipetting errors. In practice, such errors can be detected and eliminated by analyzing, as in the present study, at least n = 3 replicate samples. While no outliers were deleted in the present study to present the reader with a realistic dataset, performing for example a Grubbs outlier test can identify such anomalies. In potential future large-scale applications, a robotic pipetting system can prevent such outliers. Overall, the repeatability of the measurements of concentration series prepared individually, digested in different microwave runs, and measured on the same day was very high (Fig. 4). This demonstrates that there is no significant buildup of Si in the instrument within one run, and the selected rinsing time of 55 s (10% HNO 3 ) between samples was sufficient. We found, however, that it is necessary to clean the detector window in regular intervals and to thoroughly rinse the instrument with 10% HNO 3 and Milli-Q (18.2 MΩ · cm) after each run.
We expected the recovery of Si in ICP-OES to be proportional to the stability of free Si(OH) 4 , and inversely proportional to the fraction of re-polymerized Si(OH) 4 in the sample. The polymerization of silica is accelerated under several conditions such as pH >2, high temperature, and ionic strength >0.2 M 19,36 . Here, the pH was adjusted by the addition of H 2 SO 4 to a pH of <2 to minimize polymerization and push the equilibrium towards orthosilicic acid. Although this addition of H 2 SO 4 also increased the ionic strength, previous studies found that Si(OH) 4 polymerization in the presence of H 2 SO 4 is minimal 36 . The present results (Fig. 3) show a moderate stabilizing effect of H 2 SO 4 on dissolved Si, based on a 3.5% difference between the sensitivities of Si calibrations in H 2 SO 4 or BgS.
The higher the excess KOH concentrations, the lower the Si recovery was, which is in line with the abovementioned destabilization of Y and stabilization of Si in high KOH environments. The Si recovery dropped by 15% in samples containing 0.1 M KOH compared to 1.0 M KOH. Hence, it is important to add the same concentration of KOH to the calibration in case the samples require KOH concentrations >0.1 M for digestion to account for this matrix effect. Finally, the SiO 2 polymerization is accelerated by high Si(OH) 4 concentrations 36 . We found plasma instability starting from 4000 µg Si L −1 upwards, and therefore limited routine concentrations to <1000 µg Si L −1 .
Sample storage. Storing samples for extended periods showed that digestates could be analyzed after up to two weeks without a statistically significant loss of recovery. A 3.6% decrease of Si recovery from 101.1% to 97.5% was observed between day 1 and at day 14 ( www.nature.com/scientificreports www.nature.com/scientificreports/ dissolved Si in the sample (ANOVA p < 0.02), likely due to re-polymerization. We noted improved stability of (1) refrigerated, (2) diluted, (3) low ionic strength, and (4) low pH samples. All these three conditions are known to push the equilibrium of polymerized SiO 2 towards Si(OH) 4 36 .
Method applicability. Suspensions containing fumed SiO 2 -NPs (Aerosil ® ). The recoveries for fumed
SiO 2 -NP suspensions digested using the method KOH1.0 (Table 1) are summarized in Table 2. The fumed SiO 2 -NP stock suspensions mainly contained aggregates (hydrodynamic diameter 267 nm) of smaller primary NPs 13 ± 5 nm in diameter (Supplementary Fig. S1 and Supplementary Table S1). We chose a harsher KOH concentration of 1.0 M for fumed SiO 2 -NPs due to the expected poorer solubility of the non-porous and less hydroxylated fumed SiO 2 -NPs compared to the more porous and more hydroxylated colloidal SiO 2 -NPs 8,32,37 . While the specific surface area is, for the present particle sizes and fractal dimensions, expected to be higher for the fumed SiO 2 -NPs (200 m 2 g −1 ) than the colloidal SiO 2 -NPs (~23-32 m 2 g −1 based on literature for colloidal particles of smaller size) 32 , both the lower surface hydroxylation and lower porosity of fumed SiO 2 -NPs can hamper the base-catalyzed hydrolytic degradation due to the postulated mechanism of amorphous SiO 2 dissolution that first requires hydration and hydrolysis of amorphous siloxane networks into silanols before the nucleophilic attack of OH − 31 . Also, suspensions of pre-digested, oven-dried SiO 2 -NPs formed acidic suspensions, which partially neutralized the added KOH in initial attempts to use 0.1 M KOH for digestion. Using 1.0 M KOH, we obtained a recovery of 114 ± 25% for fumed SiO 2 -NPs, and the same digestion at RT without microwave 105 ± 1.4% (Table 2). This elevated recovery (not significantly higher than 100%, one sample T-test, p > 0.22) may be a result of slightly less stabilized free Si ions than Y ions in the digestates, which were slightly more acidic than the calibrations. In samples digested using KOH concentrations ≤0.5 M, recoveries remained <85% in ICP-OES measurements ( Table 2), confirming that significant matrix effects occur due to excess KOH, as discussed in section Repeatability and Recovery, only in SiO 2 samples that are digested in >0.1 M KOH.
SiO 2 in complex matrices. The performance of the method in SiO 2 -containing complex matrices tested is shown in Table 2 (cell culture medium, tomato sauce, potato seasoning). We observed low recoveries for the digestion of SiO 2 in complex samples using 0.1 M KOH in preliminary tests. By using the method KOH0.5 on the SiO 2 -NP-spiked cell culture medium samples, we obtained a recovery of 84 ± 20% of SiO 2 without pre-digestion ( Table 2). The large statistical scatter can be connected to the complex formulation of the cell culture media Dulbecco Modified Eagle Medium (DMEM). Among many amino acids and vitamins, DMEM also contains ~10 g L −1 of dissolved inorganic salts, of which 3.6 g L −1 is sodium, which is notorious for causing high variability in ICP-OES measurements 30,38 . The present results show that the KOH digestion of SiO 2 -NPs in a serum-free cell culture medium delivered, despite some variability, an acceptable accuracy and recovery.
For the food matrix samples, i.e. the tomato sauce spiked with colloidal SiO 2 -NPs and the potato seasoning, the matrix was first digested in HNO 3 to isolate the SiO 2 -NPs (i.e. pre-digestion) and then these NPs were dissolved by KOH (refer to Experimental Section). As with DMEM, we had to use higher KOH concentrations of 1.0 M to get satisfactory recoveries. We obtained a recovery of 124 ± 5% and 95 ± 13% for colloidal SiO 2 -NPs in tomato sauce and food grade SiO 2 (E551) in potato seasoning, respectively. The recoveries of both samples (tomato sauce, potato seasoning) were calculated relative to the mass of remaining solids after the first acid-mediated digestion step, as SiO 2 was the sole remainder detected by energy-dispersive X-ray spectroscopy (EDX) after the harsh HNO 3 pre-digestion (data not shown). According to the literature, the natural Si concentration in tomatoes is maximally ~61 mg kg −1 39 , corresponding to ~31 µg natural Si in the analyzed mass of tomato sauce. The high recovery of 124 ± 5% for colloidal SiO 2 -NPs spiked into the tomato sauce (Table 2) indicates that additional natural SiO 2 was detected in the tomato sauce. The recovery of 95 ± 13% SiO 2 found for the potato seasoning (Table 2) Figure 5. Stability of digestates containing hydrolytically degraded SiO 2 over time. The concentration is proportional to the recovery: the data can be read from both y-axes. Certified Si standard solutions digested according to method KOH0.1 (Table 1) and stored at room temperature were measured at different time points after digestion. The storage time significantly affected the concentration after sixty-one days, but not after fourteen days (analysis of variance, p < 0.02, Tukey's post-hoc test, p > 0.69).
www.nature.com/scientificreports www.nature.com/scientificreports/ corresponds to a total of 4.8 g SiO 2 kg −1 for the potato seasoning. Sodium residues from the pre-digestion can be the reason for the more variable results compared to the other tested matrices, in line with the results for DMEM, and as also reported by Frantz et al. 30 . The quantity of the anti-caking agent was not indicated on the potato seasoning package. However, our results are in good agreement with Si analyses of related products in the literature 40 .
SiO 2 -NP digestion at room temperature-ICP-OES (HF-free) vs. colorimetry (not HF-free).
For colloidal SiO 2 -NPs in a simple matrix, the microwave digestion is replaceable by an RT digestion overnight in 0.1 M or 1.0 M KOH, without much reduction in recoveries ( Table 2). Colloidal SiO 2 -NPs digested in 1.0 M KOH at RT yielded a recovery of 84 ± 5% compared to 85 ± 2% for 0.1 M KOH in the microwave (both measured by ICP-OES). This demonstrates that porous, almost entirely hydroxylated colloidal SiO 2 -NPs are digestible at RT without expensive instrumentation, and confirms reports by Tanakaa and co-workers, who found that silica gel dissolves in 0.1 M KOH without the aid of microwave irradiation 18 .
The efficiency of ICP-OES and colorimetry in detecting SiO 2 -NPs was directly compared for samples digested using the method RT + KOH1.0 (Tables 1 and 2). Using colorimetry, the recovery for fumed SiO 2 -NPs was lower (76 ± 9%) than for ICP-OES (105 ± 1.4%). Also, for the fumed SiO 2 -NPs, the recovery was only 67 ± 2% when digested in 0.1 M KOH at RT (Table 2), revealing a limitation of the digestion methods at RT for fumed SiO 2 -NPs and colorimetry that only detects fully dissolved orthosilicic acid or small Si oligomers 21 . Despite the larger specific surface area, the non-porous, less hydroxylated fumed SiO 2 -NPs were, in agreement with Zhang and co-workers 37 , harder to completely digest and required the harsher 1.0 M KOH conditions, in contrast to the more soluble porous, more hydroxylated colloidal SiO 2 -NPs. The ICP-OES method was more robust in detecting incompletely digested SiO 2 at RT: a high recovery was found for fumed SiO 2 -NPs of 105 ± 1.4% in 1.0 M KOH.
The trend in the recovery of the two detection methods for colloidal SiO 2 -NPs was inverse: despite milder digestion conditions (0.1 M KOH), colorimetry detected more Si (111 ± 7%) than ICP-OES (84 ± 5%, 1.0 M KOH). The simplest explanation for this seemingly contradictory result is that the harsh 1.0 M KOH conditions readily dissolved the colloidal SiO 2 -NPs, and because the easier to dissolve colloidal SiO 2 -NPs did not consume all of the 1.0 M KOH, the excess KOH negatively affected the ICP-OES recovery. This confirms the earlier finding that, for colloidal SiO 2 -NPs, KOH concentrations <0.5 M are sufficient for ICP-OES analysis and excess KOH should be avoided.
The present results show that the ICP-OES detection of Si is more widely applicable than colorimetry because, despite satisfactory recoveries, the quantification via colorimetric detection of Si has several limitations. First, as mentioned before, the colorimetric quantification of Si suffers from a wide variety of interferences 20,41 and exclusively detects fully dissolved Si(OH) 4 or small oligomers 21 . Second, the present colorimetric determination of Si employed a four-fold higher dilution factor (105) compared to ICP sample preparation (25). Based on the LODs in Table 2, this results in an estimated detectable concentration for the colorimetry of >15-32 mg SiO 2 L −1 , and for the ICP-OES of >1.7-7.4 mg SiO 2 L −1 , depending on the sample matrix. The high detection limit for the colorimetry makes it challenging to detect Si in samples with low SiO 2 concentrations of <15 mg SiO 2 L −1 without additional pre-concentration steps as used e.g. by Rimmelin-Maury and co-workers 6 . Future development of the KOH digestion method for colorimetry should, therefore, focus on reducing the LOD by reducing this dilution factor or including pre-concentration steps. Finally, the digestion protocol for colorimetry uses ammonium fluoride at a low pH, which raises concerns of in situ hydrofluoric acid formation due to its pK a of ~3.17.
Conclusion
Herein, we report a series of methods using basic KOH digestion to quantify Si in a broad variety of samples. Digested samples containing particulate amorphous SiO 2 or Si(OH) 4 could be quantified by ICP-OES or colorimetry (Fig. 6). The method was successfully applied in samples of low and high complexity including aqueous colloidal or fumed SiO 2 -NP suspensions, SiO 2 -NP-spiked cell culture media, SiO 2 -NP-spiked tomato sauce, and potato seasoning containing food grade SiO 2 (E551). SiO 2 dissolved at a minimum KOH: SiO 2 ratio of 1.2 at pH values >12. The complexity of the sample matrix and the manufacturing process of the SiO 2 under investigation www.nature.com/scientificreports www.nature.com/scientificreports/ both affect the Si recovery. Recovery can be improved by controlling the excess of KOH. The different optimal KOH concentrations reflect trade-offs between high excess KOH and harsh pH conditions that favor the rapid dissolution of less porous and less hydroxylated fumed SiO 2 -NPs and Si in more complex matrices; and low excess KOH concentrations, where less matrix effects occur. In case KOH concentrations >0.1 M are used, the calibration has to be prepared in the same concentration of KOH to account for these matrix effects (matrix-matched calibration). Some limitations of the method to be addressed in follow-up studies are the efficiency for larger SiO 2 particles ≥397 ± 22 nm, long term sample storage, the applicability of the method in sera (e.g. 10% fetal calf or bovine serum), and the differentiation of dissolved and particulate SiO 2 species that can be addressed by size fractionation steps prior to further analysis.
Both detection by ICP-OES or colorimetry yielded satisfactory recoveries of up to 100% for SiO 2 -NPs ≤397 ± 22 nm. This shows that our approach without HF can lead to recoveries and detection limits comparable to the state-of-the-art colorimetry method involving HF that was tested here to validate our method 42 . While colorimetry is easy and fast for simple matrices and colloidal SiO 2 -NPs and also feasible with a preceding HF-free KOH digestion, the ICP-OES method presented here is completely hydrofluoric acid-free, independent from color interferences due to matrix components such as Fe, nitrates, and sugars, and more accurate than colorimetry for incompletely digested nanoparticles (e.g. from fumed SiO 2 ). Thus, the hydrofluoric acid-free SiO 2 dissolution and quantification methods presented here are simple to implement alternatives to current standard procedures and applicable in fields such as biomedical sciences and environmental chemistry where SiO 2 -NP quantification in complex matrices is important.
Method Section
Materials, chemicals, and matrices. Commercially available fumed (pyrolytic) SiO 2 -NPs (Aerosil ® 200, 98% SiO 2 , specific surface area of 200 m 2 g −1 ) were purchased from Evonik (former Degussa). Fumed SiO 2 -NPs are produced by continuous flame hydrolysis, are reported to be non-porous by the manufacturer and Mebert and co-workers 8 , and are less hydroxylated than colloidal SiO 2 -NPs 37 . All chemicals used were per analysis grade unless it is stated otherwise. Water was pre-purified by a Milli-Q system (18.2 MΩ.cm arium 611DI, Sartorius Stedim Biotech, Germany). Dialysis membranes were purchased from Roth (Membra-Cel ™ , 14 kDa cut-off).
Both cell culture medium and food matrices are relevant chemically complex matrices that reportedly pose significant analytical challenges for NP analytics 43,44 . We selected three representative complex matrices according to the following criteria: (1) the cell culture media DMEM is widely used in in vitro NP-cell interaction studies 45 ; (2) tomato sauce is a typical food matrix containing with <61 mg kg −1 comparatively little SiO 2 39 ; and (3) potato seasoning is a foodstuff where E551, i.e. food grade SiO 2 , was listed on the packaging as an anti-caking ingredient. The potato seasoning (Qualité & Prix Country Potato Seasoning Blend, Germany) and the tomato sauce (Cirio Rustic Tomato Purée, Italy) were purchased from a local supermarket.
Colloidal SiO 2 -NP synthesis. Colloidal SiO 2 -NPs were synthesized via a co-condensation reaction adapted from Stöber et al. 46 . Briefly, ethanol (522 mL, absolute, Honeywell), ammonia (122.7 mL, 1.65 mol, 25% aqueous solution, Merck), and water (40.5 mL, MilliQ) were mixed and heated to 60 °C. The mixture was stirred at that temperature for 1 h to equilibrate. Tetraethyl orthosilicate (67.5 mL, 302 mmol, Sigma-Aldrich) was added, and the mixture was stirred at 60 °C overnight. The mixture was allowed to cool to RT, and the NPs were washed three times by centrifugation (Thermo Scientific, F15-8 × 50cy fixed-angle rotor, 5000 × g, 10 min) and redispersed in water. The final opaque SiO 2 -NP suspension (500 mL) contained 23.1 g SiO 2 kg −1 , as determined gravimetrically by drying aliquot volumes of the suspension. Due to the sol-gel manufacturing process, colloidal Stöber SiO 2 -NPs are more porous and almost fully hydroxylated compared to the fumed SiO 2 -NPs 8,32,37 . Nanoparticle characterization. The SiO 2 -NPs were characterized by TEM (primary particle diameter), and dynamic light scattering (DLS, hydrodynamic particle diameter, surface charge). The results are summarized in Supplementary Fig. S1 and Supplementary Table S1. For TEM analysis, samples were prepared by diluting NP suspension (1 μL) with ethanol (5 μL, absolute, Honeywell) for SiO 2 -NPs and water for fumed SiO 2 -NPs directly on the TEM grids (carbon film, 300 mesh on Cu, Electron Microscopy Sciences) and wicking remaining liquid using a precision wipe tissue (Kimtech Science). The TEM images were recorded in 2048 × 2048 pixel resolution (Veleta CCD camera, Olympus) on a FEI Tecnai Spirit TEM, operating at an acceleration voltage of 120 kV. The DLS samples were diluted with water (1% v/v) and measured on a Brookhaven Particle Size Analyzer Plus90 (USA) (scattering angle 90°, 1 min acquisition, 10 repetitions). The size distribution of the particles was analyzed by computer-assisted particle size analysis software (imageJ, plugin: psa-r12) 47 , applied to the TEM micrographs.
Digestion pretests to find the KOH concentration for complete SiO 2 dissolution. A series of digestion methods (throughout the text referred to as High_SiO 2 ) was tested to find the highest SiO 2 mass and lowest KOH concentration that allowed for complete solubilization of all SiO 2 nanoparticles in the sample. Colloidal SiO 2 -NP suspensions (433-2165 µL of a 23.1 g SiO 2 kg −1 suspension, equivalent to 10, 20, 30, and 50 mg of SiO 2 ) were weighed into the PTFE microwave vessels, and KOH (10 mL, 0.05, 0.1, 0.5, or 1.0 M) was added ( Table 1). The mixtures were sealed and digested in the microwave (details below). The digestates were measured by DLS (particle counts per second) and visualized using TEM to detect undigested SiO 2 -NPs. Figure 1 shows the key steps, and Table 1 the reagents and concentrations used in the different digestion protocols investigated. All microwave digestions were conducted using an Anton Paar Multiwave PRO, equipped with a 24HVT50 rotor holding 25 mL PTFE microwave vessels with pressure-activated-venting caps (PTFE-TFM, max. pressure 40 bar). All microwave runs consisted of a temperature ramp to 200 °C for 7 min followed by a temperature hold for 7 min and concluded by a cooling (2019) 9:7938 | https://doi.org/10.1038/s41598-019-44128-z www.nature.com/scientificreports www.nature.com/scientificreports/ segment until the internal temperature in all containers reached 70 °C (Supplementary Fig. S2) resulting in a total duration of the microwave digestion of ~28 min. The power limit for all runs was set to 1500 W. If not stated otherwise, digested samples and calibrations were stored at RT and analyzed by ICP-OES within 24 h. Digestates spiked with internal Y standard and stabilized in acidic BgS and were stored in the fridge. The background equivalent concentrations (BEC), the limits of the detection (LOD) and limits of quantification (LOQ) were calculated by adding three times the BEC standard deviation to the BEC (LOD), and ten times the BEC standard deviation to the BEC (LOQ).
Digestion methods investigated for ICP-OES.
KOH0.1-KOH1.0. These methods served to assess the Si recovery for (a) 120 colloidal SiO 2 -NP suspensions in the range of 1.88 to 8.53 mg L −1 SiO 2 in the course of ten experiments (method KOH0.1); (b) a different SiO 2 source (fumed SiO 2 -NPs, method KOH0.1 and KOH1.0); (c) more complex matrices spiked with colloidal SiO 2 -NPs in the concentration range of 0.4-1.7 mg L −1 SiO 2 (0.5 g of tomato sauce and 0.5 mL of cell culture medium, pre-digestion except for cell culture medium as explained below, then methods KOH0.1-KOH1.0); and (d) the pre-digested commercial potato seasoning (0.5 g) with an unknown Si concentration treated equally to the complex matrices in (c). For all methods, SiO 2 -NP stock suspensions (25 mg SiO 2 kg −1 ) were weighed into the PTFE microwave vessels and diluted with water to ~2 g. Aqueous KOH (3 mL, 0.1, 0.5, or 1.0 M, respectively, Table 1) was added, and the mixtures were prepared for the microwave run. The digestates were transferred to conical tubes (polypropylene, Falcon ® ) and acidified by H 2 SO 4 (2.25 M) to pH 1-2. Internal standard (yttrium, 50 mg L −1 solution in 2% HNO 3 /0.5% HCl, 100 μL) was spiked, and the samples were topped off with water to 10 mL for the ICP-OES analysis.
RT + KOH0.1, RT + KOH1.0. Here, the SiO 2 digestion at RT was tested, and the Si detection of ICP-OES was compared with colorimetry. For the ICP measurements, stock suspensions (25 mg SiO 2 kg −1 in water) of either colloidal or fumed SiO 2 -NPs were mixed with KOH (3.0 mL, 1.0 M) and stirred overnight at RT (Table 1). Then, H 2 SO 4 (2.25 M) was added until pH 1-2 was reached. Internal standard (yttrium, 50 mg L −1 solution in 2% HNO 3 /0.5% HCl, 100 μL) was spiked, and the resulting digestates were topped off with water to 10 mL for the ICP-OES analysis.
Pre-digestion of samples containing food matrix. The colloidal SiO 2 -NP-spiked tomato sauce samples and the food grade SiO 2 -NP containing potato seasoning were pre-digested according to a procedure for food analysis established in our laboratory. In pretests, we found that for these food matrices, the method KOH0.1 to KOH1.0 was not suitable due to the high solid content. We therefore used a two-step digestion for these samples, consisting of an acidic pre-digestion of the food matrix followed by KOH digestion of the oven-dried SiO 2 -containing residue. Briefly, for the pre-digestion, the sample (~0.5 g) was added to the PTFE microwave vessels and nitric acid (63%, 3 mL) was added. The closed vessels were heated in the microwave (700 W, 10 min at 60 °C) without previously running a ramp. After this run, the vessels were opened to release nitric oxide gases, closed again, and heated in the microwave (800 W) according to the following program: ramp (90 °C, 5 min), hold (2 min), ramp (180 °C, 6 min), hold (15 min) and cool to 70 °C. The cooled digestates were transferred into 15 mL conical tubes (polypropylene, Falcon ® ) and diluted with water to 5 mL. The digested samples were cleaned by centrifugation at 8000 × g for 10 min at 4 °C and redispersed in 1 mL of water. The centrifugation-redispersion cycle was repeated until the pH of the suspensions reached 5-6. The water was evaporated in an oven and the resulting Si-containing solids were operationally defined to consist of 100% SiO 2 , as an energy dispersive X-ray spectrometric (EDX) elemental analysis found no impurities. These solids were used to prepare stock suspensions in water for quantification experiments using the basic digestion methods KOH1.0-KOH0.1 and subsequent ICP-OES analysis.
Four types of Si calibrations with increasing complexity were prepared using the same volumes and concentrations as in the digestion method KOH0.1 to assess the effects on the Si sensitivity of the ICP-OES for samples in different acids, in KOH matrix, and digested in the microwave. The four Si calibrations were Si in water and H 2 SO 4 (short: water + H 2 SO 4 ); Si in BgS; Si in water and KOH (3 mL, 0.1 M), acidified by H 2 SO 4 (short: matrix-matched + H 2 SO 4 ); and Si in water and KOH (3 mL, 0.1 M) digested in the microwave, and acidified by H 2 SO 4 (short: matrix-matched + H 2 SO 4 + digested). The background was accounted for by subtraction of the blank concentration.
Sample preparation for colorimetric SiO 2 analysis.
To test the suitability of the KOH digestion method for colorimetry, and to cross-validate the ICP-OES results using a conventional approach involving hydrofluoric acid, we quantified the dissolved silicon dioxide according to a modified version of the colorimetric method based on the blue molybdosilicic acid complex (Fig. 1) 19 . For the digestion, lyophilized colloidal SiO 2 -NPs (2.0 mg, 33.0 µmol SiO 2 ) or fumed SiO 2 -NPs (1.7 mg, 28.3 µmol SiO 2 ) were suspended in 0.1 M KOH (20 mL) for the colloidal SiO 2 -NPs or 1.0 M KOH (20 mL) for the fumed SiO 2 -NPs and stirred overnight at RT. All the resulting digestates were then diluted to a final concentration of 0.1 M KOH. From here, we followed the colorimetric SiO 2 analysis protocol reported by Yang et al. 19 using 5 mL of the colloidal SiO 2 -NP digestate and 9 mL of the fumed SiO 2 -NP digestate (concentration: 9.2-92 colloidal SiO 2 L −1 , and 9.2-14.4 mg fumed SiO 2 L −1 , respectively). Water (5 mL), HCl (1 M, 5 mL) and NH 4 F (1 M, 1 mL) were added, and the mixtures were stirred at 25 °C in a water bath for 45 min. Mixing a 5-fold excess of HCl with NH 4 F produces HF in situ due to the pK a of HF of | v3-fos-license |
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} | pes2o/s2orc | In vitro protein expression changes in RAW 264.7 cells and HUVECs treated with dialyzed coffee extract by immunoprecipitation high performance liquid chromatography
RAW 264.7 cells and HUVECs were compared to evaluate the effects of dialyzed coffee extract (DCE) and artificial coffee (AC). Immunoprecipitation high performance liquid chromatography (IP-HPLC) showed DCE-2.5- (equivalent to 2.5 cups of coffee a day) and DCE-5-induced protein expression that was beneficial to human health, i.e., they led to significant increases in proliferation-, immunity-, cellular protection-, antioxidant signaling-, and osteogenesis-related proteins but decreases in inflammation-, NFkB signaling-, cellular apoptosis-, and oncogenic signaling-related proteins in RAW 264.7 cells, and slight decreases in angiogenesis-related proteins in HUVECs. These protein expression changes were less frequently observed for DCE-10 treatment, while AC treatment induced very different changes in protein expression. We suggest that the favorable cellular effects of DCE were derived from minor coffee elements that were absent in AC, and that the reduced effects of DCE-10 compared with those of DCE-2.5 or DCE-5 might have been caused by greater adverse reactions to caffeine and chlorogenic acid in DCE-10 than DCE-2.5 or DCE-5. IP-HPLC results suggested that minor coffee elements in DCE might play beneficial roles in the global protein expression of proliferation-, immunity-, anti-inflammation-, cell protection-, antioxidant-, anti-apoptosis-, anti-oncogenesis-, and osteogenesis-related proteins in RAW 264.7 cells and enhance anti-angiogenic signaling in HUVECs.
Figure 1. Comparison of cell number changes induced by DCE and AC in RAW 264.7 cells. Cultured cells on
Petri-dishes were directly counted in multiple digital images (x200). The results are represented as mean ± STD, n = 24 to 35. The statistical difference was analyzed by Chi-squared test. The raw data for the cell counting analysis were shown in supplement 1.
Effects of DCE and AC on the expression of cMyc/MAX/MAD signaling proteins in RAW 264.7 cells. RAW 264.7 cells treated with DCE-2.5 or DCE-5 showed slight increases in the expression of V-Myc myelocytomatosis viral oncogene homolog (cMyc, 108.9%) and Myc-associated factor X (MAX, 109.8%) but or AC (A2,B2,C2,D2) treatment in RAW 264.7 cells as determined by IP-HPLC. A distinct contrast in protein expression between DCE-5 and AC-5 treatments was found in the radial graphs of (A3,B3,C3,D3). marked decreases in MAD (90.8%) expression versus non-treated controls, whereas cells treated with DCE-10 showed similar changes in the expression of MAX and MAD but marked reductions in cMyc (Fig. 2B1). By contrast, RAW 264.7 cells treated with AC-2.5 or AC-5 showed minimal changes in the expressions of cMyc, MAX, and MAD, whereas AC-10 induced a slight increase in cMyc expression (Fig. 2B2). These results suggested that DCE enhanced cMyc/MAX signaling in RAW 264.7 cells and thus induced proliferation.
Effects of DCE and AC on the expression of translation-related proteins in RAW 264.7 cells.
RAW 264.7 cells treated with DCE-2.5, DCE-5, and DCE-10 showed slight increases in deoxyhypusine synthase (DHS) (106%) but slight decreases in eukaryotic translation initiation factor 5A1 (eIF5A1) and eIF5A2 protein levels (96.6% and 92.9%, respectively) compared with non-treated controls. However, the decreases in eIF5A1 and eIF5A2 were concurrent with the decrease in eIF2AK3 (91.3%), which could lead to a rapid reduction of translational initiation and repression of global protein synthesis (Fig. 3A1). Deoxyhypusine hydroxylase (DOHH) protein expression was also slightly decreased to 95.5% by DCE-5 and DCE-10, whereas AC slightly reduced DHS expression to 91.7% and had almost no effect on eIF5A-1 expression (98.5% to 100.2%) (Fig. 3A2). Because eIF5A-1 can function as a translation initiation and elongation factor depending on the presence of hypusination of its lysine residues by DHS and DOHH 23 , the increase in DHS expression after DCE-2.5 treatment might indicate the induction of protein translation by increasing eIF5A hypusination; however, these expression levels were reversed by AC treatment. These results suggest that DCE affects the basal protein translation level for cellular proliferation and different types of functional activation.
Effects of DCE and AC on the expression of growth factor-related proteins in RAW 264.7 cells.
RAW 264.7 cells treated with DCE-2.5, and DCE-5 showed marked increases in the expression of growth hormone (GH, 19.6%) and growth hormone-releasing hormone (GHRH, 13.6%), slight increases in estrogen receptor beta (ERβ, 107.8%) expression, and marked reductions in human epidermal growth factor receptor 1 (HER1, 86.7%), but no effect on the expression of HER2 was observed (98.8-99.7%) (Fig. 3C1). RAW 264.7 cells treated with AC-2.5, AC-5 or AC-10 showed slight increases in the expression of hepatocyte growth factor (HGF, 108%), HER1 (106.6%), and HER2 (110.3% by AC-2.5), but a gradual decrease in GHRH (88.8%), GH (84.1%), and HER2 (88.1%) by AC-10. Alternately, the expression of general growth factors, that is, transforming growth factor-β1 (TGF-β1), IGFIIR, and ERβ, changed the minimum to less than ±5% similarly to the control housekeeping proteins (Fig. 3C2). These results suggested that the increased expression of GH, GHRH, and ERβ by DCE-2.5 or DCE-5 might be associated with enhanced RAS signaling and positively impact human health and that the marked reductions observed in the expression of HER1, HER2, and IGFIIR after DCE-2.5 or DCE-5 treatment might be useful for the treatment of different human diseases, such as breast cancer and diabetes. NFkB changed the minimum to less than ±5% similarly to the control housekeeping proteins after DCE-5 or DCE-10 treatment, whereas the expression levels of MDR and JNK1 were reduced to 91.7% and 94.4%, respectively. The mitogen-activated protein kinase p38 showed enhanced phosphorylation, and consequently p-p38 expression increased slightly to 106.7% after DCE-5 treatment (Fig. 3D1). RAW 264.7 cells treated with AC showed gradual increases in TNFα (111.3%) and MDR (110.1%) expression, which became marked after AC-5 treatment. However, the expression of NFkB and ikappaB kinase (IKK) was minimally changed. Cells treated with AC-2.5 showed a slight increase in MDR expression, and this increase was lower in cells treated with AC-5 or AC-10. By contrast, the expression of pAKT (88.7%), ERK-1 (92.2%), and phosphatidylinositol-3-kinases (PTEN, 89.4%) was slightly decreased after AC treatments (Fig. 3D2). These results suggested that in RAW 264.7 cells treated with DCE, NFkB signaling was not affected and the cells were in an unstressful condition similarly to the non-treated control, whereas treatment with AC increased NFkB signaling, increased the expression of TNFα and MDR, and decreased the expression of pAKT, ERK-1, and PTEN, indicating increased cellular stress.
Effects of DCE and AC on the expression of immunity-related proteins in RAW 264.7 cells. RAW
264.7 cells treated with DCE showed significant increases in the expression of cathepsin C (121.4%), cathepsin G (124.4%), cluster of differentiation 20 (CD20, 117.4%), CD28 (109.9%), CD31 (105.4%), and CD68 (112.4%). The expression levels of cathepsin G, cathepsin C, CD31, and CD68 were markedly increased by DCE-5, but less so by DCE-10. In particular, cathepsin G was overexpressed in cells treated with DCE-5 or DCE-10. The expression levels of CD20 and CD28 were elevated by all DCE treatments and were marked after treatment with DCE-10 ( Fig. 4A1). By contrast, cells treated with AC showed less-significant changes in the expression of immunity-related proteins, although AC-5 and AC-10 slightly increased the expression of cathepsin G and cathepsin C to 109.6% and 112.3%, respectively (Fig. 4A2). These results indicated that DCE activated RAW 264.7 cells by increasing the expression of macrophage biomarkers, i.e., cathepsin G, cathepsin C, CD31, and CD68, and stimulated these cells by increasing the expression of the immunogenic proteins CD20, CD28, CD31, CD40, and CD68, thereby resulting in the cellular immunity activation of RAW 264.7 cells.
Effects of DCE and AC on the expression of FAS-mediated apoptosis-related proteins in RAW 264.7 cells. RAW 264.7 cells treated with DCE-2.5 showed slight increases in the expression of FAS (107.6%)
and FAS-associated via death domain (FADD, 106%), although cells treated with DCE-5 or DCE-10 showed a gradual decrease in the expression of FAS (97.1%) and FADD (104.2%). The expression levels of caspase 8 and caspase 3 were consistently down-regulated to 97% and 91.1% by DCE at all three concentrations, respectively. Additionally, the expression of FASL, FLIP, and BID showed a minimum change of less than ±5% similarly to the control housekeeping proteins (Fig. 4D1). Whereas RAW 264.7 cells treated with AC-2.5 showed a slight FADD up-regulation (109.8%), cells treated with AC-5 or AC-10 showed a slight up-regulation of caspase 3 (110.7%) and non-significant changes in c-caspase 3, respectively. Changes in the expression levels of other FAS-mediated apoptosis-related proteins by AC treatments were non-significant. The expression of poly-ADP ribose polymerase (PARP) was markedly increased to 106.4% by DCE-2.5 but reduced to 97.2% by DCE-5 and further reduced to 91.6% by DCE-10, whereas the expression of c-PARP was consistently reduced to 90.6% at all three DCE concentrations, which contrasted with the non-significant changes in PARP and cleaved PARP levels induced by AC at all three concentrations (Fig. 4D2). These results suggested that RAW 264.7 cells treated with DCE tended not to undergo FAS-mediated cellular apoptosis and that cells treated with AC-5 or AC-10 were more likely to undergo FAS-mediated cellular apoptosis.
Effects of DCE and AC on the expression of osteogenesis-related proteins in RAW 264.7 cells.
RAW 264.7 cells treated with DCE showed slight increases in the expression of osteoprotegerin (OPG, 105.4%), osteonection (105.9%), osteopontin (105.4%), osterix (108.4%), and alkaline phosphatase (ALP, 109.7%) compared with non-treated controls. The expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL) and cathepsin K were obviously up-regulated to 107.7% and 106% after DCE treatment, but those of osteocalcin and HSP-90 showed a minimal change similarly to the control housekeeping proteins (Fig. 5D1). RAW 264.7 cells treated with AC showed consistent up-regulation of osteogenic proteins, that is, ALP (113.9%), RUNX2 (111.4%), osterix (106.7%), HSP-90 (106.1%), osteonectin (105.1%), osteocalcin (105.9%), and RANKL (116.8%), whereas cells treated with AC showed non-significant changes in the expression of OPG and osteopontin (Fig. 5D2). These results indicated that DCE slightly up-regulated osteogenesis-related proteins to a degree in RAW 264.7 cells compared with the non-treated control, but the expression levels were lower than those observed in response to AC treatment. However, it can be assumed that RAW 264.7 cells, which have the potential to become osteoclasts, showed a slight osteogenic effect after DCE treatment compared with the non-treated control and that RAW 264.7 cells treated with DCE might play a role in the osteogenic effect, even though they had a reduced osteogenic effect than the cells treated with AC.
Effects of DCE on the expression of proliferation-related proteins in HUVECs. HUVECs treated
with DCE-2.5, -5, or -10 showed almost no change in the expression of proliferation-related proteins, that is, Ki-67, proliferating cell nuclear antigen (PCNA), and MPM2, compared with non-treated controls. These expression changes were usually within ±5% similarly to the expression changes exhibited by control housekeeping proteins (Fig. 6B1). Therefore, we concluded that DCE treatment probably had no proliferative effect on HUVECs but a marked proliferative effect on RAW 264.7 cells (Fig. 2A1).
Effects of DCE on the expression of cMyc/MAX/MAD signaling proteins in HUVECs.
HUVECs treated with DCE-2.5, -5, or -10 showed almost no change in the expression of components of the cMyc/MAX/ MAD network versus non-treated controls. The expression changes exhibited by cMyc, MAX, and MAD were usually within ±5% and were similar to those of control housekeeping proteins, although DCE-5 induced slight increases in MAD expression to 108.1% (Fig. 6B2). We concluded that DCE treatment did not activate the cMyc/ MAX/MAD network in HUVECs, which was By contrast to the marked activation of the cMyc/MAX/MAD network in RAW 264.7 cells (Fig. 2B1).
Effects of DCE and AC on the expressions of inflammation-related proteins in HUVECs.
HUVECs treated with DCE-2.5, -5, or -10 showed almost non-significant change at less than ±5% in the expression of inflammation-related proteins, including TNFα, IL-1, IL-6, IL-8, IL-10, IL-12, IL-28, and MMP-2, versus non-treated controls. These expression changes were usually similar to those observed for control housekeeping proteins (Fig. 6C2). Therefore, we concluded that DCE likely had no inflammatory effect on HUVECs, but it induced marked anti-inflammatory effects on RAW 264.7 cells, a murine antigen presenting cell lineage (Fig. 4B1).
Discussion
The protective and antioxidant effects of DCE in RAW 264.7 cells observed in the present study were similar to those reported for kahweol in SH-SY5Y, which up-regulated HO-1 and p38 levels 28,29 . It has been suggested that the anti-apoptosis effect of DCE in RAW 264.7 cells might play a role in the radiation-protective effect of caffeine by down-regulating BAX protein 30 . In other studies, the anti-inflammatory effects of DCE-2.5 and -5 were found to be closely related to the down-regulation of NFkB signaling 4,31 . Furthermore, the coffee-specific diterpene, kahweol, was found to suppress proliferation and induce apoptosis of human colorectal cancer cells and head and neck squamous cell carcinoma cells [32][33][34] . However, in the present study, DCE induced the proliferation of RAW 264.7 cells and simultaneously induced cellular immunity but reduced oncogenic protein expression, but it did not induce cellular proliferation or inflammatory reactions in HUVECs. In particular, DCE reduced the expressions of matrix inflammatory proteins (IL-6, IL-28, COX2, MMP-2, MMP-3, LTA4H, and CXCR4) and NFkB signaling proteins (p38, ERK-1, MDR, NRF2, and JNK1) independently of TNFα and NFkB expression. Therefore, it is presumed that in addition to the coffee-specific diterpene, DCE might have additional unidentified chemical elements and promote the expression of proliferative, protective, antioxidant, anti-apoptotic, innate immunity-related, and anti-inflammatory proteins in RAW 264.7 cells and thus have a net anti-carcinogenic effect.
The anti-inflammatory effects of DCE observed in RAW 264.7 cells was consistent with its strong antioxidant effect, its inactivation of NFkB signaling, and its promotion of anti-oncogenic signaling, whereas the observed increased proliferation of RAW 264.7 cells might have been due to a series of molecular signaling changes, such as increased cMyc/MAX signaling, Rb/E2F signaling, and histone activation by epigenetic modification. In particular, the marked down-regulation of p53-mediated apoptosis by DCE matched the increased expression of cellular protection-related proteins and antioxidant-related proteins, and the slight down-regulation of protein translation by DCE also matched the decreased expression of NFkB signaling-related proteins to reduce endoplasmic reticulum stress 35 . The consistent down-regulation of LTA4H and COX2 directly indicated a strong anti-inflammatory effect of DCE during the lack of COX1 expression change by DCE, and it was also remarkable that DCE induced both anti-inflammatory effect and cellular immunity-stimulating effects simultaneously 36 , which has been rarely observed for other anti-inflammatory agents.
Tea and coffee have been associated, both positively and negatively, with the risk of cardiovascular disease (CVD). Controversy still exists regarding the effects of coffee, for which there have been concerns regarding associations with hypercholesterolemia, hypertension and myocardial infarction 37 . Caffeine and kahweol are known anti-angiogenic compounds 24,27 that may function as antitumor and anti-myocardial infarct agents. The present study showed a mild anti-angiogenesis effect after DCE treatment in RAW 264.7 cells via the down-regulation of angiogenin, VEGF-A, p-VEGFR, LYVE-1, ET-1, and MMP-2. HUVECs also showed a marked anti-angiogenic effect after DCE treatment via the down-regulation of HIF, VEGF-A, LYVE-1, CMG2, and vWF. It has also been reported that higher coffee consumption is associated with a small but significant reduction in the number of teeth with periodontal bone loss. Thereby, coffee consumption may be protective against periodontal bone loss in adult males 38 . However, many controversies have arisen due to the negative or positive osteogenic effects of coffee elements [39][40][41] . The present data revealed a significant osteogenic effect after DCE treatment, which was slightly lower than that observed for AC treatment. Therefore, DCE may have consistent osteogenic potential in RAW 264.7 cells.
These wide-ranging interactions between molecules in DCE and different essential signaling proteins in RAW 264.7 cells suggest that DCE molecules derived from the coffee bean matrix are bio-inert and engulfed by cells and may act to preserve and support the functions of essential proteins in RAW 264.7 cells rather than function as specific antagonists or inhibitors. In particular, DCE usually induces different beneficial effects on both RAW 264.7 cells and HUVECs in the absence of cellular stress and apoptosis, By contrast to AC. Therefore, some DCE elements other than AC elements play are considered to play an important role as chemical chaperones to assist the functions of different signaling proteins in cells 42 and affect global protein expression in RAW 264.7 cells and HUVECs.
The non-significant changes in FAS-mediated apoptosis-related proteins induced by DCE might have been associated with the down-regulation of matrix inflammation observed in RAW 264.7 cells (almost no inflammatory signaling change was observed in HUVECs). DCE also reduced p53-mediated apoptosis signaling and enhanced cellular protection from free radical damage in RAW 264.7 cells, and it simultaneously induced cellular immunity and anti-inflammatory reactions in RAW 264.7 cells. However, it had no notable inflammatory or apoptotic effects on HUVECs. As IP-HPLC analysis revealed a series of different protein expression profiles with an accuracy of <5%, DCE appeared to have strong antioxidant, anti-inflammatory and immune stimulant, cellular proliferation and protective, anti-oncogenic and anti-angiogenic, and mild osteogenic effects on RAW 264.7 cells, and also anti-angiogenesis effects on HUVECs.
Coffee contains different polyphenol derivatives, consisting of mostly caffeine and chlorogenic acid, which have been investigated for their biological functions 43,44 . Many other coffee constituents have not been clearly identified and characterized to date. In the present study, we used DCE in the cell culture experiment rather than coffee constituents, such as kahweol or cafestol, and we examined the effects of DCE and AC at identical caffeine and chlorogenic acid concentrations. The IP-HPLC results for protein inductions by DCE or AC in RAW 264.7 cells showed that the two treatments had quite different effects on protein expression and that DCE induced more favorable effects than AC in RAW 264.7 cells. However, we also found that DCE-2.5 and -5 had more favorable effects on RAW 264.7 cells than DCE-10. AC consistently induced increases in the expression of osteogenesis-related proteins in RAW 264.7 cells with the potential to be osteoclast cells, whereas the expression of osteogenesis-related proteins was lower after DCE treatment but still slightly increased compared with the non-treated control. These findings suggest that DCE has a positive effect on bony tissue in the absence of osteoporosis 39,45 .
In summary, DCE, which contains most of the minor coffee elements as well as chlorogenic acid and caffeine, induced global protein expression for essential cellular functions in RAW 264.7 cells, while AC (1 mM chlorogenic acid and 2 mM caffeine at the identical concentration to DCE) induced a very different global protein expression pattern by IP-HPLC using 180 antisera (Fig. 7). DCE induced an up-regulation of cellular proliferation, cellular protection, and antioxidant-related proteins; a down-regulations of apoptosis-related, angiogenesis-related, and oncogenic proteins; and enhanced cMyc/MAX, Rb/E2F, and RAS, growth factor signaling as well as osteogenesis in RAW 264.7 cells. The overall protein expression revealed a signaling circuit triggered by antioxidant-related proteins and genetic/epigenetic activation induced by DCE (Fig. 7, *). Therefore, it is presumed that DCE may function as strong antioxidant and genetic and epigenetic stimulant in vitro culture of RAW 264.7 cells.
However, these effects were somewhat muted in DCE-10-treated RAW 264.7 cells. By contrast, AC induced the expression of very distinct proteins. Our results indicated that the proteins induced by DCE would have favorable SCIeNtIfIC RepoRts | (2018) 8:13841 | DOI:10.1038/s41598-018-32014-z effects on RAW 264.7 cells and HUVECs, that is, DCE increased RAW 264.7 macrophage (antigen presenting cells) numbers and the expression of proteins associated positively with cellular immunity, anti-inflammatory effects, cellular protection, antioxidant effects, and anti-oncogenic effects. Furthermore, DCE slightly decreased the expression of angiogenesis-related proteins in HUVECs, which might be helpful for the treatment of cancer and cardiovascular diseases 25,27 . Our results indicated that these favorable effects of DCE in RAW 264.7 cells were probably due to unknown minor coffee elements that were not present in AC, which was prepared at caffeine and chlorogenic acid concentrations of 2 and 1 mM, respectively. Nevertheless, the current protein expression profile induced by phytochemicals, DCE and AC cannot explain most of the biological features of RAW 264.7 cells and HUVECs using the limited dosages of DEC-2.5, 5, DEC-10, AC-2.5, AC-5, and AC-10 in vitro cell culture. Therefore, further extensive molecular biological studies should be conducted.
Methods
Production of dialyzed coffee extract (DCE) and artificial coffee (AC). First, 20 cups of coffee (20 × 150 mL = 3000 mL) were prepared from medium roasted coffee beans (Coffea arabica L., Nepal, roasted in Chuncheon, Korea, 20 g per a cup) by soaking them in hot water (90-95 °C) as usual for coffee drink. 300 mL aliquots of this extract were repeatedly dialyzed ten times using a permeable cellulose bag (<1000 Da; 131492, Spectra, USA) in 1500 mL double distilled water at 4 °C under stirring for 2 hours. The dialyzed coffee extract (DCE) may be concentrated with low molecular coffee elements more than the original coffee extract, and immediately preserved at −70 °C in a deep freezer until use.
In order to know the amount of low molecular coffee elements, non-adherent reverse phase column chromatography (YMC-Pak, Japan) at 0.25 mL/min using water as an eluent was performed using a HPLC unit (1100, Agilent, USA) and showed that the primary constituents of DCE were caffeine and chlorogenic acid. HPLC analysis of DCE revealed a caffeine concentration of ~2 mM, indicating that 150 mL DCE contained approximately 60 mg of caffeine (Fig. 8A). As 150 mL of coffee extract contained ~120 mg of caffeine, it was considered that the dialysis coefficient for minor coffee elements was approximately 50% and that 300 mL of DCE was equivalent to one cup of coffee extract (150 mL) for a human adult (mean 60 kg, 59.4 liter). Thus, 300 mL of DCE for a human adult (DCE-1) was equivalent to 0.25 mL of DCE in 50 mL of medium for RAW 264.7 cells in culture (Supplement 2).
For comparison of molecular contents between DCE and AC, the HPLC analysis using C8 column (Grace Vydac, USA) was also performed by elution with graded methanol from 0 to 100% (5-50 min) and subsequently 100 to 0% (50-70 min) in H 2 O mobile running for 80 min. The results revealed two similar peaks, chlorogenic acid and caffeine in DCE and AC, and several minor peaks that were found only in DCE (Fig. 8B-D). The total amount of these minor peaks was calculated by measuring peak areas, and then it was estimated at least 19.7 ±1.6% of the total DCE contents (Fig. 8D). In the infrared absorption using FT-IR (Spectrum Two, Perkin Elmer, USA), DCE and AC showed similar infrared absorption peaks, indicating the absence of other atypical molecules (Supplement 3). Therefore, AC consisted of 2 mM caffeine and 1 mM chlorogenic acid from chemical products (Sigma Aldrich, USA). Both DCE and AC were sterilized by filtering through 0.2-µm-pore-size membrane filter (ADVANTEC ® , Japan), and their sterilization was confirmed by a bacterial culture test using a Lysogeny broth (LB) plate.
In order to confirm lipopolysaccharide (LPS) contamination in DCE, LPS detection assay was performed through IP-HPLC using anti-LPS antibody (Santa Cruz Biotechnology, USA). 1 mL and 2 mL DCE (experiment 1 and 2), 1 mL LPS solution (1 ng/mL, Sigma Aldrich, USA, positive control), and 1 mL distilled water (negative control) were separately analyzed by the same procedures of IP-HPLC. The peak areas of experiment 1 and 2 were similar to that of negative control, while the peak area of positive control, LPS solution, was predominantly increased (Supplement 4). These results may indicate that DCE is almost free from LPS contamination. Overlapping panel B and C, the two dominant peaks were almost identical in the DCE and AC graphs, and the minor small peaks (arrows) were estimated to be at least 19.7% of the total DCE contents. Five times repeated experiments were performed, and a representative chromatograph is presented. 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (WelGene, Inc. Korea) in 5% CO 2 at 37.5 °C. Cells were not stimulated with bacterial antigen (LPS) to detect native protein expressions induced by the coffee extract.
RAW 264.7 cells were separately treated with different doses of dialyzed coffee extract (DCE) equivalent to 2.5, 5, or 10 cups of coffee (DCE-2.5, DCE-5, and DCE-10, respectively); control cells were treated with 1 mL of normal saline. Cells were incubated for 12 hours, harvested with protein lysis buffer (PRO-PREP TM , iNtRON Biotechnology, INC, Korea), and immediately preserved at −70 °C in a deep freezer until required.
During HUVEC active growth, the experimental groups were treated with different doses of DCE equivalent to 2.5, 5, or 10 cups of coffee (DCE-2.5, DCE-5, and DCE-10, respectively); control cells were treated with 1 mL of normal saline. Cells were incubated for 12 hours, harvested with protein lysis buffer (PRO-PREP TM , iNtRON Biotechnology, INC, Korea), and immediately preserved at −70 °C in a deep freezer until use.
Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40) and incubated in protein A/G agarose columns at 4 °C for 1 hour (columns were placed on a rotating stirrer during the incubation). After washing each column with sufficient phosphate-buffered saline solution, target proteins were eluted with 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using a HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Korea). Elution was performed using 0.15 M NaCl, 20% acetonitrile solution at 0.4 mL/min for 30 min and detection by UV spectroscopy at 280 nm. Control and experimental samples were run sequentially to allow comparisons. For IP-HPLC, whole protein peak areas (mAU*s) were calculated by subtracting antibody peak areas of negative controls, and protein peak area square roots were calculated for normalization to the relative level of the molecular concentration (Supplement 7).
When the IP-HPLC results were compared with the western blot data of cytoplasmic housekeeping protein (β-actin), the former exhibiting minute error ranges less than ±5% could be analyzed statistically, while the latter showed a large error range of more than 20%, and thus it was almost impossible to analyze them statistically (Supplement 8). We also performed several western blot experiments using IL-10, CD20, NRAS, etc., and compared with the results of IP-HPLC. Western blot results showed relatively irregular protein expression changes depending on the increase of DCE dose, i.e., DCE-0, DCE-2.5, DCE-5, and DCE-10, compared to the IP-HPLC results. Although western blot data were plotted similar trends of protein expression to IP-HPLC data, the protein expression changes of western blot data were not proportional and showed a feature of fluctuation in line graphs compared to those of IP-HPLC data (Supplement 9). Particularly, repeated experiment of IP-HPLC for each protein expression, 4-10 times, produced accurate IP efficiency with minimum error range (less than ±5%) (The raw data sheets of IP-HPLC analysis were presented in Supplements 10-12). Therefore, in the present study we preferred to do IP-HPLC analysis rather than western blot analysis in order to analyze the protein expression changes statistically. | v3-fos-license |
2017-06-25T21:55:42.225Z | 2014-02-26T00:00:00.000 | 17184840 | {
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} | pes2o/s2orc | Biological removal of phenol from saline wastewater using a moving bed biofilm reactor containing acclimated mixed consortia
In this study, the performance of an aerobic moving bed biofilm reactor (MBBR) was assessed for the removal of phenol as the sole substrate from saline wastewater. The effect of several parameters namely inlet phenol concentration (200–1200 mg/L), hydraulic retention time (8–24 h), inlet salt content (10–70 g/L), phenol shock loading, hydraulic shock loading and salt shock loading on the performance of the 10 L MBBR inoculated with a mixed culture of active biomass gradually acclimated to phenol and salt were evaluated in terms of phenol and chemical oxygen demand (COD) removal efficiencies. The results indicated that phenol and COD removal efficiencies are affected by HRT, phenol and salt concentration in the bioreactor saline feed. The MBBR could remove up to 99% of phenol and COD from the feed saline wastewater at inlet phenol concentrations up to 800 mg/L, HRT of 18 h and inlet salt contents up to 40 g/L. The reactor could also resist strong shock loads. Furthermore, measuring biological quantitative parameters indicated that the biofilm plays a main role in phenol removal. Overall, the results of this investigation revealed that the developed MBBR system with high concentration of the active mixed biomass can play a prominent role in order to treat saline wastewaters containing phenol in industrial applications as a very efficient and flexible technology.
Introduction
Several industries including olive oil mills, pickled vegetables, fish processing, meat canning, dairy products, tanning process and oil refining process generate wastewaters containing high salt content and high organic concentration (Lefebvre and Moletta 2006). Phenol and other phenolic compounds are common organic contaminants found in saline wastewaters formed by some of these industries such as olive oil mills, tannery and oil refinery, ranging from one to several hundred milligrams per liter (Moussavi et al. 2009;Edalatmanesh et al. 2008;Chiaiese et al. 2011).
Phenol has been classified as a priority hazardous organic pollutant regulated by the American Environmental Protection Agency (EPA) (Aravindhan et al. 2009). Thus, in order to protect human health and ecosystems from the potential toxic effects caused by exposure to phenol, its removal from saline wastewater with an efficient and environmentally benign technology is quite obligatory (Pan and Kurumada 2008). Busca et al. (2008) have recently published a short review of different technologies for phenol removal from wastewater, including physical, chemical and biological processes. However, despite its importance, few studies have been accomplished on phenol removal from saline wastewaters.
Biological processes have advantages to physicochemical processes in pollution control due to their ability to efficiently degrade the pollutants in an environmentally sound and cost effective way (Karthik et al. 2008) and offer efficient removal of wide range of pollutants in wastewater treatment. Although wastewaters containing high-concentrations of phenol are generally difficult to treat biologically due to substrate inhibition (Ho et al. 2009); the efficient biodegradation of phenol can be obtained by microbial acclimation. Phenol can be degraded by pure cultures as well as mixed bacterial consortia (Bajaj et al. 2008). In addition, biological treatment of saline wastewater usually results in low removal efficiencies because of the adverse effects of salt on microbial flora (Uygur 2006), but by a proper adaptation of the biomass to a desired salt concentration or use of halophilic microorganisms, the detrimental effects of salinity on the overall bioprocess performance can be also mitigated (Aloui et al. 2009;Moussavi et al. 2010). Among the bioprocesses invented for biological treatment of wastewater, bioreactors implying biofilm systems play important roles in the detoxification of hazardous organic contaminants such as phenol (Hosseini and Borghei 2005). Moving bed biofilm reactor (MBBR) is a highly effective biological treatment process that was introduced about 30 years ago and now it is used in largescale all over the world (Rusten et al. 2006). MBBR is a completely mixed and continuously operated biofilm reactor that is designed to offer the positive aspects of biofilm process including a stable removal efficiency of toxic pollutants, compact and simplicity of operation; without its drawbacks including high head loss, medium channeling and clogging (Chen et al. 2008;Delnavaz et al. 2010). In addition, moving bed reactors provide a better control of biofilm thickness and higher mass transfer characteristics (Moussavi et al. 2009). The concentration of biomass in MBBR can be increased either by raising the amount of moving media (Bassin et al. 2011), or using media with a high effective biofilm surface area, that enhances resistance to toxicity and consequently improves MBBR performance. As a consequence of such advantages MBBR process has been recently used for the removal of many toxic wastewaters including landfill leachate (Chen et al. 2008), aniline (Delnavaz et al. 2010), ammonium from saline wastewater (Bassin et al. 2011), coal gasification wastewater (Li et al. 2011), thiocyanate (Jeong and Chung 2006) and antibiotic fermentation-based pharmaceutical wastewater (Xing et al. 2013).
Treatment of phenol-laden saline wastewater using MBBR has not been reported yet. Reported applications that have dealt with biological process are mostly limited to the cases of single microbial species or low inlet phenol concentrations (Dosta et al. 2011;Afzal et al. 2007;Leitão et al. 2007;Kobayashi et al. 2007); both of which may have limitations in the field application that contaminant concentrations of targeted wastewater may alter from low to high. Accordingly, the basic purpose of this research was to investigate the performance of an aerobic MBBR to examine the above-mentioned benefits in treating synthetic phenol-laden saline wastewater using a mixed culture that gradually acclimated to phenol and salt. To achieve this aim, the MBBR was operated at different operational conditions including inlet phenol concentration, hydraulic retention time (HRT), inlet salt content and shock loadings.
MBBR experimental setup
The study was performed using the cylindrical MBBR reactor (see Figure 1 for more details), made from Plexiglas with internal diameter, height and wall thickness of 14, 75 and 0.5 cm respectively, equivalent to 11.5 L total volume. The effective depth of wastewater in the reactor was 65 cm (10 L working volume) filled with up to 50% the floating biofilm carrier elements composed of high density polyethylene (HDPE) with a density of 0.96 g/cm 3 and an effective surface area of 520 m 2 /m 3 . The aeration system was proceeded with the aid of central compressed air system. Fine bubbles were produced by the aeration system. These bubbles could provide a sufficient mixing to keep the carriers moving in the reactor. In order to keep the carriers in the reactor, outlet diameter was designed to be smaller than carrier's size. Synthetic wastewater was injected to the reactor by a peristaltic pump with a flow controlling mechanism. Pumping rate of the wastewater into the reactor was regulated according to the working volume and HRT. In order to take samples and monitor the performance of the system in phenol and chemical oxygen demand (COD) removal, two sampling ports were provided on the influent and effluent lines of the reactor.
MBBR operation procedure
The MBBR used in this investigation was operated at continuous mode (except in biomass acclimation phase) for 251 days. Different phases of the experiment and the range of investigated variables are presented in Table 1.
In continuous system, the MBBR was run to investigate the effects of different operational variables on phenol and COD removal at the steady state operational conditions; which it was assumed that the steady state condition occurred when changing in the removal efficiency was within ±5% for consecutive HRTs at each operational run. The reactor was operated at room temperature (23 ± 2 ºC) under dissolved oxygen (DO) concentration of 4-5 mg O 2 /L controlled by regulating the aeration rate.
Wastewater and inoculum preparation
Synthetic wastewater was prepared daily by adding phenol, nutrient stock solution and NaCl to tap water. Phenol was the sole carbon and energy source for the biomass in the MBBR. The nutrient solution consisted of Urea as a nitrogen source, (NH4)3PO 4 .3H 2 O as a nitrogen and phosphorus source and the trace elements. The COD:N:P ratio in the feed wastewater was kept at 100:5:1 throughout the experiment, where 1 mg/L of phenol is equal to 2.15 mg/L of COD. All chemicals were of analytical grade except for NaCl, which was purchased commercially. In addition, the pH of the inlet wastewater was kept at neutral range.
During the start-up, the MBBR was inoculated with an activated sludge obtained from Pars oil wastewater treatment plant. The health of the activated sludge was verified by microscopic study. Acclimation of the activated sludge to phenol and salt was lasted 90 days and was performed in the batch system. First, phenol concentration in the reactor was increased step-wise up to 500 mg/L, and then salt content in the reactor was increased step-wise up to 30 g/L. Phenol concentration remained at 500 mg/L during this stage. In each phenol and salt concentration, the reactor was operated until the removal efficiency of phenol exceeded 90% after passing 1 day. During this phase, the biofilm was gradually formed on the carriers. Microscopic observations revealed that the active and enriched salt-tolerant phenol-degrading biofilm was achieved. This biomass was used as an inoculum to the reactor.
Analytical method
To evaluate the performance of the MBBR, samples from inlet and outlet of the reactor was taken and analyzed at HRT interval. The measured parameters in inlet samples were phenol, COD, chloride, ammonia nitrogen, phosphate and pH; whereas phenol, COD and chloride were measured in outlet samples. The parameters of pH, DO and temperature of the mixed liquor were daily measured in order to control the optimum condition for bacterial growth in the reactor. For evaluating biomass characterization the parameters of mixed liquor suspended solid (MLSS), biofilm solid (BS), biofilm thickness and specific oxygen uptake rate (SOUR) were measured in the mixed liquor and the carriers samples routinely. In order to measure phenol and COD, the samples were filtered through a filter with 0.45 mm pore size before analysis. Phenol concentrations were measured spectrophotometrically, using a Unico-UV 9200 UV/VIS Spectrophotometer by the colorimetric 4aminoantipyrine according to the procedure given in the Standard Methods (APHA 2005). The pH, DO and temperature were measured using specific electrodes. The parameters of chloride, ammonia nitrogen, phosphorous, and MLSS were determined according to the Horn et al. (2003) and Moussavi et al. (2009), respectively. The morphology of the biomass was visualized using a microscope with 1000× magnification factor.
Results and discussion
Effect of inlet phenol concentration on removal efficiency The effect of inlet phenol concentration ranging from 200 to 1200 mg/L (430-2580 mg COD/L) on the performance of the MBBR in phenol and COD removal efficiency was assessed over six runs under the conditions given in Table 1. Figure 2 depicts average phenol and COD removal efficiency resulted from defined steady-state conditions versus inlet phenol concentration. As demonstrated in Figure 2 increasing inlet concentration up to 800 mg/L did not significantly affect the performance of the MBBR in phenol and COD removal and the efficiencies were over 99% for both parameters. This denotes that the part of phenol metabolized as a carbon and energy source has been completely biodegraded, although further increasing inlet concentration showed an adverse effect on the removal efficiency. Particularly, increasing inlet concentration to 1000 and 1200 mg/L resulted in decreasing phenol removal below 97.6% and 94%, respectively. Rate of decreasing COD removal was higher than that of phenol and was below 93% and 85.1% at inlet concentration of 1000 and 1200 mg/L, respectively.
The results might be explained by consideration that at low phenol concentrations no effect is noted on gross measures of metabolic activity such as specific growth rate, respiration rate, rate of synthesis, etc. By increasing phenol concentration, the biological parameters will increase due to stimulation of metabolism of the microorganisms. Eventually, the concentration is reached to a point which further increase of the concentration does not increase the biological parameters. Further increasing phenol concentration will eventually cause the physiological parameters decrease and the substrate utilization inhibition to occur (Hosseini and Borghei 2005).
Because phenol was the sole substrate the difference between COD equivalent of measured phenol and COD measured in the effluent could be explained by accumulation of organic intermediates (metabolites) that were generated during the partially phenol biodegradation caused by inhibitory effect of high phenol concentration in synthetic saline wastewater on microbial activities (Moussavi et al. 2010). The concentration of metabolites at inlet concentration of 1000 and 1200 mg/L was 97.3 and 230.6 mg/L as COD, respectively.
The results revealed that at phenol concentrations less than 800 mg/L, a complete mineralization occurred and no metabolites were generated under the given conditions of operation. Thus, this value was selected as an optimum inlet phenol concentration for the following phases of the experiment. Therefore the optimum surface loading rate based on inlet concentration (at HRT of 24 h) on the MBBR was found to be 3.08 g phenol/m 2 .day (6.62 g COD/m 2 .day). Accordingly, the MBBR could effectively remove both phenol and its COD from the synthetic saline wastewater.
These kinds of behavior and conclusions have also been shown by other researchers, although by using a pure culture (Afzal et al. 2007;Leitão et al. 2007;Kobayashi et al. 2007) or a phenol degrading mixed culture (Moussavi et al. 2010) that cannot be applicable in industrial scale. High capacity of the investigated MBBR to complete removal of phenol in saline wastewater could be attributed to use of the mixed culture of gradually acclimated active biomass to phenol and salt, using the biofilm carriers with high specific surface area available for microbial growth and high filling ratio.
Effect of hydraulic retention time on removal efficiency
In order to determine the required retention time for the efficient removal, which specifies the size of facilities in biological wastewater processes, the next phase of the experiment was designed to assess the effect of various HRT of 24, 20, 18, 16, 12 and 8 h on the performance of the MBBR in phenol and COD removal under the operating conditions given in Table 1. The reactor was operated during each HRT until defined steady-state conditions were attained. The mean phenol and COD removal efficiencies versus HRT are demonstrated in Figure 3. Figure 3 shows that phenol and COD removal efficiencies were not affected by reducing HRT down to 18 h and the removal efficiencies of both parameters were greater than 99%. Although by further reducing HRT, the removal efficiencies of both phenol and COD were reduced and at higher values of HRT, the investigated MBBR was less sensitive to reduction of HRT. By reducing HRT to 16, 12 and 8 h, the mean removal efficiency of phenol decreased to 98.9%, 93.9% and 84.4%, respectively. COD removal efficiency decreased with higher rate in comparison to that of phenol and at HRT of 16, 12 and 8 h the mean removal efficiency of COD was below 97.8%, 89.2% and 74.4%, respectively. This behavior might be explained by considering that the decrease of HRT until the retention time is enough for complete oxidation, has no remarkable effect on the removal efficiency. Further decreasing HRT leads to incomplete degradation. In addition, increase in hydraulic load speeds leads to detachment of the biofilm from the carrier elements and reduction of active biomass in the reactor (Hosseini and Borghei 2005).
Reducing HRT and consequently increasing phenol loading rate over the biodegradation capacity of the biomass in the reactor might inhibit the complete mineralization resulted in increasing metabolites concentration in the effluent. There was no considerable accumulation of metabolites down to retention time of 18 h. By reducing HRT to16, 12 and 8 h, phenol inhibition occurred and metabolites concentration in the effluent was increased to 18.8, 80 and 171.2 mg COD/L, respectively.
It can be concluded from above that an optimum HRT for the MBBR under the selected operational conditions was 18 h, at which phenol and COD removal efficiencies were above 99% and no metabolites were detected. Thus, this value was selected as the optimum retention time for the next phases of the experiment. Accordingly, the optimum surface loading rate based on HRT (at inlet phenol concentration of 800 mg/L) on the MBBR was found to be 4.1 g phenol/m 2 .day (8.82 g COD/m 2 .day). These results indicate that the MBBR inoculated with the active mixed biomass adapted to phenol and salt can efficiently remove high phenol loading rate and associated COD.
The adverse effect of HRT on COD removal efficiency in the MBBR system has also reported by other researchers (Li et al. 2011;Hosseini and Borghei 2005). Based on the available literature, no experiments were found dealing with removal of phenol from saline wastewater by using mixed active cultures adapted to phenol and salt in the MBBR. In comparison to other bioreactors, the investigated MBBR indicated a high performance in the removal of phenol and COD from saline wastewater. Moussavi et al. (2010) worked on phenol removal from saline wastewater with a granular sequencing batch reactor (GSBR) containing phenol-degrading consortia adapted to salt under operational conditions of cycle time of 17 h and inlet phenol concentration of 1000 mg/L, finding 99% removal efficiency. Dosta et al. (2011) evaluated the performance of a membrane biological reactor (MBR) for removing phenol from saline wastewater at HRT of 12-17 h, inlet phenol concentration of 8-15 mg/L and reported COD removal efficiency of over 98.5%. The great performance of the MBBR in this study could be especially due to the existence of a high concentration of the acclimated and active mixed culture of biomass and using a high percentage occupation of the carriers with a high effective surface area.
Effect of salt content on removal efficiency
In this phase of the experiment, the effect of salt content of synthetic saline wastewater ranging from 10 g/L to 70 g/L was assessed on the behavior of the MBBR under the previously optimized conditions given in Table 1. The MBBR was operated at each salt concentration until determined steady-state condition was achieved. The mean phenol and COD removal efficiencies as a function of salt concentration in the feed stream are demonstrated in Figure 4.
According to Figure 4 inlet salt content in the range of 10-50 g/L had negligible effect on the performance of the MBBR in phenol removal and the efficiency remained greater than 99%. However, further increase in salt concentration to 60 g/L and subsequently to 70 g/L, resulted in decrease in phenol removal efficiency down to 98.1% and 96.7%, respectively. The effect of salt content up to 40 g/L on COD removal was insignificant and the efficiency remained around 99%. However, when salt content was increased to 50, 60 and 70 g/L, COD removal efficiency was reduced down to 97.4%, 95.3% and 92.7%, respectively. Gradually acclimation of the biomass to specific salt concentration can mitigate the detrimental effect of salinity on microbial activity. Much more salt content causes disintegration of cells because of the loss of cellular water (plasmolysis) or the recession of the cytoplasm which is induced by an osmotic difference across the cell wall and cause of outward flow of intracellular water resulting in the loss of microbial activity and cell dehydration (Abou-Elela et al. 2010).
By decreasing microbial activity in high salt content, metabolites concentration was increased to 9.6, 31, 47.5 and 68.2 mg COD/L in salt content of 40, 50, 60 and 70 g/L, respectively. Hinteregger and Streichsbier (1997) worked on the effect of salt content (1-14%) on biotreatment of saline phenolic wastewater by a moderately halophilic strain and showed the adverse effect of salt on biotreatment. Nonetheless, Moussavi et al. (2010) showed that salt content in the range of 3-8% has no effect on the GSBR performance containing the phenol-degraded biomass adapted to salt. Stability of the MBBR against high salt content in the range of 10-40 g/L can be related to establishing the biomass containing a high concentration of salt-adapted microorganisms. It can be inferred from above that the operating MBBR with the mixed consortia acclimated biomass can attain a high performance for phenol-laden saline wastewater in terms of phenol and COD removal under the different operational conditions.
Response to shock loading
Shock loading can be applied by sudden increase of organic concentration, flow rate and salt content in saline wastewater. Therefore in this phase of the study, response of the investigated MBBR to mentioned shock loads was evaluated.
Response to organic shock loading
In order to evaluate the adverse effect of phenol shock load on the MBBR performance, a sudden increase of inlet phenol concentration from 800 to 1400 mg/L was applied to the bioreactor for a period of 4 h under the conditions presented in Table 1. During this period and after that the concentrations of outlet phenol and COD were monitored every 1 h until the steady state condition was reestablished, as shown in Figure 5. It can be seen that outlet phenolic and total COD were increased from 10 and 15 mg/L to the maximum concentrations of 68.4 and 145 mg/L, respectively. Around 5 h after shock load outlet phenolic and total COD gradually reached to the near steady state values of 10.75 and 25 mg/L, respectively.
Response to hydraulic shock loading
In order to study the reactor stability against a sudden variation of flow rate, HRT was changed from 18 to 9 h for a period of 4 h under the conditions listed in Table 1. To understand the trend of outlet phenolic and total COD concentration during shock loading and after that, the effluent was sampled and analyzed at 1-h-intervals and the results are shown in Figure 6. According to Figure 6 outlet phenolic and total COD started to increase from 10 and 15 mg/L to the maximum concentrations of 138 and 235 mg/L, respectively; then gradually started to decrease to the near steady state value of 18.7 and 37.5 mg/L about 5 h after shock load.
Response to salt shock loading
To evaluate the resistance of the MBBR against jump of inlet salt concentration, a sudden change was applied to the reactor where inlet salt concentration increased from 30 to 80 g/L for a period of 4 h under the conditions listed in Table 1. The changes in outlet phenolic and total COD concentration under this condition and after that were monitored hourly and the results are presented in Figure 7. Figure 7 depicts that outlet phenolic and total COD concentrations changed insignificantly and were increased from 10 and 15 mg/L to the maximum concentrations of 10.3 and 20 mg/L, respectively. But after switching the influent to the initial condition, the effluent was remained unchanged, which could be because of remaining a high salt content in the bioreactor. It can be inferred from the above results that the MBBR exhibited a high stability against organic, hydraulic and salt shock loadings and recovered from these changes in a relatively short time. This low sensitivity to shock loadings could be due to the existence of a high concentration of biomass containing gradually acclimated and active microbial consortia and a high filling ratio of the biofilm carriers with a high effective surface area in the reactor. High stability of the MBBR against shock loadings had previously reported by other researchers (Chen et al. 2008;Hosseini and Borghei 2005). These advantages introduce the MBBR as an effective and stable process for the removal of phenol from saline wastewaters.
Biomass characteristics
The biomass characteristics were evaluated both in suspension and biofilm during this study. The determined characteristics were MLSS, BS, biofilm thickness and SOUR. The range of measured biological parameters during this study is presented in Table 2. In the continuous system, the suspended biomass in the bioreactor was negligible in comparison to the biofilm attached to the carriers. It can be inferred that the attached biomass had the main role in the removal of phenol and COD rather than the suspended biomass in the investigated MBBR. The thickness of the biofilm formed on the media was in the range of effective biofilm thickness (the depth of the biofilm to which the substrates have penetrated) (Rusten et al. 2006). The SOUR values indicate a high activity of the biomass in the reactor which could be attributed to the moving media containing a thin biofilm that improves the oxygen and substrate transfer rate and contact between the substrate and the biomass, therefore, enhances degradation rate (Moussavi et al. 2009).
Microscopic examinations were carried out in order to observe the existing microorganisms in the bioreactor during the experiments. Photomicrograph of the biofilm and the mixed liquor are demonstrated in Figure 8. As shown in Figure 8(a) predominant species in the biofilm was yeast and some mold and bacteria also were found in the biofilm, but there was no indication of these groups of microorganisms in the liquid bulk where bacteria was the main, as shown in Figure 8(B). As demonstrated in Figure 8(A), the existence of yeast ongoing to budding and fission implied a high activity of the biofilm inside of the bioreactor. Dan et al. (2003) indicated that yeast culture is more efficient in treating high organic-high salinity wastewater compared to bacterial cultures. Hence, the high performance of the investigated MBBR in phenol removal efficiency could be attributed to the existence of the high concentration of yeast in the biofilm.
Conclusion
The present work investigated the performance of a bench scale MBBR for phenol removal from saline wastewater. The results revealed that the MBBR provides improved phenol and COD removal efficiencies. Inlet phenol concentrations up to 800 mg/L did not significantly affect the performance of the MBBR with HRT of 24 h and salt content of 30 g/L, where phenol and COD removal efficiencies were above 99%. Optimum HRT for the reactor was 18 h, such that decreasing HRT below this value led to reduction of the removal efficiencies of both phenol and COD. The MBBR exhibited low sensitivity to increasing salt concentrations up to 40 g/L. The reactor was very stable against phenol, hydraulic and salt shock loadings and performed well under various operational conditions. The active biofilm containing yeast as a predominant species performed the main role in phenol removal in the MBBR. Overall, high performance of the investigated MBBR in the removal of phenol from saline wastewater could be attributed to existence of the mixed culture of gradually acclimated biomass to phenol and salt and using a high filling ratio of the biofilm carriers with a high effective surface area. the manuscript; SMB gave technical support and conceptual advice. All authors read and approved the final manuscript. | v3-fos-license |
2018-04-03T02:10:49.876Z | 2016-08-26T00:00:00.000 | 28384164 | {
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} | pes2o/s2orc | Crystal structure of methyl 4-(4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate monohydrate
The title hydrate crystallizes with two formula units in the asymmetric unit (Z′ = 2). The organic molecules form a dimer, linked by a pair of N—H⋯O hydrogen bonds. Further hydrogen bonding together with weak C—H⋯π and π–π interactions further consolidates the packing, generating a three-dimensional network.
Chemical context
Dihydropyrimidine (DHPM) derivatives are used in the treatment of disease as antiviral, antitumor, antibacterial and antimalarial agents, as first reported by the Italian chemist Pietro Biginelli in 1893 [Kappe (2000), Nayak et al. (2010) and references therein]. We have been working on the synthesis of various DHPM derivatives for better biological activities (Narayanaswamy et al., 2013;Nayak et al., 2011) and a wide range of applications (Nayak et al., 2009(Nayak et al., , 2010. Here, we report the synthesis and single-crystal structure of the title compound, (I).
Figure 1
The asymmetric unit of the title compound with 50% probability ellipsoids. The double-dashed lines indicate hydrogen bonds.
Figure 2
Crystal structure of title compound showing the dimers formed by N-HÁ Á ÁO hydrogen bonds as well as the links to the water molecules, which donate O-HÁ Á ÁO hydrogen bonds to the ester groups. Table 1 Hydrogen-bond geometry (Å , ).
Figure 3
Three-dimensional crystal structure of the title compound showing the role of the water molecules in the hydrogen-bonding network.
role of the water molecule in the hydrogen-bonding network is shown in Fig. 3.
Synthesis and crystallization
The title compound was obtained by the reaction of three components, viz. methyl acetoacetate, 4-hydroxybenzaldehyde and urea in ethanol solution according to a reported procedure (Tumtin et al., 2010). The reaction progress was monitored by thin layer chromatography and after the completion of the reaction, the solvent was removed and the solid obtained was recrystallized from ethanol to obtain the pure product. Colorless single crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of a solution in ethanol (yield 75%, m.p. 412.
Refinement
Crystal data, data collection and structure refinement details are summarized in Table 2. All hydrogen atoms were located in difference Fourier maps and freely refined.
Computing details
Data collection: APEX2 (Bruker, 2008); cell refinement: SAINT (Bruker, 2008); data reduction: SAINT (Bruker, 2008); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL2014/7 (Sheldrick, 2015); molecular graphics: ORTEP-3 for Windows (Farrugia, 2012) and Mercury (Macrae et al. (2008); software used to prepare material for publication: SHELXL2014/7 (Sheldrick, 2015). Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. | v3-fos-license |
2019-05-07T13:07:48.735Z | 2019-05-07T00:00:00.000 | 146120264 | {
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} | pes2o/s2orc | Biogenic Control of Manganese Doping in Zinc Sulfide Nanomaterial Using Shewanella oneidensis MR-1
Bacteria naturally alter the redox state of many compounds and perform atom-by-atom nanomaterial synthesis to create many inorganic materials. Recent advancements in synthetic biology have spurred interest in using biological systems to manufacture nanomaterials, implementing biological strategies to specify the nanomaterial characteristics such as size, shape, and optical properties. Here, we combine the natural synthetic capabilities of microbes with engineered genetic control circuits toward biogenically synthesized semiconductor nanomaterials. Using an engineered strain of Shewanella oneindensis with inducible expression of the cytochrome complex MtrCAB, we control the reduction of manganese (IV) oxide. Cytochrome expression levels were regulated using an inducer molecule, which enabled precise modulation of dopant incorporation into manganese doped zinc sulfide nanoparticles (Mn:ZnS). Thereby, a synthetic gene circuit controlled the optical properties of biogenic quantum dots. These biogenically assembled nanomaterials have similar physical and optoelectronic properties to chemically synthesized particles. Our results demonstrate the promise of implementing synthetic gene circuits for tunable control of nanomaterials made by biological systems.
INTRODUCTION
Modern technology increasingly relies on integrating precision nanomaterials. Indeed, nanomaterials are at the heart of innumerable applications. For applications in catalysis, electrochemistry, biomedical engineering, ultra-strong magnets, and photonics, nanomaterials are a central component in contemporary manufacturing and consumer industries (Vance et al., 2015). To meet the growing demand for nanomaterials in industry and research, fundamentally new methods of synthesis are needed that are both scalable and efficient while at the same time offering the ability to precisely control nanomaterial properties. Current nanomaterial synthesis includes physical, chemical, and biological methods. In biogenic methods, bacteria offer a wide variety of tools to alter the redox state of many compounds and perform atom-by-atom nanomaterial synthesis (Wakatsuki, 1995;Nealson et al., 2002;Rodríguez-Carmona and Villaverde, 2010). By deploying different wild-type bacteria to facilitate nanomaterial growth, researchers may access diverse shapes and structures of varying composition, including both inorganic (Au, Ag) and semiconductive (CdTe, ZnS) materials (Naik et al., 2002;Akid et al., 2008;Hussain et al., 2016;Wu and Ng, 2017). Recent advancements in synthetic biology have developed genetic tools to program cellular activity (Ang et al., 2013;Smanski et al., 2016;Schuergers et al., 2017;Segall-Shapiro et al., 2018), including nanomaterial synthesis (Mao et al., 2003;Chen et al., 2014). By combining the natural ability of microbes to assemble nanomaterials under ambient temperature, pressure, and neutral pH with synthetic gene constructs, new routes of nanomaterial synthesis can be developed.
Previous studies developed biogenic routes of nanomaterial synthesis (da Costa et al., 2016;Hussain et al., 2016), including production of chalcogenide nanomaterials such as arsenic sulfide, zinc sulfide, cadmium sulfide, cadmium selenide, and cadmium telluride (Kershaw et al., 2013;Jacob et al., 2016). For example, researchers used a bacteriophage as a template for zinc sulfide nanomaterial nucleation (Mao et al., 2003). In another study, purified proteins were used in direct reduction and nucleation of cadmium sulfide nanomaterials (Dunleavy et al., 2016). Many of these previous studies took advantage of the natural ability of microbes to reduce starting materials and assist in the nucleation and growth of nanomaterials; however, these initial studies did not attempt to integrate natural bacterial biosynthesis with engineered genetic control circuits to tune nanomaterial properties. Recent progress in synthetic biology has developed powerful tools to regulate gene expression and cellular behavior (Spoerke and Voigt, 2007;Smanski et al., 2016;Schuergers et al., 2017). Such gene circuits may enable precise control over the properties of biogenic nanomaterials by tuning the expression level of genetic components involved in the synthesis of inorganic nanomaterials.
Here, a biological system is engineered for the synthesis of zinc sulfide nanomaterials. Zinc sulfur (ZnS) has gathered particular interest in fields of optoelectronic, photocatalytic, and photovoltaic applications (Peng et al., 2006;Son et al., 2007;Stroyuk et al., 2007). Due to a lack of photobleaching, a major drawback observed in fluorescent molecules, ZnS quantum dots have been used in bio-imaging (Deng et al., 2011). Additionally, the lower toxicity levels compared to cadmium sulfide, another common bio-imaging material, further increases the demand for ZnS for medical purposes. Furthermore, doping ZnS enable researchers to engineer a plethora of nanomaterials with different physical properties, sensitively dependent on the dopant level. Doping is a process in which a trace amount of an impurity, often a metal, imparts attractive properties to semiconductor materials (Smith and Nie, 2010). Doping zinc sulfide nanomaterials with manganese (Mn:ZnS) imparts a characteristic emission peak around 590-610 nm, a useful emission wavelength in biological imaging (Deng et al., 2011). Moreover the optical properties of Mn:ZnS nanomaterials are sensitive to the level of doping, and control of the doping level during synthesis is essential. Physical and chemical methods demand high-temperature, high-pH, or high-pressure. Herein, we report a biogenic synthesis route and tunable doping of ZnS:Mn(II) nanomaterials using an anaerobic, metal reducing bacteria Shewanella oneidensis at room temperature and pressure.
Using a regulatory genetic circuit, we modulate bacterial electron transfer involved in metal reduction of manganese to synthesize manganese doped zinc sulfide nanomaterials. In an engineered strain of Shewanella oneidensis MR-1, a Gram-negative, metal reducing bacteria, the expression of the MtrCAB cytochrome complex was regulated by an external inducer to control the level of manganese doping in ZnS nanomaterials. The properties of the biogenic nanomaterials were similar to nanoparticles used using traditional, chemical synthesis. These results demonstrate the potential for tunable control of the properties of biogenic nanomaterials using synthetic gene circuits.
Bacteria Culture Conditions
Shewanella oneidensis JG3631 strain was obtained from Jeff Gralnick's lab (University of Minnesota, Minneapolis, MN, United States). The strain has been engineered to express the multi-heme cytochrome complex MtrCAB under control of a native promoter PtorF that responds to inducer molecule trimethylamine N-oxide (TMAO). Additional information about the strain is available in Supplementary Figure S1. Previously, strain JG3631 reduced iron oxide in proportion to the concentration of TMAO inducer added to the culture. The MtrCAB expression level plateaued at 1,000 µM TMAO (West et al., 2017). Here, we induce cells with 0, 50, 100, and 1,000 µM TMAO. Shewanella oneidensis JG1486 was used for control experiments reported in the Supplementary Information, containing deletions of mtrB, mtrE, mtrC, mtrF, mtrA, mtrD, omcA, dmsE, SO4360, cctA, and recA (Coursolle and Gralnick, 2012).
Cultures of Shewanella oneidensis JG3631 were inoculated from a bacterial frozen stock into Luria-Bertani medium and grown overnight (14-16 h) at 30 • C under aerobic conditions. Cultures were then transferred to Shewanella minimal media prepared from the recipe from Bretschger et al. (2007) with 15 mM lactate as electron donor, 30 mM fumarate as electron acceptor, and TMAO (inducer) at 0, 50, 100, or 1,000 µM. Cultures were grown under anaerobic conditions. After 24 h, the cells grown in minimal medium were centrifuged, washed with 7 mM HEPES buffer, and suspended in 7 mM HEPES buffer to a final OD 600nm of 0.8-1.0. Control experiments showed that more dilute cell cultures were also capable of forming ZnS:Mn(II) particles (Supplementary Figure S2). A stoichiometric excess of lactate (10 mM) was used as electron donor in the culture, and the culture was made anaerobic by bubbling sterile nitrogen gas into the bottle. This culture was then used in the experiments for nanomaterial synthesis. Nanomaterial synthesis experiments were performed under anaerobic conditions.
Biogenic Synthesis of Mn Doped Zinc Sulfide Nanomaterials
Solid manganese (IV) oxide was prepared using the protocol described in an earlier work (Burdige and Nealson, 1985). Prepared manganese (IV) oxide was mixed with HEPES buffer and injected into anaerobic bacterial cultures described above to a final concentration of 750 µM manganese with 10 mM lactate was the electron source. After 24 h of manganese reduction by the bacteria, a filter sterilized stock solution of 2.5 mM zinc sulfate was added to the culture followed by 2.5 mM sodium sulfide. Extended manganese reduction, for 48 h total, did not result in additional manganese reduction or change the photoluminescence of the resulting particles (Supplementary Figure S3). Manganese reduction occurred at 303 • K, and samples were moved to room temperature (approximately 295 • K) after the addition zinc and sulfide for the remainder of the synthesis reaction. Samples were thoroughly mixed via vortex. The precipitation of nanomaterials started immediately and proceeded for 16 h. Continued manganese reduction was not detected after the addition of zinc sulfate and sodium sulfide, as shown in Supplementary Figure S4. As shown in Supplementary Figure S5, cell viability was maintained throughout the manganese reduction step, however, no live cells were detected 16 h after the addition of zinc sulfate and sodium sulfide.
Chemical Synthesis of Mn Doped Zinc Sulfide Nanomaterials
Chemical synthesis of Mn:ZnS nanomaterial was accomplished by adding precursors to a sterilized, anaerobic serum bottle containing 25 ml 7 mM HEPES buffer. Sterile nitrogen gas was bubbled through the buffer solution and precursor stock solutions to make them anaerobic. To synthesize Mn:ZnS, first 2.5 mM zinc sulfate was added followed by different manganese acetate at a concentration of either 0, 0.1, 0.5, 1, or 5 mM. Finally, 2.5 mM sodium sulfide was added and the bottle was thoroughly mixed using a vortex. Chemical synthesis was performed at room temperature. The addition of Shewanella oneidensis MR-1 cells during chemical synthesis did not appear to impact photoluminescent properties of the chemically synthesized cells, see Supplementary Figure S6.
Manganese Measurement Using LBB Assay
The reduction of manganese by Shewanella oneidensis JG3631 was quantified using the leucoberbelin blue (LBB) assay (Francis et al., 2002). Five hundred microliters was collected directly from well mixed anoxic serum bottles using a sterile, 20G syringe. Sample was added to LBB [0.04% (w/v) LBB in 45 mM acetic acid] to react in the dark for 15-20 min, and then centrifuged to separate the cellular material and insoluble fractions. A standard curve for concentration of was made by preparing serial dilutions of KMnO 4 and measuring the absorbance at 620 nm to quantify the concentration of Mn. To calculate the amount of Mn(II) at a given time, we subtract the initial amount of Mn(IV) from the amount of Mn(IV) remaining in solution. A calibration curve was made using solutions of potassium permanganate, see Supplementary Figure S7.
Cleaning and Sonication of Nanomaterial
Upon the completion of nanomaterial synthesis, the contents of the bottle were transferred to a 50 ml conical tube for rinsing and cleaning. Nanomaterial solutions were centrifuged at 3,800 × g for 30 min to collect the nanomaterials, the supernatant was discarded, and the nanomaterial pellet was re-suspended in DI water. This washing step was repeated four times to remove salts and cellular materials from the solution of nanomaterials. In samples where we observed excess aggregation, the final solution of nanomaterials was sonicated in an ice bath for 20 min prior to AFM and SEM.
Characterization of Nanomaterials Synthesized via Biogenic and Chemical Method
Photoluminescence Nanomaterials synthesized were tested for photoluminescence (PL) emission using a Tecan plate reader (Infinite 200 PRO, excitation wavelength: 325 nm, well mixed condition, 25 • C).
Absorbance
Nanomaterial samples were mixed and added to a cuvette with 10 mm path length and an absorbance scan was performed using Nanodrop 2000C. Background from media/buffer was subtracted.
Scanning Electron Micrograph
Cleaned nanomaterials were deposited on a silicon wafer for electron microscopy and samples were sputter coated (Cressington 108C) with gold. JEOL 7000 electron microscopy was used to image the nanomaterials and EDX was used to characterize the elemental composition of the nanomaterials.
Atomic Force Microscopy
Cleaned nanomaterials were diluted to low concentrations in DI water and deposited on graphite substrate for atomic force microscopy (AFM) imaging. Samples were imaged in AC mode in air using an Asylum Cypher ES instrument and an AC mode tip (Asylum Research, silicon probe model AC240TS-R3 with 2 N/m nominal spring constant). The images acquired were analyzed using Gwyddion software. A minimum of 100 individual nanomaterials per sample were analyzed for size calculation.
X-Ray Diffraction
Concentrated samples were deposited on a glass slide and used for XRD analysis. The X-ray diffraction (XRD) scattering profiles were obtained using a Rigaku Ultima IV Diffractometer using characteristic Cu Kα radiation = 1.54 Å.
EPMA
Concentrated samples were deposited on a silicon substrate for a quantitative elemental analysis using JEOL 8200 electron microprobe (20 kV, focused beam mode). Reference materials used were zinc sulfide and manganese sulfide (Sigma-Aldrich). A minimum of 10 spots were analyzed for quantification of manganese concentration in the zinc sulfide nanoparticles. "Spots" are usually aggregates of nanomaterials, since the sample preparation and deposition resulted in nanomaterials aggregates, therefore individual spots were composed of nanomaterial aggregates.
RESULTS AND DISCUSSION
Controlling the Dopant Concentration of Mn:ZnS Using a Genetic Circuit By utilizing a genetically engineered bacteria, we designed a biological system to synthesize semi-conductive ZnS nanomaterials doped with Mn(II). Moreover, the degree to which the gene circuit responds to an outside signal modulates the concentration of available Mn(II) for doping in ZnS, thereby adjusting the optoelectronic properties of these biogenically fabricated nanomaterials. Estimated manganese concentrations using EPMA are presented in Supplementary Table S1. These nanomaterials of biogenic origin exhibit almost identical properties to those made by non-biological, chemical methods. To begin our study we synthesized ZnS nanoparticles via chemical means. In bulk, these particles exhibited a characteristic blue emission upon excitation with UV light ( Figure 1A). Next, we introduced variable concentrations of Mn(II) during chemical synthesis and the soluble Mn(II) was passively integrated into the ZnS nanoparticles. These doped nanoparticles exhibited a characteristic orange hue upon excitation with UV light ( Figure 1A) (Beerman, 2005;Cao et al., 2009;Deng et al., 2011). The photoluminescent intensity of the Mn(II) doped nanoparticles depended on the level of doping.
To control the optical properties of Mn:ZnS nanoparticles through biogenic route, we used an engineered strain (JG3631) of Shewanella oneidensis MR-1 because of its metabolic versatility, whole genome sequence availability, and a library of characterized, engineered strains (Bouhenni et al., 2005;Nealson, 2005;Bretschger et al., 2007;Fredrickson et al., 2008). Shewanella naturally respire insoluble metal oxides of iron and manganese via extracellular electron transport protein complex MtrCAB, a multiheme cytochrome complex. This protein complex moves electrons from the periplasmic space to the exterior of the cell during respiration (Myers and Myers, 2001;Nealson et al., 2002;Bretschger et al., 2007). Expressed under anaerobic conditions, the Mtr pathway is composed of three components: MtrA, a periplasmic decaheme c-cytochrome; MtrB, an outer membrane porin; and MtrC, an outer membrane decaheme c-type cytochrome. Here, we utilize an engineered strain JG3631 in which an inducible promotor PtorF regulates expression of the mtrCAB operon ( Figure 1B) (West et al., 2017). Previously it was shown that this strain reduced external iron oxide in proportion to the amount of the inducer (TMAO) added to the culture (West et al., 2017).
First, we tested the ability of the strain JG3631 to reduce manganese in the presence of different TMAO concentrations. As shown in Figure 1C, the amount of Mn(IV) reduced by the cell culture was proportional to the concentration of inducer molecule TMAO. However, at 0 mM TMAO, some manganese reduction was observed, potentially due to a combination of leaky expression of mtrCAB and additional biochemical pathways involved in low levels of manganese reduction, such as MtrDEF cytochrome (Coursolle and Gralnick, 2010). Additional information on the engineered strain and the experimental outline is presented in the Supplementary Figure S1.
After confirming that we could control manganese reduction, and therefore the available concentration of Mn(II) via the concentration of TMAO inducer molecule, we investigated Frontiers in Microbiology | www.frontiersin.org the biogenic synthesis of Mn:ZnS. Because Shewanella prefer to express metal reduction protein complexes in the absence of oxygen, all biogenic reactions took place under anaerobic conditions, as outlined in the "Materials and Methods" section. The nanoparticles produced without Mn(IV) added to the culture appear blue under UV excitation (Figure 1D, left). By adding TMAO, however, the MtrCAB pathway is modulated in a manner reflecting the concentration of inducer molecule (Figure 1C), i.e., more TMAO results in more insoluble Mn(IV) being reduced to soluble Mn(II). As expected, nanoparticles produced in the presence of both Mn(IV) and TMAO exhibit a characteristic red-shifted emission upon UV excitation, similar to chemical synthesis. The buffer solutions and the precursor solutions did not exhibit any UV associated luminescence (Supplementary Figure S8). Altogether, by connecting a naturally occurring metal reduction route in an engineered strain of Shewanella with an inducible promotor, we made possible the controllable synthesis of nanoparticles with tunable optoelectronic properties.
Optoelectronic Properties of ZnS and Mn:ZnS Nanomaterials
Following chemical and biogenic synthesis, we assessed the optoelectronic properties (e.g., photoluminescence and absorbance) of the Mn:ZnS. Both methods yielded nanomaterials with the characteristic 600 nm photoluminescence (PL) peak associated with the presence of dopant metal Mn(II) in a ZnS lattice. The shift in PL emission due to Mn(II) doping into the ZnS nanomaterial is caused by an additional electronic transition between excited electrons and the energy levels of the Mn dopant (Bhargava et al., 1994;Karar et al., 2004). Chemically synthesized nanomaterials produced PL peaks in the 602-604 nm range and the biogenic nanomaterials produced peaks in the 604-608 nm range (Figures 2A,B). The difference in the PL peak emission between the two methods may be a result of biogenic moieties altering the emission spectrum. Earlier work showed that PL emission wavelength of ZnS nanoparticles varied according to the capping agent used during the reaction (Warad et al., 2005;Wanjari et al., 2015).
Buffers, and precursor solutions did not produce any absorbance or PL emission peaks (Supplementary Figure S8). Manganese may react with sulfide to produce manganese sulfide (MnS) precipitate. Some groups have observed PL emission on MnS films near 640 nm, which could explain the slight red shift in the PL peak with increasing Mn(II) concentration (Goede et al., 1986). We do not observe any PL optical signature of MnS nanoparticles in this region (Supplementary Figure S8). This finding leaves Mn:ZnS as the only candidate emitter at Frontiers in Microbiology | www.frontiersin.org 600 nm during PL characterization. Next, we examined the effect of biologically regulated Mn(II) concentration on the PL emission of the nanoparticles. In the chemical method, the PL intensity increased up to 2 mM Mn(II) and subsequently decreased. In the biogenic method, the PL intensity increased up to 100 µM TMAO induction and then decreased with increasing concentrations of TMAO. Recall that TMAO concentrations dictates the concentrations of soluble Mn(II). This observed effect of dopant concentrations on PL emission peak intensityan increase followed by a decrease upon reaching a critical concentration (Figures 2C,D) -is consistent with previous results (Son et al., 2007;Cao et al., 2009). This effect, known as luminesce quenching, is a common feature among doped semiconductors, of which Mn(II) doped ZnS is a classic example (Hurd and King, 1979;Sasakura et al., 1981;Katiyar and Kitai, 1992). Briefly, the probability of an electron indirectly transitioning through a Mn(II) site to the ground state increases with Mn concentration up to 1-4 wt%. As the Mn concentration increases beyond a few percent by weight, however, unfavorable interactions between adjacent Mn(II) sites, interruptions in the ZnS crystallinity, an increase in non-radiative transition processes, and a decrease the in the excitation efficiency from the ZnS lattice diminish the effect of doping. Finally, it is worth noting that where the Mn(II) ions subsume with the nanomaterial dramatically affects its optoelectronic properties. It is known that incorporating Mn(II) on the surface of ZnS quantum dots instead of the bulk results in an ultraviolet PL emission. That our nanomaterial emits in the visible region near 600 nm suggests that the majority of Mn(II) is embedded within the ZnS structure rather than the surface (Xiao and Xiao, 2008). The PL emission intensity was generally higher in chemically synthesized particles, and it is currently unclear if synthesis in the presence of cellular compounds impacts the intensity of the PL emission. Overall, the PL properties of the chemically and biogenically synthesized Mn:ZnS nanomaterials are similar to each other and with literature precedent.
Next, we measured the absorption spectrum for biogenically and chemically synthesized nanomaterials in the 300-700 nm wavelength windows. Figures 2E,F show the absorption spectrum for the nanoparticles is in the UV wavelength regions of 310-320 nm, which agrees with earlier work (Kole and Kumbhakar, 2012). Chemically synthesized nanoparticles had an absorption peak of 310 nm and the biogenic nanoparticles had an absorption peak of 315 nm. From the absorbance, one may find the band gap of the nanomaterials. The band gap energies of nanomaterials made by chemical and biogenic methods are 4.0 eV and 3.9 eV respectively. These values are higher than for bulk ZnS material, which is 3.7 eV. An inverse relationship exists between the size of the nanoparticle and its bandgap due to quantum mechanical confinement, i.e., the system resembles a particle-in-a-box. The bandgap is the energetic difference between the ground state and excited state (the bands) of the electron in an atom or bulk material. A single atom possesses a large bandgap because the allowed electron states are precisely defined. That is, the band is extremely narrow and the gap between bands (the bandgap) is large. A continuous, bulk material, however, is composed of many, overlapping electron orbitals. The effect of overlapping increases the band width with a concomitant decrease between the ground and excited state of electrons in the material. As a result, the band is wider and the gap between bands decreases (Son et al., 2007;Begum and Chattopadhyay, 2014;Marusak et al., 2016).
Crystalline Structure Analysis of ZnS and Mn:ZnS Nanomaterials
Next, we characterized the crystalline structure of Mn:ZnS synthesized by the chemical and biogenic methods via XRD, an X-ray scattering technique which measures coherent diffraction from crystalline domains within nanomaterials across an entire sample. The XRD pattern indicated there were three distinct diffraction peaks (28.7 • , 47.9 • , and 56.7 • ) with 2θ values corresponding to three planes (111), (220), and (311). These peaks confirm that the synthesized nanomaterial has a cubic phase of zinc blende consistent with previous reports and zinc sulfide standard (Supplementary Figure S9). By analyzing the XRD data using the Scherrer model (Supplementary Equation S1), one may extract the size of the crystalline domains within the nanomaterials. Briefly, the width of the diffraction peak is inversely proportional to the size of the crystalline domain. For both the chemically and biogenically synthesized nanomaterials, the crystalline domain is between 5 and 8 nm in diameter (Supplementary Table S2), slightly smaller than the particle size derived from optical methods. This is not unexpected, as the geometry and crystallinity of nanoparticles may differ due to the presence of amorphous, X-ray diffusive regions, which XRD cannot detect. Finally, as further evidence that ZnS and Mn:ZnS made the bulk of the nanomaterial, no peaks associated with MnS crystals appear in the XRD pattern of our purified nanomaterials (Figure 3 and Supplementary Figure S9). To determine if the presence of Mn(IV) during synthesis influenced nanoparticle properties, Mn(IV) oxide was added during chemical synthesis. The addition of Mn(IV) oxide did not inhibit the formation of ZnS and ZnS:Mn(II) nanoparticles, see Supplementary Figure S9B. Also, Mn(IV) oxide alone was not sufficient for manganese doping during chemical synthesis, see Supplementary Figure S10. These results are in line with earlier work and show good agreement between the chemical and biogenic route (Son et al., 2007).
Morphology, Elemental Analysis, and Size Distribution of ZnS and Mn:ZnS
Next, we leveraged energy-dispersive X-ray spectroscopy (EDX) measurements to confirm the elemental composition of the nanomaterial. In an EDX experiment, X-ray excitation induces emission spectra unique to specific atomic nuclei in the material, thereby furnishing information about the specific elemental make-up of the sample. The peaks of zinc and sulfur indicate the material formed was zinc sulfide (Supplementary Figures S11, S12) in agreement with the XRD data. The primary peaks in these data correspond to Si and O and likely arise from the sample substrate, a Si 2 wafer. The secondary peaks, however, correspond to Zn, S, and Mn (in doped samples). It is worth noting that the peaks Sizes of nanomaterials synthesized via chemical method and biogenic method using AFM. A minumum of 150 nanoparticles were analyzed (n > 150); particle clusters were omitted. The box indicates the interquartile range, which captures all the data between the first and third quartile. The horizontal line within the box represents the mean or expected value. The bars extending above and below indicate the min and max of the data, excluding outliers. The dots outside the min and max lines represent outliers, which are values 1.5 inner quartile range distance from the inner quartile. The box plot represents data from one replicate of each sample. Comparison of particle sizes using an unpaired Student's t-test revealed that all pairs of measurements were significantly different (p < 0.01) except for biogenic particles at 0 and 1 mM TMAO which had a p > 0.05. are unlikely from left over substrates as the samples were thoroughly washed to remove the initial reactants used for nanomaterial synthesis.
Next, we used scanning electron microscopy (SEM) and AFM to identify the size and shape of the synthesized ZnS and Mn:ZnS nanomaterials. SEM images showed that particles were quasi-spherical ( Figure 4A) in good agreement with earlier work (Warad et al., 2005;Chandrakar et al., 2015). SEM analysis was used for identifying the morphology of the nanoparticles, while AFM was used to quantify the size of the nanoparticles.
We measured the size of the ZnS and Mn:ZnS nanomaterials using AFM. AFM size analysis (Figures 4B,C) reveals that the size of Mn:ZnS nanomaterials synthesized via chemical method was slightly larger and polydisperse compared to nanomaterials synthesized via biogenic method. Researchers have measured the size of chemically synthesized Mn:ZnS nanomaterials in the range of 2-70 nm (Warad et al., 2005;Cao et al., 2009;Kole and Kumbhakar, 2012;Komada et al., 2012). The mean value for the chemically synthesized particles is 8.7 nm [0 mM Mn(II)], 11.5 nm [1 mM Mn(II)] and the mean value for the biogenically synthesized particles is 4.0 nm (0 TMAO, no induction) and 5.0 nm (1 mM TMAO, full induction). The difference in size and polydisperity may result from different reaction kinetics between the chemical and biogenic methods, an advantage in nanomaterial sysnthesis that merits further investigating. Altogether, EDX, SEM, and AFM reveal that the nanomaterials synthesized via chemical and biogenic routes were pure, spherical, and approximately 2-20 nm.
CONCLUSION
We synthesized manganese doped zinc sulfide (Mn:ZnS) nanoparticles using chemical and biogenic methods. Mn:ZnS nanoparticles have applications as field emission materials, field effect transistors (FETs), p-type conductors, biosensors, chemical sensors, and catalysts, and nanogenerator (Fang et al., 2011). Previous studies have shown that biogenic Mn:ZnS nanoparticles exhibit biocompatibility and lesser toxicity in biomedical imaging applications as compared to chemically synthesized nanoparticles (Hazra et al., 2013). Here, both methods yielded nanoparticles with a characteristic PL emission peak at 600 nm, although in general the PL emission spectrum of Mn:ZnS nanomaterials is known to vary with synthesis method (Cao et al., 2009;Deng et al., 2011;Komada et al., 2012;Ali et al., 2016).
To optimize the biogenic synthesis process, experimental design should consider the location of nanoparticle synthesis. For example, nanomaterials produced by bacteria may be nucleated and grown in the cytoplasmic, periplasmic, or extracellular space. These three regions, especially the cytoplasm and periplasm, contain a variety of biomolecular moieties, each of which influence synthesis. Moreover, harvesting nanomaterials from the interior of the cell may require lysing cells, which can introduce additional postproduction modifications to the nanomaterials, like capping. Designing biogenetic nanomaterial synthesis routes with optimal or minimal biomolecular blends should therefore consider focusing nanomaterials synthesis on/outside the cell. Following this line of reasoning Marusak et al. (2016) explored the relationship between stages of bacterial cell growth of E. coli on the synthesis of the CdS nanoparticles. By adding CdCl 2 to E. coli cultures after an initial 10-h growth period, CdS nanomaterial formation was largely extracellular, which reduced doping by non-specific agents. Likewise, in our system, because the MtrCAB protein complex extends from the periplasmic to extracellular space, we suspect that ZnS and Mn:ZnS nanomaterials originate outside the cell. Subsequent work will explore the origin and control of nanomaterial nucleation/growth.
Although this work focuses on Mn(II) as a dopant, the feasibility of incorporating additional dopant(s) through biogenic routes should be investigated. Other groups have reported chemically synthesizing ZnS with dopants as varied as nickel, cadmium, and copper (Biswas et al., 2006;Fang et al., 2011). Although the cell culture itself is reducing, additional control experiments shown in Supplementary Figure S13 show that mtr expression and activity were needed for manganese doping. Removal of the carbon source during the manganese reduction step was not sufficient to completely eliminate the reducing activity of cells expressing mtr. Future work should look at the wide variety of available cytochromes and their capability to reduce many transition metals, lanthanides, and actinides (Lloyd, 2003). These starting materials could also be incorporated into biogenically derived nanomaterials.
As a complement to doping, tuning the size of the nanomaterial offers a parallel and complementary level of control over the optoelectronic properties. Particle size of the manganese doped nanoparticles zinc sulfide slightly varies according to the synthesis method, which is not unexpected since each method will have specific nucleating factors and reaction kinetics which influence the size and growth of the nanomaterials. Capping agents from various sources such as plants, fungi, and bacteria cells have been used to control the size and nucleation of nanomaterials (Singh et al., 2011;Wanjari et al., 2015;Hussain et al., 2016). In our work, cellular biomolecules in the exo-, peri-, or cytoplasm may have played a role in determining the size and geometry of nanomaterials. For example, biofilm surfactins of many microbes are known to alter nanoparticle size (Singh et al., 2011(Singh et al., , 2014Rodrigues, 2015). As in controlled Mn doping, synthetic gene circuits regulating genes involved in production and secretion of biological capping agents could control nanomaterial morphology. Such biological control over multiple facets of nanoparticle synthesis may produce nanomaterials unavailable via chemical methods. Realizing the potential of biogenic nanomaterial synthesis will benefit from future developments in both synthetic gene circuits and an increased understanding of how microbes influence nanomaterial formation.
AUTHOR CONTRIBUTIONS
PC, KN, KS, JB, and ME-N contributed to the conception and design of the study. SP and MC performed some characterization and experiments. PC wrote the first draft of the manuscript. PC, KN, and JB wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version. | v3-fos-license |
2018-12-20T23:05:50.520Z | 2012-04-01T00:00:00.000 | 67757601 | {
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} | pes2o/s2orc | An Efficient Multipollutant System for Measuring Real-World Emissions from Stationary and Mobile Sources
ABSTRACT A portable dilution sampling and measurement system was developed for measuring multipollutant emissions from stationary and mobile sources under real-world operating conditions. This system draws a sample of exhaust gas from the source, dilutes it with filtered air and quantifies total volatile organic compounds (VOCs), carbon monoxide (CO), carbon dioxide (CO2), nitric oxide (NO), nitrogen dioxide (NO2), sulfur dioxide (SO2), oxygen (O2), particle size distribution, particle number and mass concentrations, and black carbon (BC) concentration at 1–6 sec interval. Integrated samples by canisters and filter packs are acquired for laboratory analyses of VOC speciation, particle mass concentration, light absorption, elements, isotopes, ions, ammonia (NH3), hydrogen sulfide (H2S), sulfur dioxide (SO2), carbon, and organic compounds. Experiments were carried out to evaluate this system. The accuracy of key real-time instruments were found to deviate < ± 12% from references. CO2 was used as the tracer gas to verify the concentration uniformity in the three measurement modules and relative concentration difference was < 5.1%. Instrument response time was tested by emissions from lighting and burning matches. The DustTrak DRX and optical particle counter (OPC) had the fastest response time, while other instruments had 3.5–21.5 sec delay from the DustTrak DRX and OPC. This system was applied to measure emissions from burning pine logs in a wood stove. The real-time data showed flaming, transition, and smoldering phases, and allowed real-time emission ratios to be calculated. Combing real-time data and laboratory analysis, this measurement system allows the development of multipollutant emission factors and source profiles.
INTRODUCTION
Real-world emissions represent effluents of multipollutant mixtures as they would appear soon after exiting the source, cooling, and equilibrating to ambient conditions (Cadle et al., 2009;Chow and Watson, 2011).Thousands of emission tests are made each year on stationary and mobile sources throughout the world for certification and compliance purposes, but these tests are of limited use for estimating real-world emission rates and chemical compositions (Chow and Watson, 2008).
The most common approach to determine particulate matter (PM) emissions from stacks for compliance uses Method 5 (Elder et al., 1981;U.S.EPA, 1991) for total suspended particles (TSP) and Method 201A/202 for PM 2.5 and PM 10 (particles with aerodynamic diameters less than 2.5 µm and 10 µm, respectively) (U.S. EPA, 1996EPA, , 1997)).Method 5 was adapted into two methods for testing Emissions are sampled through a constant volume sampling (CVS) or partial-flow dilution (PFD) system (e.g., Chien et al., 2009;Wu et al., 2010).The test cycles are intended to represent real-world operating patterns, but real-world emissions are more variable (Sawyer et al., 2000;Yanowitz et al., 2000;Cocker et al., 2004).Despite their recognized limitations, a limited number of certification measurements are often the only ones available for constructing emission estimates (Chow, 2001;Lloyd and Cackette, 2001).
To achieve a more realistic representation of actual PM emissions and chemical compositions from stationary sources, dilution sampling system are designed to simulate the diluting, cooling, and aging of the hot exhaust under similar conditions when the plume discharge to the atmosphere (Hildemann et al., 1989;Lipsky and Robinson, 2005;England et al., 2007a;Li et al., 2011).The American Society for Testing and Materials (ASTM; 2010) is developing a dilution sampling guidance for stationary source certification that better reconciles the current discrepancy between stationary or mobile source emissions and ambient PM measurements.The Emission Measurement Center of the U.S. EPA Office of Air Quality Planning and Standards (OAQPS) is also developing a Federal Reference Method (FRM) to better characterize source emissions of both filterable and condensable PM based on the dilution sampling method (Myers and Logan, 2002).The U.S. EPA Conditional Test Method (CTM) 039 uses dilution sampling method to measure PM 2.5 and PM 10 (U.S. EPA, 2004).
Portable onboard emission measurement systems (PEMS) have been developed for in-use vehicle emission characterization of pollutants such as non-methane hydrocarbons (NMHC), carbon monoxide (CO), carbon dioxide (CO 2 ), nitrogen oxides (NO x ), and PM (Durbin et al., 2007;Abolhasani et al., 2008;Zhang and Frey, 2008;Johnson et al., 2009).Other species or parameters that are important to climate or health, such as black carbon (BC) and ultrafine particles, are typically not measured.Commercial PEMS are not designed to collect gas or filter samples that would enable measurement of speciated VOCs or PM source profiles needed for speciated emission inventories and source apportionment receptor models.
A portable dilution source sampling and measurement system is described here that allows for direct measurements of real-world multipollutant emissions.This system harmonizes emission measurements for stationary (Watson et al., 2010;Wang et al., 2011) and mobile (Chow et al., 2010a) sources.Data from wood stove combustion, with a focus on continuous measurements throughout the burning cycle, are reported to illustrate how the system functions.
MULTIPOLLUTANT DILUTION SAMPLING AND MEASUREMENT SYSTEM
Fig. 1 illustrates the multipollutant dilution sampling and measurement system.A portion of the source effluent is drawn from a ducted or not-ducted plume, diluted and mixed with filtered air at near-ambient temperature, then directed to different monitors.Continuous monitors, described in Table 1, measure VOCs (aromatics), CO, CO 2 , NO, NO 2 , SO 2 , O 2 , PM size distribution, PM number, PM mass, and PM BC at 1 to 6 sec intervals.Time-integrated samples are collected in parallel with canisters, absorbing substrates, and filters for later laboratory analysis of speciated VOCs, NH 3 , SO 2 , and H 2 S gases.Off-line PM analyses include mass, multiwavelength light absorption (b abs ), elements, isotopes, water-soluble ions, organic carbon (OC), elemental carbon (EC), and specific organic compounds including polycyclic aromatic hydrocarbons (PAHs) (Chow, 1995;Chow and Watson, 2012).
This system is modularized and packaged into five modules (Figs.2(a-e)) to provide for: 1) sample conditioning; 2) continuous gas monitoring; 3) integrated gas and particle sampling; 4) continuous PM monitoring; and 5) system power.Instruments are selected that operate on 12 V or 24 V so that batteries or power supplies (when line power is available) can be easily procured at or near the measurement location.The modular nature of the system allows for quick replacement of the monitors as newer and more sensitive technology becomes available.
A Method 5 buttonhook nozzle and probe is used to extract effluent from a duct and drawn to the sample conditioning module (Fig. 2(a)) through a heated (to ~5°C above the sample temperature) or insulated tube to minimize vapor condensation and thermophoretic particle losses.The sample is rapidly diluted with clean air generated by a compressor (Model 107CDC20, Thomas Pump & Machinery, Sheboygan, WI) and filtered by activated carbon and a high efficiency particulate air (HEPA) filter.An elutriator (Fig. 2(f)) enhances mixing between the sample and dilution flows (England et al., 2007a, b) by introducing the undiluted sample flow through a center tube surrounded by 96 1.6 mm holes through which the dilution air is directed.The diluted sample passes into a 1.5 liter (L) residence chamber where gases and particles can equilibrate to reach a stable particle size distribution (Chang et al., 2004).The aging time at the flow rate of 28.7 L/min in Fig. 1 is ~3 sec, similar to the system described by Lipsky and Robinson (2005) who estimated that the equilibrium time is < 2 sec when the total particle surface area is greater than 0.01 m 2 /m 3 .A larger-volume residence chamber can be substituted when longer residence times or higher flow rates are deemed necessary.The sample stream then passes through a cyclone (Model URG-2000-30ENG, URG Corporation, Chapel Hill, NC) to remove particles larger than ~7 µm at the specified flow rates.Three separate flow streams are drawn into the continuous and integrated sample modules (i.e., Figs.2(b-d)) for quantification using inert Teflon tubing and a Teflon-membrane filter for the gas monitors and conductive silicone tubing for the PM monitors.
Three nondispersive infrared (NDIR) CO 2 analyzers measure CO 2 concentrations in the undiluted exhaust, diluted sample, and dilution flow (background) every 1.5 sec.The instantaneous dilution ratio (DR) is calculated as (Ning and Sioutas, 2010): The one-liter canister flow rate is controlled by a critical orifice.CO, CO 2 , and methane (CH 4 ) are analyzed by gas chromatograph with flame ionization detector (GC-FID), and NMHC (C 2 -C 11 ) are analyzed by GC-mass spectrometry (MS)-FID (U.S.EPA, 1999).Since different filter media are needed for comprehensive chemical speciation (Chow et al., 2008;Watson and Chow, 2011), four filter packs are sampled in parallel (Chow and Watson, 2012), with a typical configuration shown in Fig. 1.Each filter pack is preceded by a PM 2.5 impactor (Model 202-100, Airmetrics, Eugene, OR).The flow rate (5 L/min) through each filter pack is controlled by a feedback loop between the pump (Model B2736, BGI Inc., Waltham, MA) speed and flow meter (Model 41221, TSI Inc.) readings.Additional sampling media, such as a 2,4-Dinitrophenylhydrazine (DNPH) cartridge for carbonyls (Fung and Grosjean, 1981), a Tenax cartridge for heavy hydrocarbons (Zielinska et al., 1986;1994b;Zielinska and Fujita, 1994a), or filter media for additional PM speciation can be added to this module.
The continuous PM monitoring module (Fig. 2(d)) contains a DustTrak DRX aerosol monitor, which is a combination of nephelometer and optical particle counter (OPC) and simultaneously measures PM 1 , PM 2.5 , PM 4 , PM 10 and PM 15 mass concentrations (Wang et al., 2009).The condensation particle counter (CPC) measures the total number concentration of particles larger than 10 nm.Since the maximum concentration that the CPC can measure without coincidence losses is ~100,000 particle/cm 3 , a dilution bridge similar to the leaky filter method (Whitby et al., 1972) is added upstream to enable the measurement of high concentrations; the dilution ratio is measured before and after each test.The OPC measures particle size distributions in the optical diameter range of 0.3-25 µm in 15 channels based on single particle light scattering.The (Hansen and Mocnik, 2010).The power supply module (Fig. 2(e)) contains two 12 V deep cycle marine batteries (Model Odyssey PC2150S, EnerSys Energy Products Inc., Reading, PA), a voltage regulator (Model N8XJK, TG Electronics, Houghton, MI) that stabilizes the output voltage at 13.8 V, and a battery monitor (Model TM-2020, Bogart Engineering, Boulder Creek, CA) that monitors the battery output voltage, current, and battery level.These batteries can also supply 24 V when connected in series, with 12 V drawn from each battery.
Voltage regulators are used to match battery output to what is needed by each instrument.The total current is about 16 Amps (A) when all instruments are running, which allows for ~9 hours of operation with two 12 V batteries.
Data from all instruments are sent to the data acquisition computer in digital format via RS232 or USB communication in real time.A LabView (National Instruments, Austin, TX) program controls instruments and records data.
This design has benefited from and improved upon prior dilution sampling efforts (Hildemann et al., 1989;Lipsky and Robinson, 2005;England et al., 2007a) by: 1) multipollutant flexibility to meet different emission characterization needs; 2) a suite of continuous monitors that can be examined during the test while providing more information on variation during an emitter's operational cycle; 3) integrated samples that can obtain hundreds of pollutant concentrations through laboratory analysis and provide quality control redundancy for the continuous monitors; 4) modularization for convenient shipping, handling, and installation; and 5) off-line power source with commonly-available batteries.The sample conditioning module (Fig. 2(a)) described here may not adequately measure PM from clean effluents because the 1.5 L residence chamber only allows a ~3 sec residence time at a flow of 28.7 L/min.This residence time is sufficient for diesel engines without a diesel particulate filters (DPF), wood stoves, and coal and residual oil boilers (Lipsky and Robinson, 2005).Well-controlled natural gas combustion, however, contains low primary PM surface area on which vapors might condense, and longer residence time are needed with a larger volume residence chamber (Chang et al., 2004;England et al., 2007a, b).The air compressor has a maximum capacity of ~35 L/min at a pressure of 5 psi, which limits the amount of dilution and sample flow rates.A larger capacity compressor, usually requiring line power, would be substituted for higher dilution flows.Longer sampling durations of several hours would be needed for the 5 L/min filter pack flow rates to obtain sufficient sample for laboratory characterization.Samples containing high water vapor contents require higher temperatures for the transfer line, requiring more battery power which reduces the 9 hour operation time.The system described has been adapted for these purposes, but this adaptation obviates some of the advantages conferred by its portability and self-contained power source.
SYSTEM EVALUATION
The multipollutant dilution sampling and measurement system has been evaluated using laboratory-simulated concentrations and wood combustion for: 1) individual analyzer accuracy, 2) concentration uniformity before splitting to different measurement modules, 3) response time differences, and 4) variability of continuous gas and particle emissions during wood combustion.
Continuous gas and particle monitoring instruments are initially calibrated by manufacturers and need to be periodically verified with laboratory standards.The CO 2 analyzers were tested with 2% and 10% CO 2 calibration gas; the photoionization detector (PID) analyzer was verified with 100 ppm isobutylene calibration gas; and the Testo Emission Analyzer (EA) was tested with a CO, NO, and SO 2 calibration mixture.Desired concentrations were achieved by mixing calibration gases with scrubbed zero air through a gas divider (Model SGD-710C, Horiba Instruments, Irvine, CA).The TSI CPC 3007 was compared to a TSI CPC 3010 for ambient aerosol concentrations at different dilution levels.As shown in Fig. 3, instruments showed linear responses to reference concentrations, with linear regression (forced through origin) slopes typically within 1.00 ± 0.10.Raw instrument readings are divided by the regression slope to adjust for the small deviation from 1.00.PM mass concentrations by optical methods need to be calibrated by simultaneous gravimetric measurements of the same aerosol.The DustTrak DRX was zeroed with filtered air before each run, while the OPC conducted a self-test before each run to check performance integrity.The BC concentration by the micro-aethalometer depends on the mass absorption efficiency of the light absorbing particles (Chow et al., 2010b).Comparison between the BC by aethalometer and elemental carbon (EC) by the thermal/ optical carbon analysis (Chow et al., 1993(Chow et al., , 2007(Chow et al., , 2011) ) can be used to evaluate the mass absorption efficiency for specific sources.
CO 2 was measured in the different modules (Figs.2(b-d)) to verify the mixing of the sample and dilution flows.To test this, the sampling line was connected to a 7% CO 2 balanced with air standard and the dilution ratio was varied from 5 to 34 by changing the make-up flow rate.Table 2 shows the average CO 2 concentrations at each dilution ratio.Deviations relative to the mean were < 5.1%, indicating a uniform distribution to each module.
Particle losses were estimated using laboratory-generated polystyrene latex spheres (PSL) with 0.5 to 10 µm diameters through a 25 L residence chamber with a 100 L/min flow rate and a 15 sec residence time without the ~7 µm cut-point cyclone.These tests showed ~100% transmission efficiency for 0.5-5 µm PSL and 86% for 10 µm PSL.This is a worstcase scenario compared to the smaller residence chamber in Fig. 2(a) because both the gravitational deposition parameter (residence time divided by tube diameter) and diffusion deposition parameter (flow length divided by flow rate) (Kulkarni et al., 2011) are smaller than those for the 25 L chamber.This evaluation does not include losses in the sample transfer line from the particle source to the inlet of the dilution system which can be estimated from theoretical calculation of diffusion, gravimetric settling, and thermophoretic deposition based on the flow rate, length, temperatures, and inclination angle of the transfer line for a specific experimental setup (Kulkarni et al., 2011).
The response of real-time continuous instruments is delayed by the time it takes to travel from the inlet probe to the instrument inlet and by the travel time through the sensing volume within the instrument, which may vary from test to test depending on the length and diameter of the transfer line and the flow rate.These differences need to be accounted for to synchronize the real-time data so that the differences in pollutant release time from the source can be examined.The time lags are measured before and after a test by sampling smoke from a safety match lit at the inlet, as the combustion generates most gas species and particles of interest.The system first samples ambient air for a few minutes to obtain background readings.Then one match is lit near the inlet of the sampling probe.After the first match is extinguished, the system samples ambient air for a few more minutes to allow the instrument signals to return to background levels, after which a second match is lit.Fig. 4 illustrates instrument responses.In this test, the DustTrak DRX and OPC had the fastest responses, as illustrated by the vertical dash line running across all panels.Table 3 lists the delays for each instrument to detect a 10% change from background concentration and the lag time with respect to the DustTrak DRX.Most instruments experienced 10-22 sec lags, with the exception of the CPC (3.5 sec) and micro-aethalometer (7.5 sec).The delay of 20.5-21.5 sec for VOCs and CO may depend on chemical reactions as well as transport and detection (Hays et al., 2005).The CO sensor is in series with other Emission Analyzer sensors hence its transport and detection delay time is likely to be comparable (9-15 sec).Since the response times change with flow rates, plumbing configuration, and emission characteristics, they must be quantified for every test configuration.Continuous instrument responses can also be aligned using initial rise time, peaks, fall time, or other techniques such as autocorrelation of measurement data.This alignment is performed during post-sampling data processing.Fig. 3. Performance verification of the CO 2 analyzers, photoionization detector (PID), Emission Analyzer (EA), and condensation particle counter (CPC).The two tests shown for the EA and CPC were conducted before and after a field campaign.The regression for the undiluted CO 2 analyzer was for concentration up to 100,000 ppm CO 2 (data points above 20,000 ppm are not shown).
, where x i is the concentration measured by one of the three modules, and x is average concentration of all three modules at a give dilution ratio.The delay time for SO 2 sensor from Match 2 was not included in the average owing to a near-detection limit response.
A wood stove testing experiment was carried out to illustrate how the system can be used to understand an emissions process.One kilogram (kg) of pinewood logs (2.5 cm × 2.5 cm × 33 cm) was combusted in a Pineridge wood stove.A stream of the exhaust was extracted from the chimney and diluted with an average dilution ratio of 10.3:1.In parallel with the measurement modules in Fig. 1, a scanning mobility particle sizer (SMPS, Model 3936L10, TSI Inc.) measured undiluted particle size distributions in the sub-micron size range (0.01-0.29 µm) every 135 sec.The experiment started by measuring ambient background concentrations.The fire was ignited at the 12:12:50 time mark by lighting a small pile of thin woodchips underneath the pinewood with a propane torch.As shown in Fig. 5, sharp increases in all pollutant concentrations and a decrease in the O 2 concentration were observed.The transition from the woodchip kindling to the pinewood combustion shows substantial emission variability prior to the pinewood firing state.Flaming, transition, and smoldering for the pinewood burn can be distinguished from the traces in Fig. 5, as noted by the vertical dashed lines.The average concentrations in each burning stage are summarized in Table 4. Phases of biomass combustion have been described by others (Lobert and Warnatz, 1993;Koppmann et al., 2005;Calvo et al., 2011), but transition from one phase to another is usually not distinct and criteria have not been set for defining each phase.Several studies have used the modified combustion efficiency (MCE), defined as: to separate combustion phases: MCE > ~0.9 for flaming phase and MCE < ~0.85 for smoldering phase (e.g., Koppmann et al., 2005;Reid et al., 2005b).The variety of continuous measurements offers the possibility of greater precision in distinguishing among the different wood combustion phases.In the flaming phase, CO 2 and particle concentrations were relatively stable, while total VOCs, CO, and NO increased.The MCE decreased to ~0.9 during this period, indicating the combustion became less efficient when volatile fuel components near the wood surface were burned (Chen et al., 2007).Because the stove was cold at the initiation of the experiment and the pinewood pieces were too large for complete combustion, the fire entered a transition from flaming to smoldering after ~15 minutes.During this transition period, the CO 2 concentration declined, while CO and NO levels increased, resulting in declining MCE.Total VOCs, particle number, BC, and PM 2.5 mass concentrations were relatively stable during this period.Average total VOCs in this phase were 22% higher than during the flaming phase, BC was 9% lower, while particle number and PM 2.5 differed by < 8%.Around 12:45:05, the fire entered the smoldering phase, and all pollutant concentrations decreased when the MCE reached a minimum of ~0.80.A gradual increase of MCE from the second half of the smoldering phase has been observed in earlier studies (Hays et al., 2005;Chen et al., 2007;Hosseini et al., 2010) and is probably due to the decrease of CO concentrations as the smoldering temperature decreases toward the end of the burn.The BC/PM 2.5 from the microaethalometer and DustTrak DRX decreased continuously from the beginning of the transition phase, indicating a decrease in the fraction of PM that absorbs light at 880 nm.This is consistent with earlier observations that soot particles are mostly formed in the flaming phase due to insufficient O 2 in poorly mixed areas while in the smoldering phase particles are largely formed by volatile organics condensation (Reid et al., 2005a).
Table 5 shows correlations among concentrations during the wood burning test with selected parameters plotted in Fig. 6.The highest squared correlations (r 2 = 0.96) was observed between the number concentration and PM 2.5 mass concentration by the OPC, which is expected since the OPC PM 2.5 mass concentrations was calculated from number concentration.The PM 2.5 mass concentrations by the OPC and DustTrak DRX were also well correlated (r 2 = 0.78); both instruments are based on light scattering principle.However, as shown in Fig. 6(a), the linear regression slope was only 0.03, indicating the OPC readings were ~30 times lower than the DustTrak DRX.This difference is partially because the OPC PM 2.5 concentration was calculated from particle number concentrations by assuming that particles were spheres with unit density, while the DustTrak DRX derived PM 2.5 concentrations from photometric signal Fig. 5. Time series of instrument responses to wood stove emission: a) total VOCs; b) CO; c) undiluted CO 2 ; d) diluted CO 2 ; e) background CO 2 ; f) modified combustion efficiency (MCE); g) NO; h) O 2 ; i) particle number by CPC and OPC; j) black carbon; k) PM 2.5 mass by the DustTrak DRX and OPC; and l) ratio of BC to PM 2.5 (by DustTrak DRX).Response time differences among instruments were corrected by aligning the sharp rising concentrations when the fire was ignited.Gas and particle concentrations except CO 2 were corrected for dilution.SO 2 and NO 2 were near detection limits and are not plotted.Vertical dash lines separate different burning phases, and the arrows on the panel k point to the center time of the particle size distribution scan shown in Fig. 7. (Wang et al., 2009).Furthermore, the OPC experienced coincidence losses for concentrations above ~6 mg/m 3 as indicated by the decreasing slope in Fig. 6(a) at higher concentrations.
The OPC also has a lower size cut-off at ~0.3 µm, and many particles were at or below this size range.The mass concentration measured by the Teflon-membrane filter (Fig. 2(c)) can be used to adjust mass concentrations by the DustTrak DRX and OPC.Fig. 6(b) shows r 2 = 0.79 for total VOCs and CO, both formed from incomplete combustion due to low combustion temperatures, an insufficient air supply, or poor mixing of fuel and air.On the other hand, BC, another product of incomplete combustion, was poorly correlated with CO (r 2 = 0.09).As shown in Figs. 5 and 6(c), in the flaming and smoldering phase, BC and CO had fair correlations but with different slopes and intercepts, probably due to different formation mechanisms.In the flaming phase, both BC and CO form in a fuel-rich flame, while in the smoldering phase, BC and CO form due to low fire temperatures.In the transition phase, they were uncorrelated when CO continued to increase while BC was relatively stable.BC was better correlated with CO 2 (r 2 = 0.59) than with CO.
The continuous emission measurement permits calculation of emission ratios (ER) for different burning phases as listed in Table 4.The emission ratio is defined as (Andreae et al., 1998;Koppmann et al., 2005): where [X] plume and [X] background are the measured concentrations in the plume and background, respectively.Except for PM 2.5 , ERs varied for different burning phases.This observation underlines the importance of using continuous data for estimating ERs, especially when X is poorly correlated with CO 2 (Andreae, 2001).distributions provide useful information on particle evolution over the burn period.Consistent with Figs.5(i-k), the ambient background concentration was orders of magnitude lower than the plume levels.At the early stage of ignition, particle sizes peaked ~0.026 µm, smaller than during later burning phases.Since particle concentrations were relatively low at this phase and there were not many large particles in the plume, these particles had lower coagulation efficiencies and were not quickly scavenged.The count median diameter (CMD) in the ignition phase was 0.02-0.04µm.In the flaming phase, particles were larger (CMD = 0.04-0.06µm) and concentrations were higher with particles present in both nuclei (~0.005-0.1 µm) and accumulation (~0.1-2.5 µm) modes.In the transition phase, particles in the nuclei mode decreased and more particles appear in the accumulation mode, with CMD of 0.06-0.09µm.The shape of the size distribution in the smoldering phase was similar to that in the transition mode (CMD = 0.05-0.09µm), but with lower concentrations.This observation of size distribution change from flaming to smoldering phase is similar to that observed during wheat straw combustion (Hays et al., 2005).Maruf Hossain and Park (2011) observed a decrease of mode diameter from ~0.18 µm to ~0.09 µm from flaming to smoldering combustion of rice straw.They controlled the combustion so that only flaming or smoldering predominated, which differs from the natural transition from flaming to smoldering in this study.Particle sizes observed in this experiment are in the same range as a recent study using fast scan mobility analyzers (1 sec time resolution), but are somewhat smaller than most studies summarized by Reid et al. (2005b).Differences in fuel and combustion conditions contribute to these discrepancies.
CONCLUSIONS
A portable dilution sampling and measurement system was developed to measure multipollutant emissions from stationary and mobile sources.In addition to the criteria pollutants (i.e., CO, NO x , SO 2 , and PM), additional pollutants (e.g., total VOCs, CO 2 , BC, ultrafine particle number concentration, and particle size distributions) are measured continuously.Integrated canister and filter samples allow thorough analysis of VOC speciation and PM chemical compositions.This system enables measurement of continuous emission characteristics and development of multipollutant emission factors and source profiles under real-world conditions.Key component continuous instruments were evaluated in the laboratory for their accuracy.Instruments responses agree with reference concentrations with ± 12% deviation and excellent correlation (r 2 > 0.995).
The uniformity of species concentrations delivered to the three measurement modules was evaluated using CO 2 as the tracer gas.The relative error was found to be < 5.1% indicating uniform mixing.Differences in time responses of individual detectors were evaluated by measuring emissions from lighting and burning matches.It was found that the DustTrak DRX and OPC had the fastest responses to aerosol concentration changes, while other instruments showed 3.5-21.5sec delay.
This system was tested for measuring emissions from burning pinewood logs in a wood stove.Three burning phases: flaming, transition, and smoldering were observed from the emission time series.A reasonable squared correlation (r 2 = 0.78) was found between the PM 2.5 mass concentrations measured by the DustTrak DRX and OPC.However, the DustTrak concentration was ~30 times higher than the OPC, underlining the necessity of calibrating mass concentrations by optical instruments with gravimetric methods.Good correlation was also found between VOCs and CO (r 2 = 0.79) during the entire burn.Correlations between species and the modified combustion efficiency were generally poor.Continuous emission data allows evaluation of emission ratios in different burning phases, which will lead to more accurate estimation of emission inventories.Size distribution measurement of wood combustion showed that most particles are smaller than 0.2 µm, with count median diameter (CMD) 0.02-0.04 in ignition phase, 0.04-0.06µm in flaming phase, 0.06-0.09µm in transition phase, and 0.05-0.09 in smoldering phase.
Fig. 1 .
Fig. 1.Schematic diagram of the multipollutant dilution sampling and measurement system.The listed flow rates are for operation with a dilution ratio of 40.The dilution ratio can be adjusted by changing the dilution and make-up flows.
Fig. 2 .
Fig. 2. Photograph of the component modules of the multipollutant dilution sampling and measurement system: a) Module 1-Sample Conditioning; b) Module 2-Continuous Gas Monitoring; c) Module 3-Integrated Sampling; d) Module 4-Continuous PM Monitoring; e) Module 5-Power Supply; and f) Elutriator dilution air/sample mixer (Part of the Sample Conditioning Module).Modules 1-4 have the same dimensions (L × W × H) of 80 cm × 52 cm × 32 cm, and Module 5 has dimensions of 55 cm × 42 cm × 32 cm.Modules 1-4 weighs approximately 25 kg each, and the power supply module with two batteries weighs 80 kg.The total system weight is about 180 kg.
Fig. 4 .
Fig. 4. Time series of instrument responses to lighting and burning two matches: a) total VOCs by the PID analyzer; b) CO by the Emission Analyzer (EA); c) undiluted CO 2 by the CO 2 analyzer; d) diluted CO 2 by the CO 2 analyzer and the EA; e) background CO 2 by the CO 2 analyzer; f) NO and SO 2 by the EA; g) O 2 by the EA; h) particle number by the CPC (~0.01-2.5 µm) and OPC (0.3-2.5 µm); i) black carbon by the micro-aethalometer; and j) PM 2.5 mass by the DustTrak DRX and the OPC.The two vertical dash lines indicate the instant when the DustTrak DRX and OPC detected concentration change after lighting the match.
Fig. 7 .
Fig. 7. Snapshots of particle size distribution during different stages of the wood burning experiment.Time stamp corresponds to the two-minute scan period of the scanning mobility particle sizer (SMPS).The centers of scans are labeled as arrows in Fig. 5(k).
Table 1 .
Description of continuous monitors.
Table 2 .
Comparison of CO 2 concentrations measured by each module (Figs.2(b-d)) for different dilution ratios.
a Relative error (δx) calculated by:
Table 3 .
Detection rise times for match burning tests.
Table 4 .
Average pollutant concentrations and ratios to CO 2 for each pinewood burning phase. | v3-fos-license |
2019-04-06T00:44:51.443Z | 2011-12-20T00:00:00.000 | 98032534 | {
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} | pes2o/s2orc | Millimeterwave Spectral Studies of Propynal ( HCCCHO ) Produced by DC GlowDischarge and Ab Initio DFT Calculation
The ground-state (v = 0) millimeterwave rotational spectrum of propynal (HCCCHO) produced by a DC glow discharge through a low-pressure (∼10–20 mTorr) flow of propargyl alcohol (HCCCH2OH) vapor has been observed in the frequency region: 36.0–94.0 GHz. Measured rotational transition frequencies along with the previously reported microwave and millimeterwave transitions were fitted to a standard asymmetric-top Hamiltonian to determine an improved set of rotational and centrifugal distortion constant. Detailed DFT calculations were also carried out with various functional and basis sets to evaluate the spectroscopic constants, dipole moment, and various structural parameters of propynal and compared with the corresponding experimental values.
Introduction
Propynal (HCCCHO) also known as propiolic aldehyde has been found in interstellar molecular clouds TMC-1 and Sgr B2(N-LMH) [1][2][3].In 1955 Howe and Goldstein [4] reported the J = 2 ← 1 and J = 3 ← 2 microwave transitions of the most abundant isotopic species of propynal.They have determined the dipole moment of the molecule to be μ = 2.46 ± 0.04 D. Later on, Costain and Morton [5] determined the rotational constants B, C, and (A − D k ) for the parent as well as various isotopic species and were able to determine an accurate substitution structure.Winnewisser [6] has extended the analysis of the ground state of propynal to the millimeterwave region (95.0-200.0GHz) which has yielded a set of ground state rotational and centrifugal distortion constants.Reference [6] includes a set of predicted a-type Rbranch frequencies from 37.0 to 94.0 GHz covering J = 4 ← 3 to J = 10 ← 9 transitions which are of prime importance to radio astronomers for conducting search for this molecule in the interstellar space.Infrared-microwave double-resonance spectroscopic study of propynal using a Zeeman tuned He-Xe laser as the infrared source was conducted by Takami and Shimoda [7] to determine the rotational constants of the v 2 = 1 excited vibrational state.Later on, Jones [8] has reported the rotational constants of propynal in the ground as well as several excited state combination and hot bands using a CO 2 laser as the infrared source.In all the previous works propynal was either procured commercially or prepared chemically.
Production of new molecules, molecular ions, and radicals by applying a DC glow discharge through a low-pressure flow of gas or a mixture of gases inside an absorption cell has proved to be an efficient and useful technique [9].Recently, Jaman [10] has reported an analysis of the millimeterwave rotational spectra of propyne (CH 3 CCH) produced by a DC glow discharge through a mixture of toluene and acetylene vapor and carried out a detailed DFT calculation to evaluate the spectroscopic constants and molecular parameters and compared them with experimental values.In the present communication, we report the analysis of the ground-state (v = 0) rotational spectra of propynal produced by a DC glow discharge through a low-pressure flow of propargyl alcohol (HCCCH 2 OH) vapor in the frequency region 36.0-94.0GHz.Asymmetric-top K −1 K +1 -structures of different J + 1 ← J transitions which fall under this frequency range have been observed and measured.The measured rotational transition frequencies along with the previously reported frequencies were fitted to standard asymmetric-top Hamiltonian to determine an improved set of rotational and centrifugal distortion (CD) constants.A detailed quantum chemical calculation was also carried out to evaluate the spectroscopic constants, dipole moment, and the structural parameters of propynal.Finally, the experimentally determined rotational and CD constants were compared with the best set of values obtained after a series of DFT calculations.Work on the analysis of the rotational spectrum of propenal (CH 2 CHCHO) produced by DC glow discharge is underway.
Experimental Details
The spectrometer used in the present work is basically a 50 kHz source-modulated system combined with a free space glass discharge cell of 1.5 m in length and 10 cm in diameter.The cell is fitted with two Teflon lenses at each end.A high-voltage DC regulated power supply (6 kV, 1300 mA) procured from Glassman, Japan was used to apply a DC voltage through a flow of low-pressure precursor gases.The cell is connected with a high vacuum pump at one end and to the sample holder section through a glass port on the other.Klystrons and Gunn diodes followed by frequency doubler (Millitech model MUD-15-H23F0 and MUD-10-LF000) have been used as radiation sources.Millimeterwave radiation was fed into the cell by a waveguide horn and Teflon lens.A similar horn and lens arrangement was used to focus the millimeterwave power onto the detector after propagating through the cell.The output frequency of the millimeterwave radiation was frequency modulated by a bidirectional squarewave of 50 kHz [11], and the signal from the detector (Millitech model DBT-15-RP000 and DXP-10-RPFW0) was amplified by a 100 kHz tuned preamplifier and detected by a phase-sensitive lock-in amplifier in the 2f mode.The output of the lock-in amplifier was connected to an oscilloscope or a chart recorder for signal display.The uncertainty in frequency measurement has been estimated to be ±0.15MHz.A block diagram of the spectrometer is shown in Figure 1.Details of the spectrometer used have been described elsewhere [12,13].
Propynal was produced inside the absorption cell by applying a DC glow discharge through a low-pressure (∼5-10 mTorr) flow of propargyl alcohol (HCCCH 2 OH) vapor.The discharge current was maintained at around 5 mA with an applied voltage of 1.0 kV.A mechanical on/off type discharge was found to be suitable to observe good signals of propynal.A controlled flow of liquid nitrogen vapor was used to cool the cell during the experiment.The observed signals of propynal appeared as sharp lines immediately after the DC discharge was applied but started losing intensity with time.Application of fresh discharge helps in regaining the previous intensity.
Computational Method
Quantum chemical computations were performed using GAUSSIAN 03 package [14].Density functional methods with various functionals were used to calculate the structural parameters, dipole moment, and total energy (sum of electronic and zero point energy) as well as the rotational and centrifugal distortion constants of propynal.The geometry
Rotational Spectrum and Analysis
The rotational spectrum of propynal was predicted in the frequency range 36.0-94.0GHz using the rotational and centrifugal distortion constants reported earlier [6].Accordingly, J = 4 ← 3 to J = 10 ← 9 transitions along with their different K −1 K +1 components were predicted and compared with some of the predictions given in [6].Different K −1 K +1components in each series were measured.Finally, 145 aand b-type R-and Q-branch transitions consisting of all previous microwave [5], millimeterwave [6], and present data were fit to the semirigid rotor Watson's S-reduction Hamiltonian (I r -representation) [16] to determine an improved set of spectroscopic constants including five quartic (D J , D JK , D K , etc.) and three sextic (H JK , H KJ , and h 1 ) centrifugal distortion constants.Being a light molecule (mol.wt.54), the shifts in frequency of the absorption lines from their rigid rotor positions due to centrifugal distortion were found to be substantial for the higher J and K −1 transitions.The maximum shift was found to be 1049.55MHz for 19 (14, 6) -18 (14,5) transition.The assigned new transitions and those reported by earlier workers [5,6] are listed in Table 1.The inclusion of sextic constants H JK , H KJ , and h 1 were found to be necessary to reproduce the observed frequencies with high J, high K −1 millimeterwave transitions.The contribution of the sextic distortion constant H J was found to be too small to be determined.All the ground state spectroscopic constants obtained for propynal using this "global" fit are listed in Table 2.A typographical error for the CD constant d 2 (R 5 ) = −19.500± 1.59 kHz in [6] has been noticed.The value of d 2 in [6] should have been either −0.0195 kHz or −19.500Hz.The new value of d 2 in our case comes out to be −0.022± 0.002 kHz.It is quite evident from Table 2 that the standard deviations of most of the parameters have come down with this analysis as compared to their respective values reported in [6].The agreement between the derived set of spectroscopic constants and those obtained earlier [5,6] with commercial samples indicates that the newly assigned transition frequencies of Table 1 definitely belong to propynal, a discharge product of propargyl alcohol.Figure 2 shows the observed trace of the K −1 = 3 doublet of J = 9 ← 8 transition immediately after the DC discharge was applied.The trace remained visible for a couple of minutes on the oscilloscope screen with gradually diminishing intensity.
Computational Results
Propynal is a slightly asymmetric prolate top molecule (κ = −0.989).The optimization of geometry for propynal has been tested at various levels of theory and basis sets.The number and labeling of propynal molecule is shown in Figure 3. Bond lengths and angle have been computed and are shown in Table 4.
Conclusion
An efficient method of generating propiolic aldehyde (propynal) in the gas phase by applying a DC glow discharge through a low-pressure vapor of propargyl alcohol inside the absorption cell has been presented.The gas-phase rotational spectra of propynal produced in this way have been recorded and analyzed in the frequency range 36.0-94.0GHz.The asymmetric-top K −1 K +1 -components of different transitions having J values 3 to 9 have been measured.The observed transition frequencies along with the previously reported data [5,6] were fitted to a standard asymmetric-top Watson's S-reduction Hamiltonian (I r -representation) to determine an improved set of rotational and centrifugal distortion constants and compared with the previously reported values of propynal obtained using a commercial sample.A good agreement between the two sets of values confirms that propynal is one of the major discharge products of the propargyl alcohol vapor.To compare the experimental results with theory, DFT calculations were performed using various models and basis sets.However, it was found that B3LYP model with 6-311++g (d, 2p) basis set produced the best values of rotational and quartic centrifugal distortion constants which are close to the experimental values.
Figure 1 :
Figure 1: Block diagram of source modulated millimeterwave spectrometer with DC discharge facility.
cFigure 2 :
Figure 2: Optimized geometry of propynal molecule and the numbering of atoms.
Table 1 :
Microwave and millimeterwave rotational transition frequencies of propynal (HCCCHO) in the ground vibrational state (in MHz.).
b Standard deviation of the overall fit.
Table 3 :
Calculated ground-state rotational constants of propynal (HCCCHO) in various models and basis sets.
f This work.
Table 4 :
Comparison of the molecular optimized geometry, dipole moment, and total energy of propynal calculated by various methods and basis sets with the experimental values.
Table 3 .
The calculated values of rotational and centrifugal distortion constants obtained with DFT B3LYP/ 6-311++g(d, 2p) have been compared with the corresponding experimental values in Table2.The calculated energy for the optimized geometry of propynal is 5188.185eV, and the dipole moment at the optimized geometry is 3.074 D.
d, 2p) also provides the values of spectroscopic constants which are close to the experimental values.But in the later model percentage error of constant A is slightly greater than the previous one.Hence we have chosen B3LYP/6-311++g(d, 2p) configuration for comparing different molecular constants with our experimental values.Results obtained with various models and basis sets are shown in | v3-fos-license |
2019-04-02T13:13:13.270Z | 2018-02-24T00:00:00.000 | 90571519 | {
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} | pes2o/s2orc | In Vitro Studies of Antifungal Activity of Colloidal Silver against Important Plants Pathogens
Colloids and especially silver are increasingly used in a variety of worldwide applications because of their potential antimicrobial activity and their plasmotic and conductivity properties. This research reports the fungitoxic properties of colloidal silver on mycelial growth of important plant pathogens: Alternaria brassicicola, Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Fusarium culmorum, Fusarium oxysporum, Penicillium digitatum and Sclerotinia sclerotiorum. Although variable responses towards each compound were observed within the species the results revealed a clear reaction to limiting mycelium growth relative to various concentration of Colloidal silver (CS). Results were expressed as effective concentrations which inhibit mycelial growth by 50% and 90% respectively (EC50 and EC90). Efficiency of colloidal silver on mycelial growth inhibition of different isolates based on EC50 have the following values: 3.69 ppm for Alternaria brassicicola, 7.32 ppm for Botrytis cinerea, 18.21 ppm for Aspergillus flavus, 10.43 ppm for Aspergillus niger, 11.99 ppm for Fusarium culmorum, 12.27 ppm for Fusarium oxysporum, 10.82 ppm for Penicillium digitatum and 6.34 ppm for Sclerotinia. According to the obtained results the antifungal activity of colloidal silver particles as biocide has potential for using it as a non-aggressive treatment in horticulture and sustainable horticulture.
Introduction
The presence of silver in community health life dates back to the eighteenth century when silver nitrate (AgNO3) was used to treat ulcerations (Klasen, 2000).One hundred years later, the antimicrobial features of silver ions are officially acknowledged by FDA's decision to accept using silver colloids in the management of burns (1920) (Moore and Payne 2004).Nowadays, there are clinical trials covering the colloidal silver's biocidal action although standards on application procedures are scarce and the use of silver colloids as natural biocidal product in plant cropping is a brand-new orientation in the field of integrated crop (Aktar et al., 2009).Silver nanoparticles come second in the international line of international research in agriculture after emulsions/lipids and polymers (Gogos et al., 2012).
Attempts are made in the field of integrated disease and pest management to replace pesticides with natural biocides that harm pest, restrict diseases and lead to the preservation of useful fauna and flora.The research studies conducted so far follow the line outlined by the concern for the use of natural biocides to preserve biodiversity and mitigate the impact of fungicide resistance some pathogens are increasingly demonstrating (Frac Code List, 2018;Iacomi-Vasilescu et al., 2004).
In vitro studies on pathogens and on the antibacterial and antifungal activity of silver colloids are few despite their undeniable growth-inhibiting action and are generally placed in the field of nanotechnologies.Silver nanoparticles turned out to be efficient in inhibiting the mycelial growth (more than 90%) of Sclerotium cepivorum which is responsible for the white rot of onion, at a concentration of 7 ppm (Jung et al., 2010).
Assay on mycelium
Agar disks (8 mm in diameter) were cut from the margin of a 7-day-old colony growing on PDA and were transferred to PDA medium supplemented with the CS at final concentrations 3.75, 7.5 and 15 ppm.Three replicates were used per treatment.For each active ingredient and concentration, inhibition of radial growth (product efficacy) compared with the untreated control was calculated after 7 days of incubation at 24 °C, in the dark.Results were expressed as effective inhibitory concentration EC50 and EC90 (the concentration which reduced mycelial growth by 50% and 90% respectively) determined by regressing the inhibition of radial growth values (% control) against the values of the tested product concentrations.
In vitro effects of colloidal silver on mycelial growth
Craft colloidal silver (CCS) fully inhibited (100%) the mycelial growth of Sclerotinia sclerotiorum at a concentration level of 7.5 ppm.As for the isolates of Alternaria brassicicola, Botrytis cinerea, full inhibition of mycelial growth was only witnesses at 15 ppm concentrations.It is worth noticing that Alternaria brassicicola (Abra 43) colonies demonstrated distinctive lysis signs, concentric ring growth and colour variations against the control sample in the presence of colloidal silver (Fig. 1).The conidia examined under the microscope in the presence of 15 ppm CCS revealed visible changes in the spore cellular membrane and impact on their melanisation (Fig. 2).
A 76.19% inhibition of the mycelial growth on the Penicillium digitatum was noticed at 16 ppm.The two Fusarium isolates studied demonstrated a similar behaviour in the presence of CCS as the mycelial growth was inhibited by 72.22% at 15 ppm.The Aspergillus flavus and Aspergillus niger isolates were less sensitive in the presence of CCS, with a normal growth of colonies at 3.75 and 7.5 ppm concentration levels.The mycelial groth inhibition was exclusively noticed at 15 ppm (40.48% for Aspergillus flavus and 57.14% for Aspergillus niger).
Medicer Colloidal Silver (MCS) fully inhibited (100%) the mycelial growth of Sclerotinia sclerotiorum isolate at a concentration level of 15 ppm.Its efficacy reached 57.14% with the concentration going down to 7.5 ppm (Table 2).It will be noted that this pathogen was sensitive towards both colloidal products tested, efficacy going up to 100% at concentrations of 15 ppm.Nevertheless, CCS fully inhibited the mycelial growth of Sclerotinia sclerotiorum and also reached 100% efficacy at 7.5 ppm (Fig. 3).
MCS also proved its efficacy in inhibiting the mycelial growth of Botrytis cinerea with values reaching 75.73% and 94.17% at concentration levels of 7.5 and 15 ppm respectively.Lower efficacy was demonstrated on other test isolates (between 31.31% and 36.36%),even at a maximum concentration of 15 ppm.
Growth of colonies from Aspergilius si Penicillium isolates in the presence of MCS was normal, which highlighted the tested product's lack of biocidal action.
The effect of silver nanoparticles treatments on seed borne fungi of cucumber was tested (Ziedan and Moataza, 2016).It was noticed that mycelial growth of Alternaria alternata and Fusarium oxysporum in the presence of silver nanoparticles was fully inhibited at concentration levels of 10 ppm and 15 ppm respectively.The same study reveals that pre-emerging treatments based on nano-silver solutions fully annihilated Aspergillus flavus, Fusarium oxysporum and Tricoderma spp. off the cucumber seeds.
The in vitro mycelial growth of Corynespora cassicola, Cylindrocarpon destructans and Alternaria solani, as well as of Fusarium graminearum, was also inhibited from a concentration of 50 ppm (Kim et al., 2012) and 20 ppm (Soltanloo et al., 2010) respectively.
Matters related to plasmatic changes driven by silver ion absorption, inhibition or non-inhibition of evolving processes, as well as the remanence in the fruit for feed species are yet to be studied.
Under the said circumstances, the study aimed at highlighting the biological action of colloidal silver in inhibiting certain plant pathogens, being as far as we know amongst the first studies of this kind to have ever been conducted in Romania.
Materials and Methods
Research studies were conducted in the Phytopathology Laboratory, Plant Science Department, Faculty of Agriculture, University of Agronomic Sciences and Veterinary Medicine Bucharest.
Biological material Fungal isolates and growth conditions
The fungi used in this study are listed in Table 1.All the strains were purified by monospore isolation and maintained on malt agar medium (malt extract 20 g, agar 20 g in 1 L distilled water) at 4 °C.Fresh subcultures were made by transferring hyphal plugs to potato dextrose agar (PDA) medium to obtain inocula for sensitivity tests.Six Romanian fungal isolates were obtained from commercial radish seed lots (Aspergillus flavus -Af, Aspergillus niger -An 1), vegetables (Botrytis cinerea -Bc1 from tomatoes fruits, Sclerotinia sclerotiorum-Ss1 and Fusarium oxysporum -F1 from cucumbers fruits) and lemons (Penicillium digitatum -Pd) and identified using standard criteria, based on colony/conidiophores and conidia morphology.Two from our eight tested isolates, Alternaria brassicicola -Abra 43 and Fusarium culmorum -F065 were provided by Université d'Angers, France (IRHS Fungisem).
Antimicrobials
The tested compounds were Medicer colloidal silver (MCS) -as water with colloidal silver, as food supplement on market, the manufacturer's listed ingredients being CS 20 mg, 20 ppm concentration in 1 l water, 100% reverseosmosis pure water and Craft Colloidal Silver (CCS), product which was obtained using a device bought from the US based on 999 fineness silver bars, demineralized and deionized water, maximum concentration of 20 ppm l -1 .
Test isolate sensitivity towards the two CS-based compounds was also expressed with the help of indicators EC50 and EC90 (Table 3).Test isolates sensitivity varied by species and product used.The Alternaria brassicicola isolate turned out to be the most sensitive towards CCS, with EC50 and EC90 values of 3.69 ppm and 11.8 ppm respectively, followed by Sclerotinia sclerotiorum with 6.34 ppm and 11.88 ppm and by Botrytis cinereal with values of 7.32 ppm and 12.43 ppm respectively.Botrytis cinerea (with EC50 and EC90 values of 6.58 ppm and 13.19 ppm respectively) and Sclerotinia sclerotiorum (with EC50 and EC90 values of 7.9 ppm and 13.20 ppm respectively) showed most sensitivity to MCS.
Discussion
This research aimed at pointing to the fungitoxic properties of colloidal silver on important plant pathogens for horticulture.Test isolates were selected according to their major impact on cultures, their presence during vegetation or post-harvesting period, and their transmission through infected seeds or soil.Fusarium mycelium representatives (Fusarium graminearum, Fusarium culmorum) are known for their aggressive attacks and their likeliness to generate mycotoxins -highly-dangerous secondary metabolites to be found in harvests and finite products (Semple et al., 1989).
Our results highlight the fungitoxic action of colloidal silver against most test isolates, with efficacy gaps being identified between Craft Colloidal Silver (CCS) and Medicer Colloidal Silver (MCS).
As far as we know, studies on the antimicrobial action of silver colloids against fungal plant pathogens are few, there 536 being a lack of direct concern about the use of colloidal silver as natural biocide.Some studies underline the fungal/fungitoxic action of silver nanoparticles against plant pathogens, whether seedborne or found in the vegetation.
The results of our studies point to CCS efficacy in the complete mycelial growth inhibition (100%) of Sclerotinia sclerotiorum (isolated from Cucumis sativus fruits), Alternaria brassicicola (isolated from Raphanus sativus seeds) and Botrytis cinerea (isolated from Lycopersicum esculentum fruits).We believe these results are the first to reveal the fungal action of CS against fungal; plant pathogens, for current studies only tackle the biocide potential of silver nanoparticles (Jung et al., 2010;Soltanloo et al., 2010;Lamsal et al., 2011;Kim et al., 2012;Ouda, 2014;Ziedan and Moataza, 2016).
Studies are being carried out to identify the CS action mechanism and its integration in the program for the protection of sustainable horticultural crops, as treatments to be applied on seeds, during the vegetation and the post- harvest period.First signs of CS treatment efficacy have been identified in controlling Pseudomonas syringae pv.actinidiae, the causal agent of bacterial canker of kiwi fruit (Drummond, 2011) or of silver nanoparticles efficacy in controlling pathogens of citrus fruits -Alternaria alternata and Penicillium digitatum (Salaheldin, 2016).Anthracnose attacks (Colletotrichum spp.) on paprika crops (Lamsal et al., 2011) or mildew attacks (Pseudoperonospora cubensis) on protected cucumber crops (Alavi and Dehpour, 2010) could also be prevented with silver nanoparticles.These results open new paths to studies in connection with the occurrence of new fungicide generations.
The impact of silver nanoparticles as biostimulators for seed germination and plant growth was highlighted.In this context, using CS to treat the seed against seed-borne pathogens may be an alternative to the traditional treatment.Preliminary results show that applying CCS 10 ppm on artificially-contaminated radish seeds decreased the incidence and symptoms severity of Alternaria brassicicola (data not shown).
Conclusions
According to our results, CS is highly-effective in inhibiting the mycelial growth of Alternaria brassicicola, Botrytis cinerea -two pathogens with already reported resistant isolates to active molecules used in current protection programs and Sclerotinia sclerotiorum, for which the chemical control is still a challenge.Studies currently in progress open up the possibility to include CS and other colloids in the protection programs for sustainable horticultural crops due to their antifungal properties.As for their use as plant protection products, risk parameters such as bioaccumulation, toxicity and remanence as yet to be determined.
Fig. 1 .
Fig. 1.Colony and spores morphology of Alternaria brassicicola on PDA from control and CCS 15 ppm
Table 1 .
Fungal isolates used in experiments
Table 2 .
Effects of colloidal silver on the mycelial growth
Table 3 .
Responses of fungal isolates to colloidal silver | v3-fos-license |
2020-03-19T10:53:14.675Z | 2020-03-12T00:00:00.000 | 214597314 | {
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} | pes2o/s2orc | In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert
With the purpose of discovering new anticancer molecules that might have fewer side effects or reduce resistance to current antitumor drugs, a bioprospecting study of the microalgae of the Cuatro Cienegas Basin (CCB), an oasis in the Chihuahuan desert in Mexico was conducted. A microalgae was identified as Granulocystopsis sp. through sequencing the rbcL gene and reconstruction of a phylogenetic tree, and its anticancer activities were assessed using various in vitro assays and different cell lines of human cancers, including lung, skin melanoma, colorectal, breast and prostatic cancers, as well as a normal cell line. The values of IC50 of the microalgae methanolic extract using the MTT assay were lower than 20 μg/ml, except that in the lung cancer line and the normal cell line. In vitro, the microalgae extract caused the loss of membrane integrity, monitored by the trypan blue exclusion test and exhibited marked inhibition of adhesion and cell proliferation in cancer cell lines, through the evaluation of the clonogenic assay. Also, typical nuclear changes of apoptotic processes were observed under the microscope, using the dual acridine orange/ethidium bromide fluorescent staining. Finally, the microalgae extract increased the activity of caspases 3 and 7 in skin melanoma, colon, breast and prostate cancer cells, in the same way as the apoptotic inductor and powerful antitumoral drug, doxorubicin. This study shows the anticancer activity from Granulocystopsis sp., a microalgae isolated from the CCB.
INTRODUCTION
Cancer is one of the most important causes of death worldwide and is continuously stimulating the search for new bioactive molecules from natural sources. There is an urgent need of new anticancer drugs because tumor cells are developing resistance against currently available drugs, like vinca alkaloids and taxanes (Singh et al., 2011) and some
Sampling and isolation of microalgae strain Chu2
Microalgae specimen was hand collected at the intermediate Lagoon in the Churince hydrological system (2 50.830′ N 10 09.335′ W), located in CCB, Coahuila, México during period between February and July 2016 under SEMARNAT scientific permit No. SGPA/DGVS/03121/15. For isolation of microorganisms, the sample (fresh water) was homogenized in sterile water and aliquots were placed on Petri dishes containing agar based media: BG-11 (17.6 mm NaNO 3 , 0.23 mm K 2 HPO 4 , 0.3 mm MgSO 4 ·7H 2 O, 0.24 mm CaCl 2 ·2H 2 O, 0.031 mm Citric Acid·H 2 O, 0.021 mm Ferric Ammonium Citrate, 0.0027 mm Na 2 EDTA·2H 2 O, 0.19 mm Na 2 CO 3 ) supplemented with carbenicillin 50 µg/mL. Purity of strain was resolved by sequential restrikes onto new agar plates and a pure strain named Chu2 (Churince strain n 2) was inoculated in liquid BG-11 medium for culture maintenance and up-scaled growth. Cultures were kept in a climate chamber at 20 C in a 16:8 h light:dark cycle, 70% of relative humidity and 100 µmol photons m −2 s −1 .
Microalgae morphology
The microalgae Chu2 was observed using the light microscope Olympus BX-53 equipped with phase contrast and a Qimaging camera (model Micropublisher 3.3 RTV) and Q-capture pro 7 software. The morphological identification was performed using the keys for the members of the Phylum Chlorophyta (John & Tsarenko, 2011).
Molecular identification of Chu2 microalgae
Genomic DNA was extracted and used to amplify rbcL (rubisco gene) ( Table 1). The rbcL gene was chosen because it is encoded by the chloroplast genome and is considered a housekeeping gene, and therefore conserved and appropiate for family and genus level phylogenetics. PCR reactions were exposed to the following profile: 35 cycles of denaturation (94 C for 1 min), primer annealing (55 C for 1 min), and extension (72 C for 2 min). The PCR products were ligated into pCR TM 4-TOPO Ò (ThermoFisher Scientific, Waltham, MA, USA) to generate plasmids that were sequenced by LANBAMA-IPICYT, Mexico (Table 2).
Phylogenetic reconstruction
The rbcL sequences were assembled using CodonCode Aligner 5.1 software (CodonCode Corporation, Dedham, MA, USA). The resulting contigs were aligned in Bioedit to build a consensus sequence. The resulting sequence was aligned in the NCBI database (http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST) in order to identify the closest related sequences at genus-level affiliations to the Chu2 microalgae rbcL gene (GenBank MH370163). After BLAST analysis of the sequenced gene, (Kumar, Stecher & Tamura, 2016). The model selection was performed using statistical and evolutionary analysis of multiple sequence alignments TOPALi v2 (Milne et al., 2009). To construct the phylogenetic tree from the genus of Oocystaceae family, the Maximum-likelihood (ML) method was used with MEGA software v. 7 (Kumar, Stecher & Tamura, 2016) and the Generalized time-reversible GTR+G parameter as an evolutionary model. The nodes reliability was estimated using the ML bootstrap percentages obtained after 1,000 replications (Felsenstein, 1985). Bootstrap values for ML in the range from 0.7 to 1 were marked with black rhombus.
Preparation of microalgae extract
Pure cultures were inoculated in Erlenmeyer flasks with one L of fresh media (BG-11) and incubated at 25 C, under 16 h day/8 h dark cycle, in a bioclimate chamber for 2-3 weeks. The resulting media was spun down to separate the microalgae biomass from the broth. Biomass was extracted with MeOH 1:1 (m/v) (Sigma-Aldrich, St. Louis, MO, USA) for 5 days. The crude extracts were evaporated under vacuum at 50 C (Yamato RE801) to remove methanol residues. For the cytotoxicity assays, the dried methanol extract was dissolved in dimethylsulfoxide (DMSO) to obtain a final concentration of 100 mg/mL (stock) and diluted in 1Â PBS.
Cell lines and cell culture
Cell lines were cultured in RPMI or DMEM with FBS (10% v/v). The cell culture was performed in an incubator at 37 C and 5% CO 2 to ensure growth and viability. The tumor (breast (HTB-22), colorectal (HTB-38), skin melanoma (HTB-72) and prostate (HTB-81)) and Vero normal cell (CCL-81), were purchased from the American Type Culture Collection (ATCC).
Cytotoxicity assay
Cytotoxicity effects were determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assays, as previously described by Van Meerloo, Kaspers & Cloos (2011). After incubation for 24 h, cells were treated with various concentrations of Granulocystopsis sp. extract and incubated for 48 h. An MTT solution (5 mg/mL) was added to each well and further incubated for 4 h at 37 C. A medium supplemented with DMSO was used as a control. Doxorubicin (10 µg/mL) treated cells and untreated cells were used as positive control and negative control, respectively. IC 50 were calculated for each cancer cell line using the equations previously reported, plotting a linear regression curve and using the same in succeeding assays (Eskandani, Hamishehkar & Ezzati Nazhad Dolatabadi, 2014). Each concentration of the algal extract was independently assayed three times with three technical replicates.
Trypan blue exclusion test of cell viability
Different cancer cell lines were grown for 24 h. Subsequently, the cells were exposed to the microalgae extract at the concentration corresponding to their IC 50 and cell viability was evaluated at 12, 24, 36 and 48 h. After 48 h of treatment, the medium was replaced with fresh medium (without extract) and the cells were cultured for an additional 12 h and 24 h. Trypan blue test was used for the viability assay (Strober, 2015). Human cancer and normal cell lines were used without treatment, as negative control. Five technical replicates were performed for each of the three independent experiments.
Clonogenic assay of cell in vitro
Culture dishes were seeded with 100-110 cells and incubated for 24 h in order to perform the clonogenic assay as previously described (Rafehi et al., 2011). Subsequently, the cells were exposed to Granulocystopsis sp. extract for 48 h. After treatment, a medium without microalgal extract was added, and cells were cultured for 2 weeks. To determine the number of colonies per plate, the cultures were stained and analyzed using ImageJ software (Collins, 2007) and progenitor frequencies expressed as the total number of colonies obtained per 100 cells seeded. Three independent experiments were performed with three technical replicates each.
Cell morphology and adhesion assay in vitro
Cell attachment assay was carried out with some modifications (Xia, Wang & Kang, 2005). Briefly, 5 × 10 5 cells were treated with Granulocystopsis sp. extract for 48 h in a 6-well plate and then were detached and plated back on a new culture plate. After each incubation period of 6-24 h, the cell attachment status and morphology were observed, and photographs were captured by camera infinity 1-2, Luminera. As a control, cells were cultivated in the same plate without the microalgae extract.
Dual acridine orange/ethidium bromide fluorescent staining
The AO/EB double staining assay was performed as previously described (Cohen, 1993). Briefly, melanoma and prostate cancer cells were treated with Granulocystopsis sp. extract for 48 h, trypsinized and stained with AO/EB dye. A Nikon TS100 microscope was used to see and examine the cell suspensions at 400× magnifications. Results were expressed as means ± SE for three independent determinations.
Caspase assay
Cells were seeded, treated with Granulocystopsis sp. methanol extract at their respective IC 50 values, and incubated for 48 h. Caspases activity was then determined using Caspase-3/7 Fluorescence Assay Kit (Cayman cat. No. 10009135) (Martinotti, Ranzato & Burlando, 2018) according to the manufacturer's instructions. Three independent experiments were performed with three technical replicates each.
Statistical data analysis
Data from the clonogenic assay, caspase activity and AO/EB staining, were expressed as the mean ± SEM from three experiments and GraphPad Prism 7 software was used to perform Students t-Test or one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons. The significance level was set at p < 0.05.
Identification of the microalgae strain Chu2
The Chu2 microalgae isolated from in the now extinct Churince hydrological system in CCB, Coahuila, México, was examined by microscopy and it was found to be a Chlorophyta. The cells are ellipsoidal with pointed apices, granular appearance, parietal chloroplast with a pyrenoid, 10-12-micron size, with two cells or multiples of 2 (up to 8) within an expanded lemon-shaped mother cell wall (Fig. 1). Because these characteristics are present in some of the members of the Oocystaceae family, the Chu2 rbcL gene was amplified with two pairs of primers (Table 1), cloned (Table 2), sequenced and used to construct the phylogenetic tree from the genus of Oocystaceae family in order to identify the closest related homologs in genus-level affiliations to the Chu2 microalgae. Phylogenetic analysis provided the confirmation that the isolate Chu2 belonged to a member of Granulocystopsis genus (Fig. 2), and the isolate was designated as Granulocystopsis sp. (Chlorellales: Oocystaceae).
Cytotoxic activity of Granulocystopsis sp. extract on different human cancer cell lines
To evaluate the cytotoxic properties of Granulocystopsis sp. methanol crude extract, an MTT assay was performed on five human carcinoma cell cultures: lung, prostate, breast, colorectal and skin melanoma. The cytotoxic activity of the microalgae extracts is shown in Table 3. The Granulocystopsis sp. extract induced strong cytotoxicity in four cancer cell lines (<20 mg/mL), prostate cancer cells showing striking sensitivity to treatment with the microalgae extract (IC 50 , 13.74 ± 2.06 µg/mL; Table 3). Interestingly, the Granulocystopsis sp. extract had no cytotoxic effect on the lung cancer cell line. For that reason, the lung cancer cell line was discarded in the next stage of experiments. The U.S. National Cancer Institute (NCI) has established three groups of crude extracts from natural sources according to their degree of cytotoxicity: inactive (IC 50 > 100 mg/mL), moderately active (IC 50 20-100 mg/mL) and active (IC 50 < 20 mg/mL) (Skehan et al., 1990). The IC 50 of Granulocystopsis sp. microalgae extract on the four cancer lines was less than 20 mg/mL, so the extract is "active" according to the NCI, but also is three times less active in the healthy Vero cell line, showing a slight differential effect between tumor and normal cells.
Viability (time-dependent) in cells exposed to the extract of Granulocysptopsis sp The trypan blue test was performed to determine changes in the viability of each cell line after being exposed to the Granulocytostopsis sp. extract with respect to the time. The assay was performed during 48 h of treatment and 24 h of recovery time after treatment. Interestingly, the greatest decrease in the viability in prostate cells was observed between 0 and 12 h of treatment, between 12 and 24 h of treatment in those of breast cancer and between 24 and 36 h of treatment in those of melanoma and colon. Each cell line responds differently to the extract although the viability of all the cell lines decreased in a time-dependent manner during the treatment with the microalgae extract. The melanoma, colorectal, and prostate cancer cells showed 70-90% of viability after 24 h of treatment, but breast cells reached only 55% of viability over the same time. After 48 h of treatment, the melanoma, colorectal, and prostate cancer cells showed decreased viability to below 50%, whereas the viability of Vero cells just decreased to 85% (Fig. 3).
When 48 h of treatment ended, the cells were incubated with fresh media and monitored for (Fig. 3). Again, the Granulocystopsis sp. extract appears to have a cytotoxic and selective effect against prostate, breast, melanoma and colon cancer cells, but with lesser effects on the viability of normal Vero cells.
Effect of Granulocystopsis sp. extract on the proliferation of tumor cell lines It was investigated whether the microalgae extract could affect the proliferative activity (the ability to form a colony from a single cell), using the clonogenic assay. In the four cancer cell lines treated with microalgal extract, a significant proliferation inhibition was observed (Figs. 4D, 4F, 4H and 4J). The tumor cells treated with the microalgae extract reduced the ability to form colonies by at least 50%, whilst the healthy cell line (Vero) just by 20% (Fig. 4K). According to these results, the Granulocystopsis sp. extract has the potential to inhibit the formation of twice tumor colonies in vitro, compared to normal cells.
Effect of Granulocystopsis sp. extract on cell adhesion and morphology of human cancer cells
The effect of Granulocystopsis sp. extract on cell adhesion and cell morphology was evaluated by detaching the cells treated with the microalgae extract and plating them in a new plate with fresh medium (extract free). Cells that do not attach to the plate are rounded. Figure 5 shows the level of adhesion and cell morphology between prostate, melanoma, colorectal and breast cancer cell lines with or without the microalgae extract in an interval of 24 h. Vero cells were used as a normal cell. Cells without the extract changed their morphology from round to flattened and adhered to the plate 6 h after incubation (Figs. 5J, 5R and 5HH), reaching almost 100% confluence after 24 h of incubation
Granulocystopsis sp. extract and apoptosis in human cancer cell lines
To determine whether the cell adhesion, cytotoxic activity and inhibition of cell proliferation by the microalgae extract were due to the induction of apoptosis, the AO/EB staining was assessed to detect nuclear changes and apoptotic body formation. The proapoptotic activity of Granulocystopsis sp. extract was investigated with respect to nuclear condensation of cells by fluorescence microscopy. Fluorescence microscopy images clearly showed nuclear changes such as chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in the skin melanoma and prostate cancer cell lines treated with Granulocystopsis sp. extract by 48 h (Figs. 6C and 6D). Caspase-3 and -7 activities in cancer cell lines treated with Granulocystopsis sp. extract Caspases are members of the aspartate-specific cysteinyl protease family and are involved in the regulation of apoptosis and inflammation (Kaufmann et al., 1993). Therefore, to corroborate apoptosis induction by Granulocystopsis sp. crude extract on the cancer cell lines, caspase-3 and -7 were measured. Figure 7 shows that the activity of caspases 3 and 7 was increased twice in the tumor cells treated with the Granulocystopsis sp. extract, compared to untreated cancer cells. On the other hand, in Vero (normal) cells, the positive control treated with doxorubicin showed a higher activation than Vero cells treated with microalgae extract. No differences in caspase activity were observed between cancer cells treated with doxorubicin and those treated with the microalgae extract.
Together, these experiments strongly support the conclusion that Granulocystopsis sp. extract has cytotoxic activity induced by apoptotic activation mediated by caspases 3 and/or 7.
DISCUSSION
In the last three decades, more than 50,000 natural products have been discovered from marine microorganisms, many of them with biomedical applications (Newman & Cragg, 2012;Wiese et al., 2009). Analysis of molecules produced by aquatic organisms has shown that microalgae synthesize a large number of compounds with different biotechnological applications, including those with anticancer activity. Cyanobacteria, diatoms and chlorophytes are an emerging source for the discovery of new drugs because they are organisms that grow in under-explored extreme environments. In an attempt to discover new anticancer molecules that may have fewer side effects or reduce resistance to current anticancer drugs, a bioprospecting study of microalgae from CCB, an hyper-diverse oasis in the Chihuahuan desert in Mexico was conducted. A microalgae (strain Chu2) was isolated from the Churince lagoon, and its microscopic morphology coincided with a member of the Oocystaceae family. The molecular identification of the microalgae was carried out using the rbcL gene (which encodes RuBisCO, a fundamental enzyme in the process of photosynthesis), according to the recommendation of the Consortium Barcode Of Life for the identification of photosynthetic organisms (CBOL Plant Working Group, 2009). The DNA sequence was analyzed using BLAST, showing 100% coverage and percent identity with the rbcL gene previously reported for Granulocystopsis coronata. This information was confirmed by a phylogenetic analysis with other members of the Oocystaceae family. Granulocystopsis is a genus of freshwater microalgae from the Oocystaceae family with 6 names of species taxonomically accepted: G. calyptrata, G. coronata, G. decorata, G. elegans, G. reticulata and G. subcoronata (John & Tsarenko, 2011). However, research papers about this genus are limited to its taxonomy and there are no reports about its biotechnological potential. Although the most abundant photosynthetic aquatic microorganisms reported in CCB are cyanobacteria and diatoms (Pajares et al., 2012;Winsborough, Theriot & Czarnecki, 2009), the Churince lagoon used to have several green microalgae, an unexplored group of organisms which, like the Chu2 strain (identified as Granulocystopsis sp.), are adapted to live in oligotrophic conditions, possibly by modifying their metabolism and generating molecules with possible cytotoxic activity against fast-growing eukaryotic organisms in order to avoid competition and obtain phosphorous and nitrogen from the lysed cells in their surroundings. This selective cytotoxicity may explain why they target the fast-growing cancer cells in skin melanoma, colorectal, breast, and prostate cancer without damaging normal cells.
Interestingly, in the cell lines evaluated, the IC 50 value obtained was from 13.74 mg/mL to 17.44 mg/mL, whereas normal cells treated with the microalgae extract showed an IC 50 value of 57.02 mg/mL (three times higher than cancer cells). This result revealed that Granulocystopsis sp. extracts have cytotoxic activity which might be helpful in preventing the cancer's progress, especially when it is compared against the activity of other extracts of isolated microalgae from Mexico, such as, Chlorella sorokiniana (IC 50 460 mg/mL) and Scenedesmus sp. (IC 50 362 mg/mL) against lymphoma cells (Reyna-Martinez et al., 2018), or other microalgal extracts from Alexandrium minutum (IC 50 > 50 mg/mL) against melanoma cells (Lauritano et al., 2016), Haematococcus pluvialis (IC 50 27-72 mg/mL) against colon, breast and hepatocellular carcinome (El-Baz et al., 2018), Dunaliella salina (IC 50 > 400 mg/mL) against neuroblastoma cells (Atasever-Arslan et al., 2015), Scenedesmus obliquus (IC 50 24-93 mg/mL) against colon, hepatocelullar and breast cancer cells (Marrez et al., 2019) and Chloromonas reticulata (IC 50 > 50 mg/mL) (Suh et al., 2019) and Micractinium sp. (IC 50 100 mg/mL) against colon cancer cells (Suh et al., 2018). Additionally, it was corroborated that the microalgae extract has a cytotoxic effect at the level of membrane integrity, using the trypan blue vital dye, which is excluded by an intact cell membrane (Strober, 2015). When the cancer cell lines were treated for 2 days in the presence of microalgae extract, the capability to recover the viability decreased significantly, while the healthy cell line recovered 100% viability 12 h after removal of the extract. These results suggest that the extract of Granulocystopsis sp. affects the viability of cancer cells in a time-dependent manner and probably could have tumor-specific activity with minor side effects for normal cells.
The ineffectiveness of currently available treatments is mainly due to the invasive and metastatic properties of malignant cancer cells (Lee et al., 2011). Proliferation and cell adhesion are crucial steps that play a significant role in cancer progression and metastasis. The metastatic spread is determined by the cell-cell interactions of cancer cells with endothelium, due to their ability to adhere strongly before they can colonize and establish a secondary tumor in a new place (Chambers, Groom & MacDonald, 2002). Data obtained from the clonogenic assay, the adhesion and cell morphology tests, showed that extract of Granulocystopsis sp. reduced the ability of cancer cells to form colonies and decreased the attachment ability compared to untreated cells. These results suggest a potential antimetastatic activity of Granulocystopsis sp. extract, which could be evaluated through migration and cell invasion assays and elucidate possible action mechanisms where some cytoskeleton components were involved. Apoptosis is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme-dependent biochemical processes. The result is the clearance of cells from the body, with minimal damage to surrounding tissues and it is the mechanism facilitating the action of many chemotherapeutic drugs. Failure of apoptosis and the resultant accumulation of damaged cells in the body can lead to malignant transformation and result in various forms of cancer (D'Arcy, 2019). One technique used to visualize the early and late stages of apoptosis is AO/EB fluorescent staining (Ribble et al., 2005). Our results showed that the microalgae extract activated the apoptosis mechanism in tumor lines. Interestingly, the microalgae extract induced the same level of cells in early and late apoptosis with respect to the anti-cancer compound doxorubicin, suggesting that the extract might contain a more potent compound or a mixture of compounds working in synergy, and therefore, further analyses are required for chromatographic separation and identification of active compounds by NMR, mass spectrometry, etc.
The initiation of apoptosis is dependent on the activation of a series of cysteine-aspartic proteases known as caspases (Shi, 2002). Caspases can be divided into caspase-8 and -9 (initiator caspases) and caspase-3 and -7 (executioner caspases). Both initiator caspases can activate the caspase-3 or -7, which are mainly responsible for the final stages of apoptosis, which consist of chromatin segregation, nuclear condensation, and finally DNA fragmentation (Pojarova et al., 2007;Yang et al., 2006). Our results showed that apoptosis occurred in melanoma, prostate, colorectal and breast cancer cells treated with microalgal extract, activating caspase-3 and -7, which were increased manifold over the basal level of untreated cells. Again, the level of activation of caspases was similar among the cancer cells treated with the extract and the compound doxorubicin, which strengthens our proposal for the extract of Granulocystopsis sp. as a good candidate as an anti-cancer drug, which can promote apoptosis in cancer cells via the mitochondrial-dependent intrinsic pathways. The intrinsic pathway can be triggered by irradiation, oxidative stress, hypoxia or cytotoxic drugs (Jan & Chaudhry, 2019). To discover signal transduction involved in triggering apoptosis mediated extract Granulocystopsis sp., detection of intracellular reactive oxygen species level, analysis of mitochondrial membrane potential and Western blotting analysis are required to establish the mechanisms of action of the extract and the participation of Bax/Bak (pro-apoptotic protein inserted into mitochondrial membrane), Bcl-2 (inhibits production of cytochrome c), Cytochrome c (released into the cytosol), Caspase-9 (induced by cytochrome c), and other pro-apoptotic proteins from the intrinsic pathway like Smac/Diablo, Apaf-1, among others, leading to the activation of caspase-3. Because there are studies that confirm the participation of polyphenols in the induction of apoptosis in tumor cells (Sharif et al., 2010;Walter et al., 2010), more experiments are required to demonstrate if any phenolic compound present in the Granulocystopsis sp. extract could be initiating the transduction signal from the intrinsic pathway.
Based on our results, the microalgal extract may be useful for the future development of anti-metastatic therapeutic agents. The current research aimed at the description of the molecular mechanisms of the anticancer properties of the microalgae extract, as well as the elucidation of the bioactive molecule, is being performed.
CONCLUSIONS
The current study represents the first report showing the anticancer activity derived from Granulocystopsis sp., an isolated microalgae from the Chihuahuan desert. The microalgae methanolic extract inhibited cell proliferation, showed time-dependent cytotoxic activity, modified morphology, decreased cell adhesion and induced apoptosis by activating caspases-3/7 in breast, colon, prostate and skin melanoma cancer cell lines, but showed less pronounced effects on normal cells. | v3-fos-license |
2020-04-24T13:06:17.005Z | 2020-04-24T00:00:00.000 | 216082135 | {
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} | pes2o/s2orc | Enhanced Circularly Polarized Luminescence Activity in Chiral Platinum(II) Complexes With Bis- or Triphenylphosphine Ligands
Distinct circularly polarized luminescence (CPL) activity was observed in chiral (C∧N∧N)Pt(II) [(C∧N∧N) = 4,5-pinene-6′-phenyl-2,2′-bipyridine] complexes with bis- or triphenylphosphine ligands. Compared to the pseudo-square-planar geometry of chiral (C∧N∧N)Pt(II) complexes with chloride, phenylacetylene (PPV) and 2,6-dimethylphenyl isocyanide (Dmpi) ligands, the coordination configuration around the Pt(II) nucleus of chiral (C∧N∧N)Pt(II) complexes with bulk phosphine ligands is far more distorted. The geometry is straightforwardly confirmed by X-ray crystallography. The phosphines' participation enhanced the CPL signal of Pt(II) complexes profoundly, with the dissymmetry factor (glum) up to 10−3. The distorted structures and enhanced chiroptical signals were further confirmed by time-dependent density functional theory (TD-DFT) calculations.
Synthesis
The precursors mononuclear complexes (-)-(C ∧ N ∧ N)PtCl and (+)-(C ∧ N ∧ N)PtCl were prepared according to previous procedures (Zhang et al., 2014(Zhang et al., , 2015b. The target dinuclear and mononuclear Pt(II) complexes have been facilely synthesized though the coordination reaction between different phosphine ligands and precursors in the proper proportion at room temperature (Scheme 1). The new obtained complexes were fully characterized by NMR and MS spectra. The preparation of their enantiomers was done using the same procedure.
Absorption and Emission Properties
As shown in Figure 2 and Figures S2-S4, all of the chiral dinuclear Pt(II) complexes show characteristic absorption bands (ε > 10 4 L mol −1 cm −1 ) in the UV region similar to those of bis-(diphenylphosphino)alkane bridged dinuclear Pt(II) complexes. The mononuclear Pt(II) complex (-)-7 also exhibits a similar intense absorption below 400 nm. According to previous studies, the intense bands (<400 nm) are attributed to intraligand π-π * transitions. In addition, weak absorptions in the region of 400-450 nm are designated as a mixture of metal-to-ligand charge transfer ( 1 MLCT) and ligand-to-ligand charge transfer ( 1 LLCT) transitions (Lu et al., 2002(Lu et al., , 2004Shao and Sun, 2007;Zhang et al., 2018). From the crystal structures of (-)-3-OTf, (-)-4, and (-)-7, it can be found that effective intramolecular/intermolecular Pt···Pt interactions are absent and that two [(C ∧ N ∧ N)Pt] + units manifest like two separated moieties (Sun et al., 2006). Correspondingly, the absorptions of all the complexes only extend to ∼470 nm, which agrees well with the spectrum of (-)-2, demonstrating the nonexistence of metal-metal-to-ligand charge transfer transition ( 1 MMLCT) . All of the chiral dinuclear and mononuclear Pt(II) complexes are highly emissive in solution. For all the dinuclear Pt(II) complexes, a broad and structureless emission band at 546 nm is seen, which resembles that of the mononuclear relative (-)-7 ( Figure S5). Similar to the absorption spectra, the emission energy of all the complexes also reflects the absence of effective intramolecular/intermolecular Pt···Pt interactions. The emission of all the complexes can be ascribed to a triplet metal-to-ligand charge transfer ( 3 MLCT) excited state (Lu et al., 2002(Lu et al., , 2004Shao and Sun, 2007;Zhang et al., 2018). At 77 K, the emissions are significantly blue-shifted and evolve to be more structured (Figure S5), a characteristic nature for 3 MLCT excited states.
An intense emission peak and a shoulder are observed at 515 and 550 nm, respectively, and the spacing of about 1,100 cm −1 correlates to the characteristic skeletal stretching of the free C ∧ N ∧ N ligand.
TD-DFT Calculation
Time-dependent density functional theory (TD-DFT) calculations were carried out, shedding light on the differences in the structural parameters and frontier molecular orbitals of optimized configurations. The optimized configurations of all the chiral dinuclear and mononuclear Pt(II) complexes are shown in Figure S6. Also, the calculated results of the reference mononuclear compounds (-)-(C ∧ N ∧ N)PtCl, (-)-(C ∧ N ∧ N)PtPPV, and (-)-(C ∧ N ∧ N)PtDmpi have been provided. The bond angles around the metal nucleus of chiral Pt(II) complexes coordinated with bis-or triphenylphosphine ligands are further away from linearity than those of the reference mononuclear compounds (Table 2), which is consistent with the results of the crystal structures. In optimized configurations with phosphine ligands, the angles of C1-Pt1-N2 and C2-Pt2-N4 are in the range of 157.10-158.08 • , and the angles of N1-Pt1-P1 and N3-Pt2-P2 range from 170.97 • to 176.82 • . It is further confirmed that the Pt(II) nucleus in bulk bis-or triphenylphosphine systems adopts a more distorted coordination geometry.
CONCLUSION
In summary, we introduced bulky bis-or triphenylphosphine ligands into the phosphorescent pinene-containing (C ∧ N ∧ N)Pt(II) complexes and their structures were determined by single-crystal X-ray analysis. The geometries around the Pt(II) nucleus upon coordinating with bis-or triphenylphosphine were more distorted than those in chloride, phenylacetylene, and 2,6-dimethylphenyl isocyanide systems, which was further verified by DFT calculations. Enhanced CPL activity was observed, with g lum up to 10 −3 order. This study may pave a new way for the preparation of CPL-active phosphorescent metal complexes by introducing bulky ligands.
DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the Cambridge Crystallographic Data Centre (https://www.ccdc. cam.ac.uk/structures/) under the identifiers 1984372-1984374.
AUTHOR CONTRIBUTIONS
The preparation and characterization of all the complexes were done mainly by Q-YY, X-LH, and J-LW. The spectra measurement was done mainly by Q-YY, H-HZ, and YC. The TD-DFT calculation was done mainly by S-DW, L-ZH, and Z-FS. The manuscript was written by Q-YY with the guidance of X-PZ. | v3-fos-license |
2020-11-15T14:01:34.643Z | 2020-11-01T00:00:00.000 | 226947118 | {
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} | pes2o/s2orc | Enhancing Stability and Tooth Bleaching Activity of Carbamide Peroxide by Electrospun Nanofibrous Film
Carbamide peroxide (CP) possesses a strong tooth bleaching activity, however, its clinical application is limited because of its instability in aqueous formulations. This study explores the improvement of CP stability and bleaching activity by loading CP in electrospun nanofibrous film (ENF). Polyvinylalcohol, polyvinylpyrrolidone, and silica were used as components for core-based nanofibers of ENF. Electrospinning feed aqueous solutions (EFASs) were developed for preparing CP loaded ENF (CP-ENF). Stability of CP in EFASs is significantly higher than in pure water. The highest stability of CP is found in PPS-CP3, composed of 0.5% CP, 5.5% polyvinylalcohol, 3% polyvinylpyrrolidone, and 1% silica. The results from X-ray diffraction indicate that CP is dispersed as a non-crystalline form in CP-ENFs. CP and the compositions of EFASs play a major role on characteristics and bleaching efficiency of CP-ENFs. Drug release of CP-ENFs is the first order kinetics. CP-ENF obtained from PPS-CP3 shows the highest drug entrapment efficiency, high adhesion, and suitable sustained release. Drug release mechanism is along with anomalous transport according to Korsmeyer–Peppas model. In an ex vivo study using human teeth, it shows the highest bleaching efficiency among the others. Therefore, CP-ENF obtained from PPS-CP3 is a promising ENF for clinical use.
Introduction
Hydrogen peroxide and carbamide peroxide (CP) are strong oxidizing agents and have been used as tooth bleaching drugs for a long time [1]. Hydrogen peroxide however, easily decomposes. It can react with any present oxidizable compound and catalytic agents resulting in the formation of oxygen and water [2]. CP is known as urea peroxide or hydrogen peroxide-urea as it is composed of hydrogen peroxide and urea in a solid state and is more stable than hydrogen peroxide [3]. Therefore, CP is Viscosity and conductivity of EFASs are two important physical properties that influence the electrospinnability [30]. Our results as shown in Table 2 demonstrate the viscosity and conductivity of EFASs. The result of the present study indicates that polymer concentration plays a significant role on the viscosity of EFASs. For example, in P-BL which contains 10% PVA and without CP, showed significantly higher viscosity than P-CP, the solution contained 9% PVA and 1% CP. This result indicates that only 1% decrease of PVA concentration can cause a significant decrease of EFAS viscosity. The conductivity of P-BL is lower than that P-CP, indicating that low concentration of PVA can cause high conductivity of the solution. Replacing 3% PVA of P-CP with PVP yielded PP-CP and this led to an increase in the viscosity and conductivity of the solution. This result demonstrates the effects of PVP on the viscosity and conductivity of the system. However, replacing 1% PVA of P-CP with silica yielded PS-CP and this led to a decrease in viscosity whereas the conductivity was not altered. It was previously reported that silica could break down the gelling structure of the polymer [31]. Therefore, adding silica resulted a decrease the viscosity of EFASs. Effects of CP and silica on conductivity can be clearly seen in EFASs containing PVA, PVP, silica, and CP, i.e., PPS-CP1, PPS-CP2, and PPS-CP3. Replacing 0.5% PVA of PPS-CP3 with CP yielded PPS-CP1 with lower viscosity but this did not affect conductivity. Replacing 1% PVA of PPS-CP1 with silica yielded PPS-CP2 and this led to a decrease in viscosity and conductivity. The decrease in the conductivity is due to the fact that silica has a poor electrical conductivity [32].
Stability of CP in EFASs
Interaction of CP and the excipients in EFASs may occur and can lead to the loss of CP during fabrication of ENFs. Therefore, the stability of CP in EFAS is crucial before selection of a suitable EFAS. The remaining CP in each EFAS over a period of 8 h at 25 • C is shown in Figure 1. It is seen that the stability of CP in P-CP is the lowest in comparison among the others. Previous studies have shown that PVA is oxidized by a strong oxidant such as peroxide [33,34]. Oxidation of PVA in the presence of peroxide takes place via an initial cleavage of 1,2-glycol units in the PVA chain [35]. Thus, the rapid degradation of CP in P-CP is considered likely due to the oxidation reaction between PVA and CP. Replacing some part of PVA with silica, such as in PS-CP, yielded better CP stability. Silica is an inert material with a large surface area suitable for drug adsorption [29]. CP molecules might have been adsorbed to the silica and thereby are partly protected from direct contact with PVA. Blending PVA with PVP, for example in PS-CP and P-CP, also shows significant difference in improvement of CP stability. PVP can act as a good stabilizer and retard CP degradation because the carbonyl oxygen atoms of PVP can form hydrogen bonds with hydrogen peroxide molecules in the solution [28]. Therefore, replacing 3% PVA in P-CP with PVP yielded PP-CP and led to significantly higher stability of CP. The result of the present study also indicates that the potential of PVP on enhancing CP stability is much more effective than silica. This effect can be clearly seen when comparing CP stability in PP-CP (without silica) and PPS-CP1 in which 1% PVA in PP-CP was replaced with silica. It is found that in these two formulations with the same amount of PVP and different amount of silica, the enhancing stability of CP is similar.
Fabrication and Characterization of ENFs
ENFs from the developed EFASs were successfully prepared via the electrospinning process employed in this study. The obtained ENFs are white and possess a smooth surface appearance. The scanning electron microscope (SEM) images, as shown in Figure 2, reveal that the nanofibers obtained from P-BL, a PVA solution without drug, is a non-woven structure with an average nanofiber diameter of 421 ± 68 nm. It is noted that after an addition of CP, as in P-CP, some parts of the fibers are merged and overlapped. The diameters of the nanofibers from P-CP are close to that of PP-CP with average values of 319 ± 69 nm and 321 ± 45 nm, respectively. The fibers of PP-CP are smoother than those of P-CP. This result is due to the ejected solution of PP-CP being a more homogenous and continuous jet than that of P-CP. Addition of silica to the feed solution can increase in the diameter of the fibers. This can be seen in PS-CP fiber that its average diameter is 401 ± 54 nm, significantly larger than that the fibers obtained from P-CP. PVP has been used as a polymer carrier and mixed with various compounds that are difficult to spin via electrospinning [36]. Our results show that the addition of PVP, at the expense of PVA, to the solutions containing PVA and silica, as in PPS-CP1, can yield fine nanofibers with smooth surfaces and an average diameter of 261 ± 73 nm. Increase silica content to the formulations, as in PPS-CP1 with PPS-CP2, some beaded nanofibers can be seen. Interestingly, the beaded nanofibers are absent in PPS-CP3 samples, and the obtained fibers are very fine with an average diameter of 159 ± 33 nm. Comparing PPS-CP2 and PPS-CP3, both solutions have the same ratio of silica to drug (2:1 w/w), however PPS-CP2 has a lower polymer concentration than PPS-CP3 leading to an obvious decrease in viscosity. This effect can influence the formation of highly beaded fibers from PPS-CP2. From these results, it is postulated that besides the excipient type, the concentration of the excipients is one of the important factors that influences the transformation of the feed solutions into nanofibers. It has been reported that a low polymer concentration can result in electrosprayed particles or beaded fibers, whereas a high polymer concentration can limit the fiber
Fabrication and Characterization of ENFs
ENFs from the developed EFASs were successfully prepared via the electrospinning process employed in this study. The obtained ENFs are white and possess a smooth surface appearance. The scanning electron microscope (SEM) images, as shown in Figure 2, reveal that the nanofibers obtained from P-BL, a PVA solution without drug, is a non-woven structure with an average nanofiber diameter of 421 ± 68 nm. It is noted that after an addition of CP, as in P-CP, some parts of the fibers are merged and overlapped. The diameters of the nanofibers from P-CP are close to that of PP-CP with average values of 319 ± 69 nm and 321 ± 45 nm, respectively. The fibers of PP-CP are smoother than those of P-CP. This result is due to the ejected solution of PP-CP being a more homogenous and continuous jet than that of P-CP. Addition of silica to the feed solution can increase in the diameter of the fibers. This can be seen in PS-CP fiber that its average diameter is 401 ± 54 nm, significantly larger than that the fibers obtained from P-CP. PVP has been used as a polymer carrier and mixed with various compounds that are difficult to spin via electrospinning [36]. Our results show that the addition of PVP, at the expense of PVA, to the solutions containing PVA and silica, as in PPS-CP1, can yield fine nanofibers with smooth surfaces and an average diameter of 261 ± 73 nm. Increase silica content to the formulations, as in PPS-CP1 with PPS-CP2, some beaded nanofibers can be seen. Interestingly, the beaded nanofibers are absent in PPS-CP3 samples, and the obtained fibers are very fine with an average diameter of 159 ± 33 nm. Comparing PPS-CP2 and PPS-CP3, both solutions have the same ratio of silica to drug (2:1 w/w), however PPS-CP2 has a lower polymer concentration than PPS-CP3 leading to an obvious decrease in viscosity. This effect can influence the formation of highly beaded fibers from PPS-CP2. From these results, it is postulated that besides the excipient type, the concentration of the excipients is one of the important factors that influences the transformation of the feed solutions into nanofibers. It has been reported that a low polymer concentration can result in electrosprayed particles or beaded fibers, whereas a high polymer concentration can limit the fiber production because of too high viscosity [37]. In the present study, with a working voltage of 10 kV and a spinning distance of 10 mm, it is found that a concentration of 5-10% PVA solution is the most suitable for fiber production and no fibers could be collected when the PVA concentration was <4%. Therefore, the obtained nanofibers contained at least 50% of PVA and the highest amount of CP that could possibly be loaded in EFASs is 50%. However, from preliminary development of 50% CP loaded nanofibers, it was found that the obtained CP-ENF was very brittle and easily tears due to the low amount of polymer (data not shown). The reduction in the fiber diameter of the formulations containing CP and PVP can be attributed to the increase in the conductivity of polymer solutions. These phenomena can be explained by the net charge density. A higher net charge density can increase the electrical force exerted on the jet and lead to a decrease in the fiber diameter [38,39].
could possibly be loaded in EFASs is 50%. However, from preliminary development of 50% CP loaded nanofibers, it was found that the obtained CP-ENF was very brittle and easily tears due to the low amount of polymer (data not shown). The reduction in the fiber diameter of the formulations containing CP and PVP can be attributed to the increase in the conductivity of polymer solutions. These phenomena can be explained by the net charge density. A higher net charge density can increase the electrical force exerted on the jet and lead to a decrease in the fiber diameter [38,39].
Solid State of ENFs
The X-ray diffraction (XRD) patterns of the different samples are shown in Figure 3. CP is a crystalline material with characteristic X-ray diffraction 2θ angles of 14 • , 23 • , and 28 • . However, the halo pattern is obtained after CP is loaded in the nanofibers. This indicates that CP is dispersed as a non-crystalline form which may be molecular dispersion or amorphous form. This result suggests that during the electrospinning process, the solid fibers were rapidly formed on the collector by removing the solvent instantly. The time of solidification of the nanofibers was too short for the drug to recrystallize. crystalline material with characteristic X-ray diffraction 2θ angles of 14°, 23°, and 28°. However, the halo pattern is obtained after CP is loaded in the nanofibers. This indicates that CP is dispersed as a non-crystalline form which may be molecular dispersion or amorphous form. This result suggests that during the electrospinning process, the solid fibers were rapidly formed on the collector by removing the solvent instantly. The time of solidification of the nanofibers was too short for the drug to recrystallize.
Adhesive Property and Entrapment Efficiency of ENFs
Nanofibers with high adhesive property can provide prolonged residence time on the desired site of action and thereby exert a better bleaching effect. Due to the good adhesive properties of PVA and PVP, previous studies have used these polymers in mucoadhesive dosage forms [40,41]. Among the seven samples, ENF from PP-CP possesses the highest adhesive force with a value of 0.81 ± 0.02 N followed by PPS-CP3, PPS-CP1, PPS-CP2, P-CP, and PS-CP, respectively (Table 3). These results suggest that PVP can enhance the adhesive properties of PVA. The result of the present study confirms that these two polymers are suitable as adhesive materials for CP-ENF. It has previously been reported that ENF of polycaprolactone using PVP as an exterior sheath, helped the fibers to rapidly adhere to wet biological surfaces [42]. In the present study, it is observed that CP-ENF containing PVA or a combination of PVA and PVP are non-adhesive under dry conditions but can
Adhesive Property and Entrapment Efficiency of ENFs
Nanofibers with high adhesive property can provide prolonged residence time on the desired site of action and thereby exert a better bleaching effect. Due to the good adhesive properties of PVA and PVP, previous studies have used these polymers in mucoadhesive dosage forms [40,41]. Among the seven samples, ENF from PP-CP possesses the highest adhesive force with a value of 0.81 ± 0.02 N followed by PPS-CP3, PPS-CP1, PPS-CP2, P-CP, and PS-CP, respectively (Table 3). These results suggest that PVP can enhance the adhesive properties of PVA. The result of the present study confirms that these two polymers are suitable as adhesive materials for CP-ENF. It has previously been reported that ENF of polycaprolactone using PVP as an exterior sheath, helped the fibers to rapidly adhere to wet biological surfaces [42]. In the present study, it is observed that CP-ENF containing PVA or a combination of PVA and PVP are non-adhesive under dry conditions but can readily adhere to wet mucosal tissues. This result confirms that the adhesive power of these polymers dramatically increases when they are subjected to a high humidity or moist environment. According to the adhesive properties of PVA and PVP in a wet environment [43], CP-ENFs can strongly adhere to the non-uniform tooth surfaces in the oral cavity surrounded by saliva. For drug loading capacity, the results show that entrapment efficiency (EE) values of CP-ENFs are in the range of 59-98% depending on the type and concentration of the components in each formulation. EFASs containing various compositions of PVA, PVP, silica, CP, and water (as presented in Table 1) were subjected to the electrospinning process, the water evaporated and ENFs formed, therefore the obtained ENFs contained 90-95% polymer with and without silica and the final concentration of CP in the ENFs ideally was 10% for ENFs obtained from 1% CP concentration EFASs and 5% for ENFs obtained from 0.5% CP concentration EFASs. The actual CP amount in the obtained CP-ENFs from various EFASs was 5.9 ± 0.2%, 7.4 ± 0.1%, 7.7 ± 0.2%, 8.3 ± 0.2%, 8.8 ± 0.1% for P-CP, PP-CP, PS-CP, PPS-CP1, and PPS-CP2, respectively, and 4.9 ± 0.1% for PPS-CP3. Therefore, ENF obtained from P-CP possesses the lowest EE with a value of 60 ± 2%, whereas that from PPS-CP3 demonstrates the highest EE with a value of 98 ± 2%. The lowest EE value of P-CP fibers is due to the instability of CP in P-CP. Decomposition of CP can occur before and during the electrospinning process. Ideally, the amount of drug entrapped in the nanofibers should be equal to the amount of drug added before the electrospinning process. However, CP is unstable, particularly when exposed to oxygen and light. From the stability results mentioned above, it is shown that the highest amount of CP remaining can be found in the solution containing PVP. This result supports that PVP plays an important role on stabilization of CP in EFASs. For the systems containing silica, it is considered that silanol groups of silica can form strong hydrogen bond with CP [29]. Therefore, the highly porous property with large surface area of silica can entrap and protect CP from decomposition, leading to the high drug content in the fibers. The results show that EE values of the silica containing ENFs obtained from PPS-CP1, PPS-CP2, and PPS-CP3 are significantly higher than from P-CP, PP-CP, and PS-CP, respectively (p < 0.05). Moreover, it is noted that EE values of ENFs from PPS-CP1, PPS-CP2, and PPS-CP3 are also different. Among them, ENF from PPS-CP3 demonstrates the highest EE value. During the electrospinning process, it was found that the jet streams of PPS-CP1 and PPS-CP2 were broken, resulting in the formation of beaded nanofibers. This phenomenon indicates that composition ratios of PPS-CP1 and PPS-CP2 in EFASs are important. The inappropriate ratio can hardly provide a stable electrospun fibers. Kundrat et al. reported the effects of viscosity on EE values of levofloxacin loaded poly3-hydroxybutyrate nanofibers [44]. Our experiment also shows that EE values of the obtained CP-ENFs are dependent on viscosity of EFASs. It is found that increase in viscosity can affect the formation of a stable jet leading to a difficulty in stable and uniform nanofiber formation since the solution can easily dehydrate at the tip of the needle thereby the components can adhere to the surfaces of the needle and the flow of components through the tip is disrupted. We, therefore, conclude that the composition ratio of EFAS plays an important role on the EE value of the obtained fibers by affecting conductivity and viscosity of EFASs.
In Vitro Drug Release and Drug Release Kinetics
Drug release profiles of CP-ENFs obtained from the developed EFASs with and without silica are presented in Figure 4. A burst release of CP is observed from CP-ENF obtained from P-CP. Almost 100% CP is released into the dissolution medium within 30 min. CP-ENF obtained from PP-CP shows a slower drug release compared to that obtained from P-CP, indicating the effect of PVP on retardation of drug Pharmaceuticals 2020, 13, 381 9 of 20 release. However, almost the total amount of CP (98.7 ± 1.8%) is released after 4 h. The influence of silica on drug release can be seen among ENFs from EFASs with different amount of silica. At the release period of 1 h, drug release from CP-ENFs obtained from PVA-PVP-silica core-based ENFs such as PPS-CP1, PPS-CP2, and PPS-CP3 ENF was found to be 61.1 ± 1.2%, 88.3 ± 1.1%, and 35.0 ± 2.0%, respectively, indicating that silica can causes a fast drug release. However, at the same period of 1 h, CP released from CP-ENF obtained from P-CP, a film without silica, is faster than that from PS-CP, a film with silica. This result shows the effect of brittleness and ununiformed drug distribution of the film. During the experiment, it was observed that P-CP film was easily broken into small pieces and dispersed into the dissolution medium, leading to an extreme increase of contact surface area of the film to the medium hence a faster release of the drug can be obtained. Therefore, the effect of silica on increasing drug release from PS-CP film is obscured.
based ENFs such as PPS-CP1, PPS-CP2, and PPS-CP3 ENF was found to be 61.1 ± 1.2%, 88.3 ± 1.1%, and 35.0 ± 2.0%, respectively, indicating that silica can causes a fast drug release. However, at the same period of 1 h, CP released from CP-ENF obtained from P-CP, a film without silica, is faster than that from PS-CP, a film with silica. This result shows the effect of brittleness and ununiformed drug distribution of the film. During the experiment, it was observed that P-CP film was easily broken into small pieces and dispersed into the dissolution medium, leading to an extreme increase of contact surface area of the film to the medium hence a faster release of the drug can be obtained. Therefore, the effect of silica on increasing drug release from PS-CP film is obscured.
The results from the drug release study also suggest that release of CP from the nanofibers is influenced by the viscosity of EFASs and morphology of the nanofibers. High viscosity can prolong drug release. This can be seen in the release behavior of ENF obtained from EFASs containing PVA and PVP mixture, which possesses high viscosity. However, for the ENF from P-CP, which also possesses high viscosity, a burst release is observed from its fibers. This result might be due to the ununiformed drug dispersion and the destabilization of PVA polymer chains induced by CP, leading to a high amount of drug being located on the fiber surface. The results demonstrate that replacement of silica and PVP to PVA can decrease brittleness of the films and hence the slow drug release can be observed. Among the others, the ENF obtained from PPS-CP3 shows the most sustained drug release within 8 h. Kinetic study of drug release can provide a meaningful parameter to understand the drug release mechanism for the appropriate purpose and the results can be utilized to assess the effect of formulation factors on the drug release profile [45]. Several mathematical models can be used to describe the kinetic drug release from the developed formulations, such as the zero-order and the first-order models [46]. The Korsmeyer-Peppas model has been used to describe the type of drug release mechanism from polymeric systems [47]. In the present study, the release data were analyzed by these kinetic models. The zero-order and the first-order kinetic models are expressed in the following equations, The results from the drug release study also suggest that release of CP from the nanofibers is influenced by the viscosity of EFASs and morphology of the nanofibers. High viscosity can prolong drug release. This can be seen in the release behavior of ENF obtained from EFASs containing PVA and PVP mixture, which possesses high viscosity. However, for the ENF from P-CP, which also possesses high viscosity, a burst release is observed from its fibers. This result might be due to the ununiformed drug dispersion and the destabilization of PVA polymer chains induced by CP, leading to a high amount of drug being located on the fiber surface. The results demonstrate that replacement of silica and PVP to PVA can decrease brittleness of the films and hence the slow drug release can be observed. Among the others, the ENF obtained from PPS-CP3 shows the most sustained drug release within 8 h.
Kinetic study of drug release can provide a meaningful parameter to understand the drug release mechanism for the appropriate purpose and the results can be utilized to assess the effect of formulation factors on the drug release profile [45]. Several mathematical models can be used to describe the kinetic drug release from the developed formulations, such as the zero-order and the first-order models [46]. The Korsmeyer-Peppas model has been used to describe the type of drug release mechanism from polymeric systems [47]. In the present study, the release data were analyzed by these kinetic models. The zero-order and the first-order kinetic models are expressed in the following equations, respectively, where Q 0 is initial amount of drug in the solution, k 0 is the zero-order release rate constant, k 1 is the first-order release rate constant, and t is time of drug release. A suitable mathematical model is chosen by value of a correlation coefficient (r 2 ) from linear regression analysis. The results are reported in Table 4. Comparing the r 2 values between the zero-and the first-order kinetics, the results described that the release of CP from all CP-ENFs followed the first-order kinetics, which the linear relationship can be obtained as shown in Figure 5, indicating that the release rate of CP is dependent on drug concentration. respectively, where Q0 is initial amount of drug in the solution, k0 is the zero-order release rate constant, k1 is the first-order release rate constant, and t is time of drug release. A suitable mathematical model is chosen by value of a correlation coefficient (r 2 ) from linear regression analysis.
The results are reported in Table 4. Comparing the r 2 values between the zero-and the first-order kinetics, the results described that the release of CP from all CP-ENFs followed the first-order kinetics, which the linear relationship can be obtained as shown in Figure 5, indicating that the release rate of CP is dependent on drug concentration. Drug release mechanism was analyzed using Korsmeyer-Peppas model, as the following equation where M t /M ∞ is a fraction of drug released at time t, k kp is the release rate constant of Korsmeyer-Peppas, and n is the exponent of release in function of time t which indicates the mechanism of drug release. When n is ≤0.45, the drug release mechanism follows a Fickian diffusion mechanism. When 0.45 < n < 0.89, it follows non-Fickian diffusion or anomalous transport. When n is 0.89, it follows case II transport, and when n is >0.89, it follows super case II transport mechanism [48,49]. In general, Korsmeyer-Peppas model is a mathematical model suitable for sustained release kinetics and the most valid prediction using this model should be performed on M t /M ∞ ≤ 0.6 of drug release [48,50]. Therefore, this model is not suitable for ENFs of P-CP, PS-CP, and PPS-CP2 due to their fast release behavior but may be suitable for ENFs from PP-CP, PPS-CP1, and PPS-CP3 due to their sustained release property. According to the Korsmeyer-Peppas model, log cumulative drug release against log time of these three formulations was plotted as shown in Figure 6. The kinetic parameters evaluated from these graphical plots are shown in Table 4. The correlation coefficient by linear regression analysis which closed to 1 was considered as the fitted model. It is found that the r 2 values of PP-CP, PPS-CP1, and PPS-CP3 from the Korsmeyer-Peppas model are greater than 0.95. The rate constant of CP-ENF from PPS-CP1 was the highest compared to that from PP-CP and PPS-CP3, possibly due to low amount of PVA. The drug release from CP-ENF from PP-CP, PPS-CP1, and PPS-CP3 is through non-Fickian diffusion as their n values are between 0.45 and 0.89. This indicates anomalous transport which is a combination of Fickian diffusion and case II transport. During these processes, rearrangement of polymeric chains caused by swelling and erosion can be occurred and the diffusion process simultaneously cause the time-dependent anomalous effects [51]. Therefore, the release of CP from ENFs is controlled by the swelling and erosion of ENFs and the diffusion of CP. The results of this experiment also indicate that the different amount of the compositions of ENFs can influence the kinetics of drug release from the films. where Mt/M∞ is a fraction of drug released at time t, kkp is the release rate constant of Korsmeyer-Peppas, and n is the exponent of release in function of time t which indicates the mechanism of drug release. When n is ≤0.45, the drug release mechanism follows a Fickian diffusion mechanism. When 0.45 < n < 0.89, it follows non-Fickian diffusion or anomalous transport. When n is 0.89, it follows case II transport, and when n is >0.89, it follows super case II transport mechanism [48,49]. In general, Korsmeyer-Peppas model is a mathematical model suitable for sustained release kinetics and the most valid prediction using this model should be performed on Mt/M∞ ≤ 0.6 of drug release [48,50]. Therefore, this model is not suitable for ENFs of P-CP, PS-CP, and PPS-CP2 due to their fast release behavior but may be suitable for ENFs from PP-CP, PPS-CP1, and PPS-CP3 due to their sustained release property. According to the Korsmeyer-Peppas model, log cumulative drug release against log time of these three formulations was plotted as shown in Figure 6. The kinetic parameters evaluated from these graphical plots are shown in Table 4. The correlation coefficient by linear regression analysis which closed to 1 was considered as the fitted model. It is found that the r 2 values of PP-CP, PPS-CP1, and PPS-CP3 from the Korsmeyer-Peppas model are greater than 0.95. The rate constant of CP-ENF from PPS-CP1 was the highest compared to that from PP-CP and PPS-CP3, possibly due to low amount of PVA. The drug release from CP-ENF from PP-CP, PPS-CP1, and PPS-CP3 is through non-Fickian diffusion as their n values are between 0.45 and 0.89. This indicates anomalous transport which is a combination of Fickian diffusion and case II transport. During these processes, rearrangement of polymeric chains caused by swelling and erosion can be occurred and the diffusion process simultaneously cause the time-dependent anomalous effects [51]. Therefore, the release of CP from ENFs is controlled by the swelling and erosion of ENFs and the diffusion of CP. The results of this experiment also indicate that the different amount of the compositions of ENFs can influence the kinetics of drug release from the films.
Ex Vivo Tooth Bleaching Efficiency
The bleaching protocol used in the present study was modified from the dental bleaching treatment recommended by American Dental Association [52]. A period of 8 h bleaching per day is the suggested duration for bleaching formulations having a drug concentration not exceeding 10%
Ex Vivo Tooth Bleaching Efficiency
The bleaching protocol used in the present study was modified from the dental bleaching treatment recommended by American Dental Association [52]. A period of 8 h bleaching per day is the suggested duration for bleaching formulations having a drug concentration not exceeding 10% (classified as a low drug concentration) [53]. The concentration of CP in all ENF was not higher than 10%, therefore, they are regarding as low concentration formulations [54]. Thus, the protocol with 8 h of bleaching per day was used in the current study with a period of 14 days. ENFs were placed on the tooth surface and the teeth were kept in the controlled environment. After 8 h of bleaching, it was found that the remaining films still were attached on the tooth surface as presented in Figure 7. The films were more transparent and of smaller sizes than before the test.
10%, therefore, they are regarding as low concentration formulations [54]. Thus, the protocol with 8 h of bleaching per day was used in the current study with a period of 14 days. ENFs were placed on the tooth surface and the teeth were kept in the controlled environment. After 8 h of bleaching, it was found that the remaining films still were attached on the tooth surface as presented in Figure 7. The films were more transparent and of smaller sizes than before the test. The results demonstrate that after complete bleaching period of 14 days, the tooth color of the treatment groups, i.e., P-CP, PP-CP, PS-CP, PPS-CP1, PPS-CP2, and PPS-CP3, was whiter than the initial day as seen in Figure 8a. The visual color change (ΔE) value progressions of each formulation are presented in Figure 8b. All CP-ENFs cause a significantly potential bleaching effect as compared to the negative control groups for day 0, day 1, day 3, day 7, and day 14 (p < 0.05). It is noted that on day 14, the mean ∆E value of ENF obtained from PPS-CP3 is the highest (p < 0.05) among the tested ENFs with a value of 3.84 ± 0.08. Since ENF obtained from PPS-CP3 possesses the highest EE value and high adhesion, this formulation could efficiently serve as a reservoir for drug and increase drug residence time on the teeth. As shown in the in vitro release tests, this formulation provided sustained release profiles of CP, resulting in the enhanced bleaching efficiency of the drug at the site of action.
Current literature indicates that ∆E values from using CP vary due to differences in staining procedure, type of product, duration of application, and the applied amount. It has been reported that ∆E values of at least 2.6 are visually perceptible [55], ∆E values of ENF obtained from PPS-CP3 were 2.51 ± 0.21 at day 3, and of PPS-CP1 and PPS-CP2 were above 2.6 at day 14. Therefore, these formulations were effective for tooth bleaching. However, it was observed that the entire tooth was bleached. A possible explanation for this result is that after CP-ENFs were hydrated by artificial saliva, the nanofibers were swelling, and erosion of the polymer occurred. ENFs transformed into a hydrogel film formulation. CP can diffuse from ENFs and dissolve in saliva that surrounded the teeth, leading to an increase of the contact area. After 8 h, some ENFs showed small parts remaining on the tooth surface whereas most of ENFs were dissolved. Moreover, the placement of ENF obtained from PPS-CP3 was in two directions; therefore, it covers more area of the tooth compared to the others. In addition, it was difficult to control and localize CP delivery especially when teeth not cut into flat surfaces were used. However, this provides an issue in the use of ENFs that needs to be further be explored especially for in vivo study. Further research of CP-ENFs in order to prevent the drug from contacting an unwanted area such as gingival is needed.
Another aspect of CP-ENFs that should be addressed in future studies are the treatment time, as in this study, the protocol was selected based on CP concentration in the formulations without considerations on the dosage form. The developed CP-ENFs have shown satisfactory results of bleaching efficiency in 8 h per day for 14 days. However, commercial products such as strips typically require an application of only 30 min, twice a day, for 14 days [56]. As ENFs are introduced for tooth bleaching here for the first time, the suitable treatment regimen still needs to be investigated in order to compare efficiency with commercial products.
In addition, the obtained ENFs could possibly retard CP degradation compared to hydrophilic formulations, especially for long-term storage. CP-ENFs avoid the use of organic solvents, stabilizers, The results demonstrate that after complete bleaching period of 14 days, the tooth color of the treatment groups, i.e., P-CP, PP-CP, PS-CP, PPS-CP1, PPS-CP2, and PPS-CP3, was whiter than the initial day as seen in Figure 8a. The visual color change (∆E) value progressions of each formulation are presented in Figure 8b. All CP-ENFs cause a significantly potential bleaching effect as compared to the negative control groups for day 0, day 1, day 3, day 7, and day 14 (p < 0.05). It is noted that on day 14, the mean ∆E value of ENF obtained from PPS-CP3 is the highest (p < 0.05) among the tested ENFs with a value of 3.84 ± 0.08. Since ENF obtained from PPS-CP3 possesses the highest EE value and high adhesion, this formulation could efficiently serve as a reservoir for drug and increase drug residence time on the teeth. As shown in the in vitro release tests, this formulation provided sustained release profiles of CP, resulting in the enhanced bleaching efficiency of the drug at the site of action. and other additive substances, such as glycerin which is commonly used in CP gels and may lead to tooth dehydration and sore throat [57,58].
Materials
Hydrophilic fumed silica (Aerosil ® 380) was obtained from Evonik (Essen, Germany). CP, PVA having a molecular weight range of 85,000-124,000 and 87-89% degree of hydrolysis, PVP K-90, and Current literature indicates that ∆E values from using CP vary due to differences in staining procedure, type of product, duration of application, and the applied amount. It has been reported that ∆E values of at least 2.6 are visually perceptible [55], ∆E values of ENF obtained from PPS-CP3 were 2.51 ± 0.21 at day 3, and of PPS-CP1 and PPS-CP2 were above 2.6 at day 14. Therefore, these formulations were effective for tooth bleaching. However, it was observed that the entire tooth was bleached. A possible explanation for this result is that after CP-ENFs were hydrated by artificial saliva, the nanofibers were swelling, and erosion of the polymer occurred. ENFs transformed into a hydrogel film formulation. CP can diffuse from ENFs and dissolve in saliva that surrounded the teeth, leading to an increase of the contact area. After 8 h, some ENFs showed small parts remaining on the tooth surface whereas most of ENFs were dissolved. Moreover, the placement of ENF obtained from PPS-CP3 was in two directions; therefore, it covers more area of the tooth compared to the others. In addition, it was difficult to control and localize CP delivery especially when teeth not cut into flat surfaces were used. However, this provides an issue in the use of ENFs that needs to be further be explored especially for in vivo study. Further research of CP-ENFs in order to prevent the drug from contacting an unwanted area such as gingival is needed.
Another aspect of CP-ENFs that should be addressed in future studies are the treatment time, as in this study, the protocol was selected based on CP concentration in the formulations without considerations on the dosage form. The developed CP-ENFs have shown satisfactory results of bleaching efficiency in 8 h per day for 14 days. However, commercial products such as strips typically require an application of only 30 min, twice a day, for 14 days [56]. As ENFs are introduced for tooth bleaching here for the first time, the suitable treatment regimen still needs to be investigated in order to compare efficiency with commercial products.
In addition, the obtained ENFs could possibly retard CP degradation compared to hydrophilic formulations, especially for long-term storage. CP-ENFs avoid the use of organic solvents, stabilizers, and other additive substances, such as glycerin which is commonly used in CP gels and may lead to tooth dehydration and sore throat [57,58].
Preparation of EFASs
EFASs with different concentrations of CP and major components for core-based nanofibers as shown in Table 1 were prepared as follows. Solutions of PVA or PVA and PVP in distilled water were prepared under constant stirring of 800 rpm at 70 • C for 12 h and then cooled down to room temperature. These solutions were added into a 1% N,N-dimethylformamide solution containing CP or CP and silica. The obtained mixtures were gently stirred at 100 rpm at room temperature to obtained homogenous spinning solutions without air bubbles. For EFASs without CP such as P-BL, the polymer was dissolved in distilled water and then adjusted to weight with the water.
Stability of CP in EFASs
The freshly prepared CP-containing EFASs were continuously stirred at 100 rpm at 25 • C throughout the experiment. Samples of 1.0 mL were withdrawn periodically at 30 min, 1, 2, 4, 6, and 8 h after preparation. Quantitative analysis of CP from each withdrawn sample was immediately performed using a high-performance liquid chromatography (HPLC).
HPLC Analysis
Quantitative analysis of CP can be done using the oxidation of triphenylphosphine by CP into triphenylphosphine oxide which can be detected in HPLC chromatogram at a different retention time from that of triphenylphosphine. Therefore, decrease of triphenylphosphine or increase of triphenylphosphine oxide can be calculated and converted to the amount of CP. In the current study, the determination of CP was done by quantification of triphenylphosphine oxide. The analysis was carried out using HPLC (DionexTM ChromeleonTM HPLC System, Thermo Fisher Scientific Inc., Waltham, MA, USA) and a reversed phase column (5 µm, 150 × 4.6 mm I.D, YMCTM Pack Pro C18, YMC Co., Ltd., Kyoto, Japan) as the stationary phase. HPLC condition used was according to a method previously described [59] with some modification. Briefly, an exact volume of 1.0 mL of the sample was added to an equal volume of 0.1 M triphenylphosphine and constantly stirred for 2 h at room temperature. The resulted mixture was filtered through a 0.22 µm filter membrane prior to injecting in HPLC with an injected volume of 10 µL. The HPLC mobile phase consisting of acetonitrile (A) and water (B) was used at a flow rate of 1.0 mL/min with gradient conditions as follows: 40% A and 60% B for 6.5 min, 100% A for 10 min, and 40% A and 60% B for 3.5 min. Detection was performed by means of a UV detector at a wavelength of 225 nm. The temperature of HPLC was maintained at 25 • C. Calibration curve was prepared using CP solution at a concentration range of 30-150 µg/mL and the obtained linear standard curve with r 2 = 0.9995 was used.
Viscosity and Electrical Conductivity of EFASs
Viscosity of EFASs was measured using a plate-plate Brookfield rheometer (Rheometer R/S-CPS, Middleboro, MA, USA). The gap between the two plates was 1 mm, a shear rate ranging from 1 to 360 s −1 was employed. Electrical conductivity of the solutions was measured using a pH-conductivity meter (D-24 Horiba, Kyoto, Japan). The samples were maintained at 25 • C throughout the tests. The measurements were carried out by directly inserting an electrode (9625-10D, 3-in-1 Electrodes, Horiba, Kyoto, Japan) into the samples. The temperature was maintained at 25 • C throughout the tests.
Fabrication of the ENF
The developed EFASs were loaded into a 2.5 mL syringe connected with a stainless-steel needle (Hamilton 2.5 mL, Model 1005 TLL SYR, Hamilton Metal Hub Needles, Hamilton Company, Bonaduz, Switzerland). The syringe containing EFAS was placed in a syringe pump (Harvard Apparatus Pump 11 Elite Syringe Pumps, Holliston, MA, USA) and horizontally pumped at a flow rate of 10 µL/min. The electrospinning process was performed with an applied voltage of 15 kV provided by a high voltage power supply (FC Series-Glassman High Voltage Regulated DC Power Supplies, High Bridge, Hunterdon, NJ, USA). The ENF was deposited on a stationary metal collector (VWR International, Radnor, PA, USA) covered with aluminum foil. The distance between the syringe tip and the collector plate was 10 cm. The electrospinning process was conducted at room temperature in an enclosed fume hood. After this fabrication process, the CP-containing EFASs could yield CP-ENFs. The solution without CP e.g., P-BL could yield a blank-ENF. All ENFs obtained were stored in a desiccator for further study.
Morphology Study
The micro-structure of the nanofibers was investigated by using an SEM (JEOL JSM-6610LV, Tokyo, Japan) as follows. The obtained ENF were cut into a rectangle shape of approximately 5 mm × 5 mm and placed on a carbon tape. Then, the sample surface was coated with gold for 15 s using a 40-mA sputter coater (JEOL JFC-1100E, Tokyo, Japan). The SEM images were taken at an accelerating voltage of 15 kV with 3000× and 10,000× magnifications. The average diameters of the nanofibers in the obtained ENF were measured from the SEM images of each sample by using Image J software (US National Institutes of Health, Bethesda, MD, USA).
Investigation of Internal Solid State
An XRD was used to investigate the internal solid state of the obtained CP-ENFs in comparison with CP intact and the other excipients used as major components of the core-based ENF. A Miniflex II desktop XRD (Rigaku, Japan) was used and the XRD diffractograms were recorded in continuous mode over a Bragg angle (2θ) range of 5-60 • at the scanning rate of 12 • /min.
Investigation of Adhesive Property
The adhesive property of the obtained CP-ENFs and blank ENF was investigated using a method previously described [60], with some modification. Briefly, a texture analyzer (TA.XT PlusTexture Analyzer, Surrey, UK) was calibrated with a 5-kg load cell and equipped with a stand before determining the adhesive forces of the samples. The ENF samples were cut into a circular shape with a diameter of 1 cm and wetted with 100 µL of water before fixing on a probe (P 0.5 Perspex, 0.5-inch diameter). A piece of porcine intestinal mucosa having an inner surface of 2 cm × 5 cm was attached to a glass slide and then placed on the stand. An exact amount of 1 mL artificial saliva [61] was dropped on the surface of the mucosa. The probe was lowered to contact the mucosal surface with the contact force of 0.2 N and contact time of 60 s. After that, the probe was pulled up with the rate of 1 mm/s. Once the probe was completely separated from the mucosa, the data were recorded and calculated by Exponent software (Stable Micro Systems, Surrey, UK) to obtain the adhesive force.
Determination of CP in CP-ENFs
The amount of CP entrapped in the obtained CP-ENFs was investigated by dissolving 0.05 g of each CP-ENF in 50 mL deionized water. The solution was subjected to centrifugation (Beckman Avanti 30 High Speed Compact Centrifuge, Beckman Coulter Life Sciences, Indianapolis, IN, USA) at a speed of 10,000 rpm for 15 min. The supernatant was immediately collected and determined for CP by HPLC analysis as described above. The amount of CP entrapped in the CP-ENF, expressed as EE value, was obtained from the following equation where D L is the amount of CP in the ENF and D A is the amount of CP initially added.
Drug Release Kinetic
The developed CP-ENFs were cut into a rectangular shapes of an approximate area of 1 cm × 5 cm to obtain an average weight of 0.1 g for each film and then placed in a 30 mL artificial saliva solution at a constant stirring rate of 20 rpm and at a controlled temperature of 37 ± 1 • C. Samples of 1.0 mL were withdrawn from the dissolution medium at time intervals of 30 min, 1, 2, 4, and 8 h. Fresh medium with the same volume was added into the dissolution medium after each withdrawal. The amount of CP was determined by HPLC analysis as described above. The percentage cumulative release of CP at time t (Q T ) is calculated by the following equation.
where M T is the cumulative amount of CP released in the release medium at time T and M 0 is the initial amount of drug in the CP-ENF. The obtained results were analyzed for drug release kinetics using a Microsoft Excel 2010.
Ex Vivo Tooth Bleaching Assessment
Human teeth were collected by dentists (Faculty of Dentistry, Chiang Mai University). This study was approved by the Human Experimentation Committee, Faculty of Dentistry, Chiang Mai University (No. 58/2016). The teeth were cleaned and stored in a saturated thymol solution at 4 • C until testing. Forty normal teeth without any surface stains and imperfections were selected. The baseline color at the middle surface of averaged areas of 3 mm 2 circular centers of the collected teeth was measured. The teeth were randomly allocated into eight groups, five teeth per group, according to the six CP-ENFs obtained from six different CP-containing EFASs and two negative control groups (a blank ENF and artificial saliva). Each day, the fresh ENF samples were cut into rectangle shapes having an area of 5 mm × 10 mm to obtain an average weight of 0.01 g for each film. The obtained films were placed on the enamel surface of the teeth. The CP-ENFs obtained from P-CP, PP-CP, PS-CP, PPS-CP1, and PPS-CP which CP concentration was 1% were placed horizontally whereas those from PPS-CP3 which CP concentration was 0.5%, double films were placed, one in horizontal and the other in vertical directions, as shown in Figure 9, in order to receive the same amount of CP as in CP-ENFs obtained from 1% CP concentration EFASs. After that, the films were wet with 0.05 mL artificial saliva for each tooth and kept in a close container in a controlled temperature of 37 ± 1 • C and relative humidity of 100% for 8 h daily. After 8 h of each day, the films were removed. The teeth were cleaned with distilled water before color measurement. After that, the teeth were stored in the closed containers with sufficient artificial saliva during waiting for the new ENFs on the next day. These procedures were repeated for 14 days.
Pharmaceuticals 2020, 13, x FOR PEER REVIEW 16 of 20 until testing. Forty normal teeth without any surface stains and imperfections were selected. The baseline color at the middle surface of averaged areas of 3 mm 2 circular centers of the collected teeth was measured. The teeth were randomly allocated into eight groups, five teeth per group, according to the six CP-ENFs obtained from six different CP-containing EFASs and two negative control groups (a blank ENF and artificial saliva). Each day, the fresh ENF samples were cut into rectangle shapes having an area of 5 mm × 10 mm to obtain an average weight of 0.01 g for each film. The obtained films were placed on the enamel surface of the teeth. The CP-ENFs obtained from P-CP, PP-CP, PS-CP, PPS-CP1, and PPS-CP which CP concentration was 1% were placed horizontally whereas those from PPS-CP3 which CP concentration was 0.5%, double films were placed, one in horizontal and the other in vertical directions, as shown in Figure 9, in order to receive the same amount of CP as in CP-ENFs obtained from 1% CP concentration EFASs. After that, the films were wet with 0.05 mL artificial saliva for each tooth and kept in a close container in a controlled temperature of 37 ± 1 °C and relative humidity of 100% for 8 h daily. After 8 h of each day, the films were removed. The teeth were cleaned with distilled water before color measurement. After that, the teeth were stored in the closed containers with sufficient artificial saliva during waiting for the new ENFs on the next day. These procedures were repeated for 14 days. For tooth color measurement, the color of the teeth was measured by light reflection using a colorimeter (FRU WR10 portable precision colorimeter, Shenzhen Wave Optoelectronics Technology Co., Ltd., Shenzhen, China). Before measurement, the colorimeter was validated for color by means of a spectrocolormeter (UltraScan XE, Hunter Lab, Reston, VA, USA). The scale of the Commission International de l'Eclairage (International Commission on Illumination: CIE) was applied as follows. The measurement reflects three color parameters: L*, a*, and b*. The L* indicates the lightness ranging from black (L* = 0) to white (L* = 100). The a* indicates red-green color, where a positive a* indicating red and a negative a* indicating green color. The parameter b* indicates yellow-blue color, where a positive b* indicates yellow and a negative b* indicates blue color. The ΔE value is often used in order to indicate the perceptible tooth color changes after treatment and can be calculated by using the CIE L*a*b* system and the following equation. ∆E = [(∆L*) 2 + (∆a*) 2 + (∆b*) 2 ] ½ where ΔL* = L* baseline − L* after bleaching, Δa* = a* baseline − a* after bleaching, and Δb* = b* baseline − b* after bleaching [62]. The data were collected and calculated for bleaching efficiency evaluation.
Statistical Analysis
Descriptive statistics for continuous variables were calculated and reported as mean ± standard deviation. The analysis of experimental data was performed using SPSS statistics software version 22 with a one-way analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05). For tooth color measurement, the color of the teeth was measured by light reflection using a colorimeter (FRU WR10 portable precision colorimeter, Shenzhen Wave Optoelectronics Technology Co., Ltd., Shenzhen, China). Before measurement, the colorimeter was validated for color by means of a spectrocolormeter (UltraScan XE, Hunter Lab, Reston, VA, USA). The scale of the Commission International de l'Eclairage (International Commission on Illumination: CIE) was applied as follows. The measurement reflects three color parameters: L*, a*, and b*. The L* indicates the lightness ranging from black (L* = 0) to white (L* = 100). The a* indicates red-green color, where a positive a* indicating red and a negative a* indicating green color. The parameter b* indicates yellow-blue color, where a positive b* indicates yellow and a negative b* indicates blue color. The ∆E value is often used in order to indicate the perceptible tooth color changes after treatment and can be calculated by using the CIE L*a*b* system and the following equation. where ∆L* = L* baseline − L* after bleaching, ∆a* = a* baseline − a* after bleaching, and ∆b* = b* baseline − b* after bleaching [62]. The data were collected and calculated for bleaching efficiency evaluation.
Statistical Analysis
Descriptive statistics for continuous variables were calculated and reported as mean ± standard deviation. The analysis of experimental data was performed using SPSS statistics software version 22 with a one-way analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05).
Conclusions
The present work is the first study on stabilizing CP by entrapping in the solid state of nanofibrous films using an electrospinning technique. The study demonstrates that the ENF in suitable concentrations of PVA, PVP, and silica is a promising substrate to entrap CP for improving its stability and tooth bleaching activity. PVA mainly acts as a core of the nanofibers while PVP and silica are the important substances for enhancing CP stability and drug loading efficiency of the obtained ENFs. CP, PVP, and silica also affect the viscosity and conductivity of EFASs, and this can influence the morphology and size of the obtained ENFs. The adhesive property of the ENFs is mainly influenced by PVA and PVP. The ENF obtained from PPS-CP3, composed of 0.5% CP, 5.5% PVA, 3% PVP, and 1% silica, is a promising system for CP as it provides good morphology with the highest drug entrapment efficiency and desired controlled release profiles. The release of CP from this CP-ENF is the first-order kinetics. The mechanism of drug release is a non-Fickian diffusion or anomalous transport according to the Korsmeyer-Peppas model. This ENF provides excellent ex vivo tooth bleaching efficiency and is suitable for further in vivo investigation to evaluate the potential efficacy of this innovation. | v3-fos-license |
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"year": 2016
} | pes2o/s2orc | Atomic Force Microscopy Protocol for Measurement of Membrane Plasticity and Extracellular Interactions in Single Neurons in Epilepsy
Physiological interactions between extracellular matrix (ECM) proteins and membrane integrin receptors play a crucial role in neuroplasticity in the hippocampus, a key region involved in epilepsy. The atomic force microscopy (AFM) is a cutting-edge technique to study structural and functional measurements at nanometer resolution between the AFM probe and cell surface under liquid. AFM has been incrementally employed in living cells including the nervous system. AFM is a unique technique that directly measures functional information at a nanoscale resolution. In addition to its ability to acquire detailed 3D imaging, the AFM probe permits quantitative measurements on the structure and function of the intracellular components such as cytoskeleton, adhesion force and binding probability between membrane receptors and ligands coated in the AFM probe, as well as the cell stiffness. Here we describe an optimized AFM protocol and its application for analysis of membrane plasticity and mechanical dynamics of individual hippocampus neurons in mice with chronic epilepsy. The unbinding force and binding probability between ECM, fibronectin-coated AFM probe and membrane integrin were strikingly lower in dentate gyrus granule cells in epilepsy. Cell elasticity, which represents changes in cytoskeletal reorganization, was significantly increased in epilepsy. The fibronectin-integrin binding probability was prevented by anti-α5β1 integrin. Thus, AFM is a unique nanotechnique that allows progressive functional changes in neuronal membrane plasticity and mechanotransduction in epilepsy and related brain disorders.
INTRODUCTION
Epilepsy affects ∼3 million people in the U.S. Currently there is no cure to epilepsy, nor a way of preventing epileptogenesis, the process by which a normal brain develops epilepsy due to a variety of risk factors including injury or genetic predispositions. Extracellular matrix (ECM) proteins in the brain are produced and secreted by neuronal cells. During neural development, ECM molecules play a critical role in neuroplastic events in the hippocampus, a key region involved in epilepsy. Neuroplasticity is described as the adaptive changes to extrinsic or intrinsic encounters, such as epileptogenic injury or brain injury by the nervous system (Browne and Holmes, 2001). Integrins are a large family of transmembrane heterodimeric receptors (α,β subunits) that provide a connection between ECM and the intracellular focal adhesion complex (FAC) molecules including non-receptor tyrosine kinases (NRTKs) focal adhesion kinase (FAK) and Src, as well as cytoskeleton proteins, including talin and actin (Wu et al., 1998(Wu et al., , 2001Pinkstaff et al., 1999;Wu and Reddy, 2012).
Neuronal membrane elasticity, plasticity, and related dynamics can greatly affect the neuronal responses to network discharges and epileptic seizures that can be studied by sophisticated atomic force microscopy (AFM). The AFM, invented in 1986 (Binnig et al., 1986), has emerged to be a powerful instrument for studying ligand-receptor and cell-cell interactions, as well as the mechanical properties of living cells in the neuronal and other biological research. The AFM passively senses the localized forces between AFM scanning probe and molecules on the cell surface under unique three-dimensional (3D) movements through an extremely fine sharp cantilever tip (size-nanometer). AFM can provide information regarding adhesion/binding force between molecules up to piconewton (pN) and high resolution 3D surface structural imaging up to nanometer. In addition, AFM can directly measure the association between cell mechanical properties (e.g., elasticity) and intracellular cytoskeleton proteins and organelles. Future direction in these mechanotransduction studies points to the combination of AFM technology with patch-clamp technique, confocal microscopy, and total internal reflectance fluorescence for probing cellular structure, function and signaling (Kassies et al., 2005;Trache and Meininger, 2005;Sun et al., 2009;Wu et al., 2012a). In this study, we describe an optimized AFM protocol and its application for measurement of membrane plasticity and mechanical dynamics of individual hippocampus neurons in mice with chronic epilepsy.
MATERIALS AND EQUIPMENT Neuronal Cell Isolation Materials
All equipment and materials for hippocampus slice preparation, single cell isolation and AFM probe coating preparation are commercially available (e.g., VWR and Fisher Scientific). In order to easily duplicate this experiment, equipment and materials have been listed with their company's name, comments, as well as catalog number or model number in the Table 1. All chemicals, except if specifically stated, were obtained from Sigma-Aldrich. ECM protein and antibody were purchased from BD Biosciences.
Brain Slice Preparation
(1) Transverse hippocampal slices (thickness = 400 µm) were prepared from adult C57BL/6J strain male mice (2-4 months old) and were utilized for dissociation of neuronal cells such as dentate gyrus granule cells (DGGCs) (Reddy and Jian, 2010;Wu et al., 2013;Carver et al., 2014). All procedures were performed in strict compliance with the guidelines of National Institutes of Health Guide for the Care and Use of Laboratory Animals under a protocol approved by the university's Institutional Animal Care and Use Committee. (2) Mice were anesthetized with isoflurane (Notes 1 in Box 2).
Then, the brain was rapidly removed and kept with 4 • C in ACSF bubbled with carbogen gas (95% O 2 + 5% CO 2 ) (Notes 2). Animals were euthanized after decapitated. (3) Several 400 µm slices were cut with a Vibratome. Brain slices were equilibrated at 23 ± 1 • C in ACSF on a mesh surface in a small beaker or Brain Slice Keeper in a water bath continuously with carbogen gas (Notes 3).
Dissociation of Subfields of Hippocampus
(1) The hippocampi including the DG region (similar protocol for CA1 or CA3 region) were microdissected carefully under the dissecting microscope (Wu et al., 2013). (2) The isolated subfield slices were then incubated in Brain Slice Keeper in ACSF for 1 h at 24 • C. For non-enzyme procedure, skip next step 3.2.3. (3) The slices were transferred into ACSF with protease XXIII (3 mg in 1.0 ml), and incubated for 24 ± 1 min at 23 ± 1 • C. Then, the DG slices were washed by 1 ml ACSF for 3 times. It scans areas about 90 µm in x-y-and 6 µm in z-axis. A sharp AFM probe tip (nanometers in diameter) and AFM probe with bead (micrometers in diameter) are shown in the insert. (B) "a and b," screws to fine-tune laser position on the back of AFM probe cantilever; "c and d," screws to fine-tune laser position in the photodiode detector; "e," O-ring to secure the fluid holder from the liquid; "f," AFM probe holder. AFM probe includes supporting chip, cantilever and the tip. "g," supporting chip in the AFM probe in the cartridge of the AFM holder (f); "h," "V"-shaped cantilevers in the AFM probe, which contain the pyramidal tips in the end, and one pyramidal tip is shown in (A) insert and Figure 2; "I," Screws in the AFM stand for AFM Scanner (loose the screw to secure the AFM head and tighten the screw to release the AFM head); and "j," laser position and intensity indicators (red color bar) in the AFM Scanner.
(4) Triturated the slices to separate the cells. (Notes 4). The suspended cells were carefully pipetted into the glass bottom dish for AFM (Wu et al., 2013).
The Principle of AFM
Conventional optical microscopy can examine live sample morphology and remodeling following time, as well as proteins specificity and density if combined with fluorescence. AFM is based on a laser tracking the deflection of a sharp nanometer cantilever tip, while simultaneously sensing the local force, energy, loading, and stiffness between the molecule on the tip and sample surface in real-time (Figure 2A). The advantages of AFM include: (1) the data are recorded by sensing the sample surface and underneath without using the light, even though most AFM systems are integrated with optical imaging; (2) the nano-sensor on the tip is able to probe single molecular events in living cells. It is the only tool that enables us to visualize the sub-molecular resolution of the major and minor grooves of the DNA double helix under physiological conditions. This is essential for considering the structure-function relationship of biomolecular systems in vivo and for in situ analysis of DNA-based nano-devices (Ido et al., 2013;Pyne et al., 2014); (3) the probe serves as nano-manipulation tool for pressing, pulling and rolling on cell surface; and (4) that AFM is the only microscopic method available to provide both functional and structural information at a high resolution. However, AFM has following limitations: (1) the resolution will depend on the tip radius and cantilever spring constant. The physical AFM probe used in imaging is not always associated with the cell geometrical features. Consequently, the AFM image will not reflect true cell topography. In addition, a blunt or contaminated tip may cause imaging deformations, especially in freshly isolated cells. These types of artifacts can be easily avoided by using sharp clean AFM tips with more sensitive spring constant cantilevers, as well as selecting high resolution and slower scanning on the cell surface; (2) the measurement is only at the apical cell surface; (3) evaluations of stiffness and imaging are occasionally difficult because of the irregular topography and complex mechanical properties in cell membrane; and lastly, it requires open configuration for the research on living cell and very good vibration or acoustic insulation to avoid the noises. AFM tips/probes come in different shapes and sizes, such as sharp pyramidal nanometer tip made from silicon or silicon nitride or glass/polystyrene micrometer beaded tips (Figure 1A, insert). The AFM probe sensitivity depends on the property of the flexible cantilever. The tip diameter governs the spatial resolution, the smaller in the apex size of AFM probe and the higher the resolution in imaging and force measurement. The closed loop feedback piezo-control system in the AFM program permits for the monitoring of binding forces between the tip and cell surface, and through digital/analog converter, and also controls the piezoelectric scanner that monitors movement of AFM probe on the surface of the cell. The movement of the laser beam that detects deflections of cantilever will be noticed by segmented photodiode detector (Figure 2A). The photodiode detector then sends back the signal to AFM program through analog/digital converter system.
The Maneuver of AFM
The AFM recording can be achieved through tapping (intermittent) and contact modes.
The Tapping Mode
During the mapping of the sample, the cantilever in AFM probe is oscillated at its resonant frequency (bouncing up and down) under an external electrical excitation, lightly "taps" on the cell surface, and contacts the surface at the bottom (z-axis) of each swing at each given xy-point. By maintaining constant oscillation amplitude, a constant tip-sample interaction is maintained and an image of the surface is obtained as described by Wu and colleagues (Wu et al., 2012b). The advantages of tapping mode include (a) allowing high nanometer resolution of samples; (b) the ability to be used for freshly isolated cells that are loosely held to the bottom of a dish; and (c) the ability to record the height of the image. The disadvantages of tapping mode are that (a) it does not offer a good image in a liquid; (b) the samples are possibly damaged and (c) slower scan speeds are needed for the tapping mode of operation, otherwise it will be too noisy in the data recorded in the liquid. Recent PeakForce quantitative nanomechanical mapping (PeakForce QNM) technique is based on tapping mode and the acquisition frequencies up to 1 kHz in liquid. PeakForce QNM simultaneously generates height, adhesion, and modulus data, whereas Tapping Mode yields only height and phase data.
The Contact Mode
In this mode, the spring constant of cantilever in the AFM probe is much less than the spring of the cell surface, so the cantilever will bend when it attaches to the cell. The force between the probe and the cell stays constant through the closed piezofeedback loop control system and the surface image is obtained by moving the z-scanner for each xy point. The "height" in the image reflects the true height data of the cell. The advantages of contact mode are that it is optimal for cultured cells, faster at scanning than tapping mode, and useful for rough samples FIGURE 2 | Diagram representation of the AFM and constant force curve. (A) AFM holder is rigidly connected to a 3-dimensions (3D or x-y-z-) piezoelectric component. The deflection of the cantilever will be detected by a laser beam and displayed position changes in the segmented photodiode while AFM tip moving at cell surface with xyz-axes. The NanoScope software is a feedback piezo-control system. It will control and record the cantilever deflection and the interaction forces. A PEG-ECM protein fibronectin (FN)-coated AFM probe and integrin receptor is also indicated. A force curve or 3-D image will be collected by the system. The AFM probe attaching a DGGC is showed in the inserted image. (B) Original force curves data recorded from FN-coated AFM probe on DGGC. FN-coated AFM probe is controlled to repeatedly (z-axis movement: 800 nm and scan rate = 0.5 Hz) approach/attaching (black trace) and retract/withdrawal (red trace) from single DGGC at a given "x" and "y"-axes. The stages of attaching and withdrawal are showed in the points 1-6. The insert image shows force volume imaging for mapping elastic features over axon hillock of living DGGC. While simple forces curve (B) records the force felt by the tip as it approaches and retracts from a point on the cell surface, the study of cellular mechanics often requires characterization of the distribution or variance of these forces over 3-D structures. A force volume contains an array of force curves over the selected cell area. Each force curve (z-axis movement) is measured at a unique x-y position in the area, and force curves from an array of x-y points are combined into a 3D array, or "volume" of force data. Dark region (label "2" within blue frame) represents less stiffness than light regions (label "1"). The quantization of normalized mean intensity will be used to analyze the difference of elasticity in different regions or different cells. Bar = 100 nm in (B). PEG, Polyethylene glycol; ECM, extracellular matrix protein.
and imaging analysis as well as obtaining more fine details of the sample in the "deflection" image presented. The disadvantages are (a) these images lose the true cell height information, (b) damage or deformation can occur to soft samples by movement on the sample surface and (c) contact mode is not optimal for imaging freshly isolated cells because of loosely held to a dish bottom. Contact mode is easier to manipulate and operate than tapping mode, and more convenient for switching between constant force and imaging mode. In this article, we describe a detailed protocol using AFM to perform integrin-extracellular matrix interactions in neuronal cells in constant force mode.
Constant Force Mode
In this mode, the AFM tip treated with ECM protein is brought into contact with the neuronal cell surface. The piezo-control system records the deflection signal of the cantilever by moving the z scanner over a predefined distance at each given xy point. In most cases, the xy-axis scan size is fixed, and the position of the probe is controlled in order to repeatedly contact and retract from the cell surface. The deflection signal from the cantilever tip's indentation is recorded and drawn as zposition vs. deflection of the cantilever tip, called as "force curve" (Figure 2B, Video 1). The indentation in the contact point is related to the shape of the tip, the cantilever spring constant and the cell mechanical properties.
While single force curves (z-axis movement, Figure 2B) record the force sensed by the tip at a point of the cell surface, the study of cellular mechanics often requires the characterization of the distribution or variance of these forces over 3-D structures. A group of force curves across a selected cell area is reconstructed into a 3D array and called force volume, i.e., "volume" of force data (Figure 2B, insert). Here, a force volume elasticity map was constructed from axon hillock of DGGC in control mice. The dark pixel regions ( Figure 2B, label 2) represent less stiffness than the light regions (label 1). The disadvantage of force volume is that it is quite time consuming (hours needed for one 512 × 512 resolution). Current Fast-Force volume (acquisition frequencies: from <1 Hz to 300 Hz) measurement will include data of adhesion, force modulus, stiffness, and height.
Stepwise Procedures of AFM on Constant Force Mode
The AFM has been used to study in a wide variety of samples including biological samples Wu et al., 2010aWu et al., ,b, 2012bTangney et al., 2013a). In biological samples, the AFM technique has also been successfully applied in cardiomyocytes (Wu et al., 2010a;Tangney et al., 2013a), vascular arteriolar smooth muscle cells , arteriolar endothelial cells , and neuronal cells (Parpura et al., 1993;Pasternak et al., 1995;Kirmizis and Logothetidis, 2010;Wu et al., 2012a;Spedden and Staii, 2013). In the next section, we discuss the AFM contact mode on DGGCs (Box 1).
Labeling of AFM Probes with ECM Protein Fibronectin (FN)
For adhesion force measurement, the AFM probes are usually coated with the ligands of interest (e.g., FN) to permit the study of ligands and their specific surface receptor interaction (Wu et al., 1998(Wu et al., , 2010aSun et al., 2005Sun et al., , 2008Tangney et al., 2013b). The silicon nitride cantilever tip is first rinsed with acetone and let to air-dry for 1 min. Then the probe was installed on AFM cantilever holder (Figure 1, f). The polyethylene glycol (PEG, Sigma) was used to cross-link proteins onto silicon nitride tip at room temperature. The next step is to incubate the AFM probe tip with 30 µl 10 mg/ml PEG for 5 min, and then carefully remove the PEG, wash with phosphate buffered saline (PBS) 4 times, and incubate again with 20 µl 1 mg/ml FN for 60 s, then wash the tip with PBS 4 times. For biotin-labeled borosilicate bead AFM probe, 20 µl FN is added to the probe for 5 min, then wash the probes 5 times with PBS (Wu et al., 1998).
AFM Maneuver with Nanoscope III Software
(1) Put a 60 mm glass bottom culture dish with one drop of the freshly isolated DGGCs in 2 ml physiological bath solution for at least 30 min on the inverted microscope stage (equipped with 32 × lens). All AFM experiments were performed at 22-24 • C.
BOX 1 | Flow chart shows the general outline of contact mode.
(2) Start Nanoscope software (version 5.12. Notes 5). Select the "microscope icon" in the "NanoScope control" window ( Figure 3A). Turn on AFM system Conditioner, optic illuminator and the video camera box controller ( Figure 1A). (3) Install the AFM probe (Figure 1B, h) to the clear plastic AFM probe holder (Figure 1B, f). (4) Prepare the AFM probe cantilever with 1 drop of acetone, dry it, wash it with PBS 3 times, and coat the AFM probe with extracellular matrix protein fibronectin (FN) (see Labeling of AFM Probes with ECM Protein Fibronectin (FN)). (5) After labeling with FN, mount the plastic probe holder onto the AFM Scanner (AFM head in Figure 1A) with the Oring seal on (Figure 1B, e) to prevent a short circuit by the PSS solution (Notes 6). (6) Mount and secure the AFM Scanner to the position for AFM in microscope stage through one adjustable screw ( Figure 1B, i). (7) Align the laser beam, so that any deflection of AFM cantilever will be detected by photodiode detector through movement of the laser beam. To bring the laser onto the cantilever (Figure 1B, h; Figure 3B) and bring the bright red laser light indicator shown in the (photodiode detector) window (Figure 1B, j; Figure 2A), gently turning the top two screws (Figure 1B, a, b) on the AFM Scanner. In addition, a white bar will show the strength of laser light signaling in the "Sum" in "NanoScope image" window of the program (Figure 3B). Carefully adjust the two screws FIGURE 3 | Representative program screen shot illustrations. (A) AFM scan control in main NanoScope control screen with manual engage window (inside blue frame). (B) Laser signal changes during alignment of laser beam on AFM cantilever. Laser signal will be stronger when laser spot focuses on the center of cantilever and center of photodiode. (C) AFM probe main control and raw force curve during recording. The yellow line is drawn for measuring the AFM tip deflection sensitivity and sensitivity will be shown in the deflection sensitivity cell with blue frame in Channel 1 setting.
( Figure 1B, c, d) on the side of AFM Scanner to bring laser onto center of cantilever and center of the photodiode diagram that displayed with maximum signal bar ("Sum" normally >3, Figure 3B). (8) Select "Microscope" icon in "NanoScope control, " click "reset." The 4 red lights on electronic box ( Figure 1A) will be off now, and the green light will start flickering. (9) Fine-tune the focus to observe the cell, and then move the focus to well above the cell surfaces. (10) Click the green "DOWN" arrow button in "NanoScope control" to lower the AFM Scanner down to the liquid, select "Manual" and press on "Approach" to lower the cantilever down into the buffer, and finally lower it into the focal plane ( Figure 3A, insert in blue frame) (Notes 7). Use the x-or y-axis knob (in the stage of microscopy) to bring the tip of cantilever to the blue crosshair in the center of video monitoring ("vision system") window in the program. (11) Make sure the laser spot is still in the center of the photodiode diagram with fine adjustment of screw "c" and "d" in Figure 1B. (12) Before collecting the cell force curve data, the AFM probe sensitivity has to be checked using AFM tip to touch and withdraw from the non-cell region (Notes 8). The operating steps are similar to step 13 through 19. Check the sensitivity by drawing a line parallel with the force curve in points 2-3 as described in Figure 2B. The deflection sensitivity should change automatically in the "Channel 1" frame of the "NanoScope Control" Window ( Figure 3C, blue frame). Record this number in your notebook for future data analysis. (13) Fine-tune the focus until the cells are clearly seen using the joy stick ( Figure 1A, stage controller). Use the x-or y-axis knob (in the stage of microscopy) to move a cell into the center of the view. (14) Change configurations as Figure 3A for contact force mode on the AFM "NanoScope control" window of the program.
The example values are scan size = 0, scan rate = 0.5 Hz, and deflection setpoint = −0.2 to vertical deflection. (15) Repeat step 10, and keep clicking on "Approach" button until the AFM probe is lowered to the cell surface, but the cantilever still appears unfocused. (16) Select "OK" in "Manual Engage" window, the step motor will automatically bring the tip down to the cell. AFM will automatically start scanning the cells in Contact Mode when the AFM tip senses the deflection equal to deflection setpoint. (17) To generate a topographic image of the cell surface, set the instrument to apply a constant force on the cell. In each horizontal line scan, record both height data (z axis) and the position of the probe (deflection data) in the "NanoScope image" window. At this point, we skip this step and proceed to next step since we only discuss constant force mode in this article. (18) Press on the "Scale" button in "NanoScope control, " the force curve mode operation will be started. The real-time force curves will be shown in the window of "NanoScope image" (Figure 3C). (19) Press on "Setpoint 0" to reset the force curve into the center of y-scale, adjust the force curve position on the x-scale by moving the "Z-Scan Start" in "Main Controls" window back and forth. Modify the Scan rate and ramp size (e.g., 800 nm) as needed (Figures 3A,C). (20) To continuously record force curves (Video 1), press "Capture" in the menu bar on "NanoScope control" window and click "continue." To stop recording, click "Capture" menu again, and then click "abort." (21) Press the "Eye" icon on the window of "NanoScope control" to return AFM into contact mode imaging. Kimwipes, a nonabrasive and low-lint paper. Leave the AFM probe holder dry on the black holder stand for next time use.
Data Analysis
To measure cell membrane stiffness (i.e., elasticity. Figure 2B), the approach force curves will be used (black line). Fit the approach force curves with the Hertz Model between points 2 and 3 using MATLAB software (Mathwork, Inc.) or use NForceR software (copyright, 2004) to calculate the cortical stiffness based on tip displacement and membrane indentation as described by Wu and colleagues (Wu et al., 2012b). Use the retraction or withdrawal curve (red-line) to analyze the specific adhesion forces related to bonding between AFM tip and cell surface as described previously (Wu et al., 2010a). During retraction, if a specific adhesion event occurs, it will be detected as small sharp shift (bond rupture, point 5) in the deflection curve obtained during probe retraction from the cell surface. No adhesions will be apparent as a smooth retraction curve similar in appearance to the approach curve. These deflection shifts in withdrawal curve, referred to as snap-offs, will be recorded. The "snapoffs" or rupture force represents the force expected to cause adhesive binding breakdown between the ligands coated at AFM probe and receptor in a cell surface (e.g., FN and α5β1-integrin receptor), and is termed as adhesion force (Shroff et al., 1995;Sun et al., 2005). All snap-offs or adhesion force between FN and its integrin receptor on DGGCs will be collected and charted as a function of the rupture events. Hooke's Law will be used to determine the adhesion force (i.e., rupture forces or unbinding force): Where k is the spring constant of the AFM probe cantilever and d is the height of the snap-offs in the withdrawal curve (in Figure 2B, point "5").
Data Analysis
Group averaged data are represented as means ± S.E.M. Statistical evaluations are achieved with repeated-measures analysis of variance (ANOVA) followed by post-hoc tests, or with independent two-tail t-tests, as appropriate. The results are considered to be statistically significant if p values are < 0.05.
ANTICIPATED RESULTS AND DISCUSSION
In this protocol, we successfully utilized and optimized the AFM technique for the measurements of neuronal membrane elasticity, and related dynamics that are found to be drastically altered in epilepsy. AFM probe coated with FN is employed to the DGGCs to measure the adhesion force and the cell membrane elasticity between FN and integrin receptor in the DGGC surface. Force curves are collected through continual approach and withdrawal cycles of the AFM probe at certain scan rate (e.g., 0.5 Hz) and z-axis movement as described by Wu and colleagues (Wu et al., 2012b). During the FN-coated AFM probe travels to reach the DGGC cell membrane (Figure 2B, black "approach" line, point 1-2), the curve remains flat. After contacting the cell surface, the cantilever will be bent because of the cell membrane elasticity and the position of laser beam will be changed on detector (point 2-3). Point 2 represents a "reflection point or contact point." Data in the region of points 2-3 are used to fit using Hertz model to calculate the cell cortical stiffness/elasticity. The stiffer the cell, the less the indentation and the steeper the upslope of the force curve (such as 3 ′ represents glass surface). As the probe retraction starts (red "retraction" line), the resistance force will be decreased (point 3-4). The snap-off that represents a bond rupture, termed adhesion force, between AFM tip and the DGGCs is shown in the retraction line (red-line point 5). As seen in Figure 2B, the example trace shows 2 adhesion events (bond rupture) that occurred when the FN coated-probe retracted. When all adhesions between the FN-coated probe and DGGC have been broken, the retraction curve again overlies the initial approach curve level (point 6). The data shown here was recorded while the AFM tip located 25% away from the boundary of the neurons body. Some additional technical notes are listed in Box 2.
To calculate adhesion force between FN and its integrin receptor, the distribution of adhesion force and the observed "snap-offs" events in the retraction curve were analyzed with Gaussian distributions. The data revealed a good covenant between the original data (gray histogram) and the fitting line ( Figure 4A, red line). The initial peak of the FN-integrin single bond unbinding force (i.e., adhesion force; Sun et al., 2005;Wu et al., 2010a) was about 50.7 ± 1.4 pN (n = 10 cells from 3 to 4 mice). Previous works in our laboratories and by others have reported that the adhesion force between FN and α5β1integrin is between 35 and 80 pN (Li et al., 2003;Sun et al., 2005;. The bar graph in the right margin of the Figure 4A showed the probability of adhesion between FNintegrin receptor. The probability of adhesion events, expressed as the percentage of the force curves with snap-offs divided by total curves collected, was 66% in control mice group. The α5β1-integrin has been documented to bind FN (Wu et al., 1998(Wu et al., , 2010a. Since α5-integrin subunit was associated only BOX 2 | Additional technical notes on AFM protocol.
Protocol Notes:
(1) When working with isoflurane, always work under a fume hood.
(2) For a great yield of healthy cells: (a) remove the brain tissue from the anesthetized mouse as quick as possible and then put in ice-cold solution; (b) the incubation time 23-25 min at a temperature of 24 • C should be strictly followed; (c) young adult mice (2-3 months) are better than older animals (>6 months).
(3) Carefully isolate hippocampal subfields under dissecting microscope and transfer brain slices to Brain Slice Keeper.
(4) Don't over triturate because it will damage the freshly isolated cells.
(5) Always turn on computer first in AFM system.
(6) Avoid overfilling the bath solution, there should be no liquid past the AFM probe O-fluid cantilever holder in order to avoid burning the AFM head by short circuit. If the poles touch the liquid, clean and remove the liquid immediately.
(7) Do not bring the AFM tip all the way down to the cell during the manual approach; you may damage the probe and/or the cell.
(8) Test probe cantilever sensitivity before collecting data by moving among different cells and validating that the AFM probe was not damaged or contaminated during the preparation or approaching processes.
(9) Backup your data and put into another secure place for data protection. PC with RAID-enabled system is recommended.
with the β1-integrin subunit (Hynes, 1992), the anti-α5-integrin monoclonal antibody (60 nM) was used to block FN binding to α5β1-integrin subunits. The initial peak of the adhesion force had no significant change after application of anti-α5-integrin in the bath solution (47.7 ± 0.4 pN, n = 10; Figures 4B,C). However, the adhesive probability was decreased by 31% after application of anti-α5-integrin ( Figure 4B). The integrated force value was verified as averaged force from all adhesion events. Pretreatment with antibody exhibited less spread under the adhesion force density distribution curve than with FN alone (i.e., reduced the area). It indicated less total adhesion events (Figures 4A,B). The integrated adhesion force between FN and the DGGCs was 63.8 ± 1.2 pN and was reduced by 20% in the presence of α5-integrin monoclonal antibody ( Figure 4D). The data from this study indicated that the adhesion probability to integrins significantly declined, but not the adhesion force in the presence of α5β1-integrin monoclonal antibody. These results are similar to our previous observation in cardiomyocytes (Wu et al., 2010a). The results above also indicated that α5-integrin monoclonal antibody, as a competitive inhibitor reduced the availability of integrin to the FN, presumably acted by inhibiting FN from interacting with integrin on the cell surface. Therefore, the adhesion probability and integrated force between FN-integrin were decreased. These data supported the α5β1-integrin specificity of the binding to FN.
As a non-specific protein control, bovine serum albumin (BSA)-coated FM probes were examined. BSA showed a significantly reduced adhesion probability and adhesion force with cell membrane compared to FN (−59% and −55%, respectively; n = 10; Figure 5). This confirms that the adhesion force between FN and integrin receptor in the cell membrane is specific binding.
In DGGCs from stage 5 epileptic mice, the adhesion force and adhesive probability between FN and cell surface were significantly lower (−49% and −23%, respectively) when compared to control mice (n = 10; Figure 6). The integrated force was decreased by 42% in DGGCs from epileptic mice. To quantitatively calculate the cell stiffness or membrane elasticity, the portion between points 2 (deflection point or contact point) and 3 in the approach curve ( Figure 2B) was analyzed. Figure 7A FIGURE 4 | Summary of adhesion force results with the FN coated AFM probe in mice DGGCs. (A) Analysis of adhesion force-adhesion event plots during FN-coated probe retraction. The observed adhesion force and corresponding number of events in the experiments (50 curves/cell for a total of 500 curves) were plotted as histograms. Red line represents the results that fitted with multiple Gaussian distributions. Insets: integrin-FN binding probability (solid bar). (B) Force-adhesion event plots and integrin-FN binding probability (solid bar) in the presence of function-blocking antibody against α5-integrin (60 nM). (C) Summary of the adhesion force that represents the first peak force. (D) Summary of the integrated force that represents the total area under the force-events distribution curves. Adhesion force was not changed in the presence of α5-integrin monoclonal antibodies. Integrated force, which provides a metric reflecting the average overall adhesiveness, was decreased by α5-integrin monoclonal antibodies. *P < 0.05 vs. DGGCs in control (FN-coated probe alone). n = 10 for each group.
FIGURE 5 | Specificity of adhesion force in DGGCs by FN. The peaks of adhesion force and binding probability using bovine serum albumin (BSA)-coated AFM probes as non-integrin ligands were significantly smaller than that using FN-coated AFM probes. *P < 0.05 vs. DGGCs in FN. n = 10 for each group.
showed the continuous changes in the stiffness/elasticity values during time course for cells from control and epileptic mice. The stiffness in epileptic mice was high in all given time and no-time dependence. The average value of cell stiffness after FN coated probe approached the cell membrane at 1.77 ± 0.03 kPa ( Figure 7B). In epileptic mice, the cell stiffness showed a significant increase (2.96 ± 0.07 kPa). It has been suggested that increase in stiffness is associated with changes in integrin expression, [Ca 2+ ] i levels and activation of cytoskeletal filaments (Paul et al., 2000;Rueckschloss and Isenberg, 2004;Wu et al., 2010a). The changes of cell elasticity might be associated to cell remodeling, dispersion of the DGGC layer and the appearance of neurons in ectopic locations during development of epilepsy.
In epilepsy, increased integrin expression and increased apoptotic cell death and neuronal proliferation in the DGGCs, principal excitatory phenotype neurons, cause hyper-synchronization leading to development of epilepsy (Gall and Lynch, 2004;Kokaia, 2011). During epileptogenesis in both human and rodent, DGGCs undergo extensive remodeling, including reorganization of mossy fibers, dispersion of the DGGC layer, and the appearance of DGGCs in ectopic locations within the dentate gyrus. Integrin and FAC recruitment, integrin-ECM detachment and attachment have been reported to dynamically change their position at leading and trailing edges in migrating cells (Becchetti and Arcangeli, 2010;Huttenlocher and Horwitz, 2011). In order for DGGCs to spread or relocalize within the hippocampus they need to modify their anchoring positions to the ECM (binding change) and their cytoskeletal architecture (cell elasticity change). Cleavage of adhesive connections (i.e., unbinding) and changing cell shape for migrating (i.e., elasticity change) are early steps in the formation of new synaptic configurations (Chang et al., 1993). To gain a fundamental understanding of epilepsy related changes in DGGCs at nanoscale resolution, it is necessary to first ask how cells attach, spread, and migrate through dynamic integrin receptor activation. Our results indicate that changes in adhesion force and probability, as well as cell membrane elasticity, may contribute to epileptogenesis.
In conclusion, these results suggest that the AFM is a cuttingedge nanotechnique for studies of dynamic membrane plasticity and its progressive alteration with brain injury or disease. Our AFM data showed that FN-integrin interactions in DGGCs drastically modulate adhesion force and membrane elasticity in epilepsy mice. Thus, the AFM method provides a unique tool for molecular investigations of neuronal membrane dynamics in a variety of neurological diseases and brain injury models.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnagi.
2016.00088
Video 1 | Represents contact force mode recording (available in online version). Raw force curves generated using FN-coated AFM probe (also see Figure 2 for details). FN-coated cantilever (1 mg/ml) were controlled to repeatedly approach (black trace) and retract (red trace) from cell while xy-axes fixed (right panel). The snap-off that represents bond rupture, termed adhesion force (left panel raw red traces). The example trace shows 3 adhesion events (bond rupture) between FN ligand (gradient brown circle under gray AFM probe in right panel) and 3-integrin receptors (gradient blue Y-shape) in the cell that occurred when the FN coated-probe retracted. | v3-fos-license |
2019-03-08T14:16:04.489Z | 2019-02-01T00:00:00.000 | 67790222 | {
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} | pes2o/s2orc | Alteration of renal Na,K‐ATPase in rats following the mediastinal γ‐irradiation
Abstract Na,K‐ATPase represents the key enzyme that maintains the homeostasis of sodium and potassium ions in the cells. It was documented that in directly irradiated organs the activity of this enzyme is decreased. The aim of present study was to clarify the remote effect of irradiation in mediastinal area on the activity of the Na,K‐ATPase in kidneys in rats. Ionizing radiation in single dose 25 Gy resulted in consequent decrease of the body weight gain as well as the size of kidneys in Wistar rats. In addition, radiation induced alterations in the oxidative status of blood plasma. Irradiation also decreased the activity of renal Na,K‐ATPase. Measurements of enzyme kinetics that were dependent on the concentration of energy substrate ATP or cofactor Na+ indicated that the lowered enzyme activity is probably a consequence of decreased number of active molecules of the enzyme, as suggested by lowered V max values. Immunoblot analysis confirmed the lowered expression of the catalytic alpha subunit together with decreased content of the glycosylated form of beta subunit in the renal tissue of irradiated rats. The ability of the enzyme to bind the substrate ATP, as well as Na+ was not affected, as shown by unaltered values of K m and KN a. Irradiation of the body in the mediastinal area despite protection of kidneys by lead plates during application of X‐ray was followed by significant decline of activity of the renal Na,K‐ATPase, what may result in deteriorated homeostasis in the organism.
Introduction
Radiation therapy is commonly used therapeutic procedure in oncology. The effects of radiation therapy are mediated, in addition to their direct impact on DNA, by the production of free radicals. Proper functioning of membranes is unavoidable for biological systems, and their integrity is essential for normal cell functions. Radiation-induced increase in free radicals results in lipid peroxidation, leading to structural and functional damage to cellular membranes (Purohit et al. 1980). The damage to membrane organization is an initial step in cell death. During radiotherapy, formation of hydroperoxides in membranes would result in the damage of membranebound enzymes. One of these enzymes is the Na,K-ATPase or so called sodium pump. In all living cells, this enzyme keeps the intracellular balance of Na + and K + by transporting three ions of Na + out of the cell in exchange for two ions of K + into the cell utilizing the energy from ATP hydrolysis. In our previous study, using enzyme kinetic measurements of the cardiac Na,K-ATPase in 20weeks-old male rats, we observed impairment in the affinity of the Na + -binding site together with decreased number of active Na,K-ATPase molecules 6 weeks after mediastinal gamma-irradiation at a dose of 25 Gy. These changes most probably result in deteriorated efflux of the excessive Na + from the intracellular space in hearts of irradiated rats (M eze sov a et al. 2014). This enzyme having a key role in maintaining the cellular homeostasis of sodium ions throughout the organism was affected by irradiation in various other organs like intestine (Lebrun et al. 1998), kidney (Balabanli et al. 2006) and erythrocytes (Moreira et al. 2008;Chitra and Shyamaladevi 2011). The Na,K-ATPase represents the main consumer of intracellularly produced energy in the renal tissue (Welch 2006). It indicates the possible importance of the enzyme complications induced by radiotherapy. Our study was oriented to the investigation of probable indirect abscopal effect (a phenomenon where the response to radiation is seen in an organ/site distant to the irradiated area) of mediastinal irradiation of rats on functional properties of the enzyme in kidney. This organ is highly sensitive to radiation-induced late effects resulting in the so called radiation nephropathy which occurred dramatically during second to sixth weeks after local direct irradiation of porcine kidneys (Cohen et al. 2000;Cohen and Robbins 2003). However, there is still lack of information concerning the molecular mechanism of Na,K-ATPase disturbances in the above tissue after irradiation. Investigations of the enzyme properties in such condition bring new insight into the processes involved in maintenance of sodium homeostasis in kidney after radiotherapy.
Animal model and radiation
All procedures in this study were approved by the Institutional Animal Care Committee. Male Wistar rats were obtained from Velaz Praha (Czech Republic) and maintained in our animal care facility on a 12:12-h light/dark cycle with free access to food and water. At the age of 14 weeks, animals (n = 12) were anesthetized with thiopental (65 mgÁkg À1 b.w.). Rats were irradiated with 5 MeV/1 kW electron linear accelerator UELR 5-1S with tungsten converter to X-rays at the dose rate 10 Gy/ min (Producer NIIEFA St. Petersburg, RF). A single dose of 25 Gy was given locally on mediastinal area. Irradiation was directed transversally, crossing the chest of the rat at the heart level while the rest of the animal was shielded with 20 cm thick lead plates (Fig. 1). A single dose of 25 Gy to the heart corresponds to the cumulative dose of irradiation commonly used in patients. Six weeks after irradiation the rats were anesthetized with thiopental (65 mgÁkg À1 b.w.) and sacrificed by heart excision. Agematched Wistar male rats from the same breeding facility served as controls (n = 12). After excision of the heart, blood from the chest cavity was immediately collected. Sodium-salt heparin was used as anticoagulant. Kidneys were quickly removed, rapidly rinsed with ice-cold physiological saline, weighed, frozen in liquid nitrogen, and stored until use.
Biochemical analysis of oxidative status
Markers of protein oxidation were measured by spectrophotometric analysis of advanced oxidation protein products (AOPP) (Witko-Sarsat et al. 1996). Briefly, plasma samples (diluted 1:4 in phosphate-buffered saline (PBS), pH = 7.2) were mixed with glacial acetic acid. For the calibration curve construction, chloramine T with potassium iodide was used. The absorbance was measured at 340 nm.
The markers of carbonyl stressadvanced glycation end products (AGEs) and fructosamine were determined. AGEs were measured spectrofluorometrically. Samples were diluted with PBS in the ratio 1:4. Fluorescence was measured at kex = 370 nm and kem = 440 nm (M€ unch et al. 1997). For fructosamine measurement, 20 lL of samples and standards (1-deoxy-morpholino-D-fructose) were added to the microtiter plate. Thereafter, nitro blue tetrazolium was added, and the reaction was shortly mixed and incubated at 37°C for 15 min. The absorbance was measured at 530 nm (San-Gil et al. 1985).
The following markers of antioxidant status were measured: total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP). For TAC assessment, the plasma was mixed with acetate buffer (pH = 5.8). The initial absorbance was measured at 660 nm as blank. When ABTS solution (2.2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid with acetate buffer) was added, the absorbance (660 nm) was measured again. FRAP was measured after the addition of FRAP reagent (warmed to 37°C, composed of acetate buffer (pH = 3.6), tripyridyl-striazine, FeCl 3 .6H 2 O, and water) to the microtiter plate. Afterward, initial absorbance was measured as blank. The samples were added to the reagent and measured again at 593 nm (Benzie and Strain 1996).
The concentration of proteins was measured by bicinchoninic acid kit (Sigma-Aldrich, Munich, Germany), according to the manufacturer's instructions. The bovine serum albumin was used as standard. All measurements were performed on a Tecan Sapphire II Instrument (Gr€ odig, Austria) and reagents used for these measurements were obtained from Sigma-Aldrich (Munich, Germany).
Evaluation of markers of kidney function
Biochemical markers (creatinine and urea) were measured at certified biochemical laboratory (Medirex, University Hospital Ru zinov, Bratislava, Slovakia) using analyzer Olympus AU400 (Beckman Coulter, California, USA).
Histology of kidney
Kidney tissue was fixed in 10% formalin and routinely processed in paraffin. Histological sections were stained with hematoxylin and eosin and with phosphotungstic acid hematoxylin. The change in the size of the kidney glomeruli was morphometrically evaluated in slides stained with hematoxylin end eosin by measuring the cross-section area in pixel number with ImageJ 1.42q program (National Institute of Health, USA). The results were statistically analyzed by GraphPad Prism (GraphPad Software, USA). From each kidney several slices were analyzed amounting to more than 80 in controls and to more than 120 in irradiated group.
Phosphotungstic acid hematoxylin stain that identifies basic proteins was used for identification of mitochondrial matrix proteins (Silverman and Glick 1969). Bluestained grains in the cytoplasm of proximal tubules epithelium were isolated as an area by digital color extraction and morphometrically evaluated by the same program.
Assay of Na,K-ATPase activity
The plasmalemmal membrane fraction from kidney was isolated according to (Jorgensen 1974). Amount of protein was determined by the procedure of (Lowry et al. 1951) using bovine serum albumin as a standard.
All enzyme assays were carried out at 37°C using 10 lgÁmL À1 of membrane protein. The Na,K-ATPase activity was estimated in an assay buffer containing (in mmolÁL À1 ): 4 MgCl 2 , 100 NaCl, 10 KCl, and 50 TRIS (pH = 7.4). Subsequently, after 20 min of preincubation in substrate-free medium, the enzyme reaction was initiated by adding increasing amount of TRIS-ATP in the range of 0.16-8.00 mmolÁL À1 . The reaction was stopped after 20 min by adding 12% ice-cold trichloracetic acid. The liberated inorganic phosphorus originating from ATP hydrolysis was estimated according to the method of (Taussky and Shorr 1953). In order to establish the Na,K-ATPase activity, the ATP hydrolysis that occurred in the presence of Mg 2+ only was subtracted. The enzyme kinetics for sodium activation was determined by the same way. The concentration of NaCl varied in the range of 2-100 mmolÁL À1 and the amount of ATP was constant (8 mmolÁL À1 ). The kinetic parameters were evaluated by direct nonlinear regression of the obtained data.
Preparation of tissue fractions for electrophoresis and immunochemical western blot analysis
The tissue samples from kidneys were resuspended in icecold buffer containing (in mmolÁL À1 ): 50 Tris-HCl, 250 sucrose, 1.0 dithiothreitol, 1.0 phenylmethylsulfonylfluoride (pH 7.4) and homogenized with a glass-teflon homogenizer. The homogenates were centrifuged at 800 g for 5 min at 4°C; pellets obtained after this centrifugation were discarded and the supernatants were centrifuged again at 9300g for 30 min. Following the second centrifugation, the pellets were resuspended in homogenizing buffer supplemented with 0.2% Triton X-100 and centrifuged at 9300g for 1 min. The Triton X-100-soluble supernatants represented the particular fractions. The protein concentrations were estimated by the method of (Bradford 1976).
Electrophoresis and immunochemical western blot analysis
Samples of particular protein fractions (for a1 and b1 separated by sodium dodecyl sulfate-polyacrylamide gel (12%) electrophoresis (SDS-PAGE). For western blot assays separated proteins were transferred from gel to a nitrocellulose membrane overnight at 4°C. The quality of the transfer was controlled by Ponceau S staining of nitrocellulose membranes after the transfer. Specific antibodies against a1 (mouse monoclonal antibody from Sigma; product number A-277, in dilution 1:250) and b1 (mouse monoclonal antibody from Santa Cruz; C464.8: sc-21713, in dilution 1:200) subunits of Na,K-ATPase were used for the primary immunodetection. Peroxidase-labeled antimouse (from Cell Signaling; #7076, in dilution 1:1000) immunoglobulin was used as the secondary antibody. Bound antibodies were detected by the enhanced chemiluminescence detection method (Amersham Imager 600). Densitometrical quantification of protein levels was performed by comparison to loading control beta-actin (mouse monoclonal antibody [AC-15] from Abcam; ab6276, in dilution 1:1000 and corresponding anti-mouse secondary antibody) and using an ImageJ program.
Statistical analysis
All investigated parameters are expressed as means median, 10th, 25th, 75th, and 90th percentiles as vertical boxes with error bars. Mann-Whitney Rank test were used for statistical analysis. The differences were considered to be significant when the P-value was less than 0.05.
Health condition of animals
At the end of the experiment, the irradiated animals showed significantly lower (by 28%) body weight as compared with control rats. The importance of pathophysiological complications in our experiment was emphasized also by lower body weight gain in the irradiated group compared with control and also by mortality amounting 17% in the group of irradiated animals. The kidney weight was also proportionally decreased, resulting in similar kidney weight/body weight ratio in both groups (Table 1).
Plasma protein concentration and biochemical analysis of oxidative status
Total protein concentration in plasma was lower in irradiated rats compared with control (84 AE 5 control vs. 59 AE 5 irradiated, in g/L, P = 0.004). We observed statistically significant differences between the control and the irradiated group in the following parameters of oxidative stress and antioxidant status in plasma: TAC (530 AE 23 control vs. 264 AE 28 irradiated, in lmol/L, P < 0.0001), FRAP (670 AE 75 control vs. 345 AE 57 irradiated, in lmol/L, P = 0.005), and AGEs (5.15 AE 0.38 control vs. 9.77 AE 0.97 irradiated, in relative fluorescence units/L, P = 0.001). The experimental groups did not significantly differ in fructosamine plasma levels (0.46 AE 0.05 control vs. 0.49 AE 0.05 irradiated, in lmol/L, P = 0.66) and AOPP (56.8 AE 7.9 control vs. 46.3 AE 7.7 irradiated, in mmol/L, P = 0.36). All the results are summarized in the (Fig. 2).
Markers of kidney function
Creatinine and urea concentrations in plasma samples were not affected significantly by irradiation of rats in mediastinal area. The concentration of creatinine slightly decreased from 48.4 AE 8.9 to 40.6 AE 6.1 lmol/L, but this alteration was not statistically significant. Concentration of blood urea nitrogen was similar in both experimental groups amounting 7.4 AE 1.2 mmol/L in controls and 7.7 AE 0.7 in irradiated rats.
Histology of kidneys
The visual comparison of kidneys from irradiated rats compared with those from control rats already indicated some alterations. The kidneys from irradiated rats were smaller and ruddier (Fig. 3). Indeed, the kidney weight of irradiated rats was lowered by 21% but the kidney weight/body weight ratio was similar in both experimental groups (Table 1) indicating that the observed alteration does not represent any hypotrophy, but it is simply a consequence of lowered body weight gain. Kidney tissue in histological slides stained with routine hematoxylin and eosin did not show any remarkable morphological differences. Morphometric measurement revealed a significant decrease in the size of glomeruli in the irradiated animals (Fig. 4). Staining with phosphotungstic acid hematoxylin revealed a conspicuously increased density of blue-stained tiny grains representing mitochondria in the cytoplasm of epithelial cells of proximal tubules in the kidneys of irradiated animals (Fig. 4). This increase proved to be highly significant (Fig. 4).
Kinetic measurements of Na,K-ATPase activity
Irradiation reduced the renal Na,K-ATPase activity throughout the investigated concentration range of substrate as compared with controls. During the whole ATP concentration range, the decrease in activity represented 32-34% (Fig. 5). These changes in activities were reflected in statistically significant decrease in V max by 33% with no significant alterations of K m in irradiated rats (Fig. 6).
Comparing the response of Na,K-ATPase to increasing concentrations of the cofactor Na + we observed in the irradiated group again a considerable impairment of the enzymatic activity as witnessed by 14-17% decrease in the enzyme activity throughout the concentration range of NaCl (Fig. 7). These changes in activities were manifested in statistically significant decrease in V max by 14% with no significant alterations of K Na in irradiated rats (Fig. 8).
Western blot analysis of renal Na,K-ATPase
Analysis of Na,K-ATPase subunits by western blot (Fig. 9) showed a tendency to lower presence of a1 subunit in irradiated rats in comparison with control animals. Evaluation of b1 subunit expression revealed distinct results for glycosylated and unglycosylated forms of this subunit. On the one side, the presence of glycosylated form was lower by more than 30%, on the other side the expression of the unglycosylated form was higher by 20% in the renal tissue of irradiated rats when compared with controls ( Fig. 9). However, it should be mentioned that the cumulative presence of unglycosylated and glycosylated forms of b1 subunit remained unchanged after irradiation.
Discussion
It is known that various anticancer therapies including radiotherapy can lead to cardiovascular complications and their severity depends on many factors including the site of action, the applied dose, and the method of administration. Cardiotoxicity can occur immediately upon administration of the anticancer therapy or it may have a delayed onset (months or years after the treatment) (for review see Rygiel 2017). Experimental studies oriented to local irradiation of the heart/thorax of rats using doses of 15-30 Gy resulted in cardiotoxic effects during the time interval of 60-240 days after application (Wondergem et al. 1991). Our previous study concerning the cardiac Na,K-ATPase showed deterioration of the enzyme function 6 weeks after 25 Gy irradiation via the mechanism of lowered ability to bind sodium as well as the lowered number of active enzyme molecules (M eze sov a et al. 2014). In the present study, we focused our attention to possible remote effect of irradiation, i.e. if irradiation of the body in one locality can affect another, unexposed part of the body. For maintenance of sodium homeostasis throughout the organism, the appropriate functionality of Na,K-ATPase in renal tissue is very important. Therefore, we tried to bring new information concerning the effect of irradiation of rats in the mediastinal area on the properties of Na,K-ATPase in kidneys.
Plasma protein concentration and biochemical analysis of oxidative status
We were able to detect lower plasmatic concentration of proteins as a consequence of mediastinal irradiation. Hypoproteinemia post radiotherapy was already observed in other animal experiments (Nandchahal et al. 1990;El-Gazzar et al. 2016). Lower concentrations of plasma proteins usually reflect low albumin concentrations. Preoperative radiation for retroperitoneal sarcoma was associated with more frequent hypoalbuminemia in humans (Bartlett et al. 2014). The observed hypoproteinemia after irradiation may be the consequence of liver injury, as liver is the major contributor of plasma proteins. It was shown that radiation-induced liver disease involved hypoalbuminemia in patients who underwent radiotherapy for hepatocellular carcinoma (Furuse et al. 2005). The measurement of protein concentration postradiation is significant in clinics, since hypoalbuminemia was recognized as an independent predictor of poor outcome of selected malignities (Wang et al. 2009;Kang et al. 2016).
Together with alteration of protein concentration, negative impact of irradiation was also reflected in antioxidant status and oxidative stress in plasma. Lowered antioxidant status measured in the plasma after irradiation (TAC and FRAP parameters) is in concordance with human (Khalil Arjmandi et al. 2016) as well as animal (Manda et al. 2007) studies. Together with worsening antioxidant defense, we observed increased AGE formation that may be a consequence of increased production of reactive oxygen species (ROS), a potential cause of acute and chronic toxicity from irradiation (Alikhani et al. 2007;Yang et al. 2016).
Histology of kidneys
The seriousness of pathophysiological complications in our experiment was emphasized by lower body weight gain and also by mortality amounting 17% in the group of irradiated animals. The lower size of glomeruli could be related with the decreased size of kidneys as part of the whole reduction in body weight of the irradiated animals. Similar correlation has been described also in humans (Nyengaard and Bendtsen 1992). The functionality of kidneys seems to be altered as indicated by our histological observations showing decrease in the size of kidney glomeruli together with increased optical density of blue-stained tiny grains representing mitochondria in the tissue of irradiated animals. Surprisingly, our histological data providing information about increased density of mitochondria in the cytoplasm of epithelial cells of proximal tubules in surviving animals may be of physiological relevance. In this context, it is important to mention that only 83% of irradiated animals survived. So it might be suggested that altered presence of mitochondria in kidneys of irradiated rats may represent a possible compensatory effect to irradiation. It is known that renal epithelial cells contain high densities of mitochondria necessary to produce sufficient ATP for the active transport of Na + ions. Approximately 90% of oxygen extracted by mitochondria in the kidney is used for Na + reabsorption in the nephron (Welch 2006). From this point of view the alteration of renal mitochondria may be followed by alteration of functionality of Na,K-ATPase and extrusion of superfluous sodium out from cells.
Na,K-ATPase discussion
Even if, plasma creatinine and urea, the commonly used markers of renal function did not indicate significant functional alterations of kidney, the functionality of renal Na,K-ATPase was influenced by irradiation. Evaluation of our enzyme kinetic data provides two basic information about the mechanism of Na,K-ATPase alterations as a consequence of irradiation. Concerning the qualitative properties, the ability of the enzyme to bind the substrate ATP, as well as Na + was not affected, as shown by unaltered values of K m and K Na . Concerning the quantitative alterations, our study revealed new interesting findings. Based on the data of our present study the deterioration of the renal Na,K-ATPase may be hypothesized as a remote effect of irradiation of rats in mediastinal region (Fig. 10). The decreased Na,K-ATPase activity in irradiated rats is probably caused by lower number of active enzyme molecules as indicated by decreased value of V max when compared to the control group. This difference was significant in both types of kinetic studies, it means during the activation of the enzyme with increasing concentration of both -substrate ATP and cofactor sodium. So the Na,K-ATPase extrudes less effectively the superfluous sodium out from cells in the kidneys of irradiated rats. The above hypothesis based on enzyme kinetics is supported also by our western blot analysis results documenting a tendency of lower expression of the a1 subunit in the Na,K-ATPase in the renal tissue of irradiated rats. This subunit is generally recognized as the catalytic subunit of the Na,K-ATPase, because it contains the binding sites for substrate ATP, as well as for cofactors sodium and potassium, thus securing the transmembrane transport of the above ions against their concentration gradients. The alterations in the expression of the glycososylated and unglycosylated forms of the b1seem to be very important. This subunit is responsible for correct embedding of the a-subunit onto the surface membrane of the cell. The b-subunit of renal Na,K-ATPase is a sialoglycoprotein containing three potential N-glycosylation sites (Miller and Farley 1988;Ataei and Wallick 1992).
Previously it was documented that glycosylation of the Na,K-ATPase b1 subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation in the kidney cells (Vagin et al. 2006). In addition, it was shown that inactivation of Na,K-ATPase by deglycosylation is affected by interaction with surrounding lipids. It was documented that in the presence of dioleoylphosphatidylcholine, the deglycosylated enzyme was inactivated, whereas dioleoylphosphatidylserine protected the deglycosylated enzyme, and the Na,K-ATPase activity was preserved (Cohen et al. 2005). So, the increased presence of unglycosylated b1 on the expense of the glycosylated b1 subunit may be the reason of worse incorporation of the catalytic a1 subunit into the plasmalemma resulting in decreased number of active Na,K-ATPase molecules in the renal tissue of irradiated rats. The above deterioration of the Na,K-ATPase function in kidney as a consequence of remote irradiation in the mediastinal area may be mediated by reactive oxygen species as indicated by worsened oxidative status in the plasma of irradiated rats. This proposal is in agreement with previous observations that oxidative stress in other pathological situations like hypertension or diabetes reduced electrolyte transport efficiency (Welch et al. 2001) and caused mitochondrial uncoupling (Friederich et al. 2009). Reduction of tubular electrolyte transport efficiency was hypothesized as a result of an interplay of several different mechanisms, including altered paracellular electrolyte permeability, direct effects on Na,K-ATPase, and the shift of Na + transport to lessefficient nephron segments (Hansell et al. 2013). The increased presence of unglycosylated b1 subunit in the renal tissue of irradiated rats may be a consequence of deterioration of the nuclei forasmuch as in the kidneys of mice subjected to radiation dose 13-15 Gy, tubular cells with abnormally large nuclei were observed. For this abnormality it was suggested that it may belong to steps involved in cell loss in proximal tubules after irradiation (Otsuka et al. 1988).
Comparison of cardiac and renal Na,K-ATPase
The difference in the response of Na,K-ATPase depending on the distance from the site of irradiation seems to be an interesting fact. In the heart, which was subjected directly to irradiation, the enzyme showed decrease in the number of active molecules together with lowered ability to bind the energy substrate ATP and also sodium ions (M eze sov a et al. 2014). On the other hand, the present study showed that in kidneys which were during the irradiation procedure protected by lead shield, the enzyme revealed lowered presence of active molecules without significant effects on the ability to bind ATP and sodium.
Conclusion
It may be summarized that irradiation by 25 Gy in the mediastinal area of rats induced remote deteriorating effects in other parts of the body despite their protection by lead shield. In the mediation of this process, probably oxygen radicals are responsible as documented by worsened oxidative status of blood plasma. The functionality of the main consumer of the intracellular ATP in renal cells, the Na,K-ATPase, was also disturbed as documented by lowered number of active molecules as a consequence of worse incorporation of the catalytic alfa subunit into the plasmalemmal membrane. This system localized in the surface membrane of cells may represent one of the first systems injured by irradiation. | v3-fos-license |
2018-12-18T05:35:52.870Z | 2016-06-29T00:00:00.000 | 56365075 | {
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} | pes2o/s2orc | Effects of Extruded Soy Protein on the Quality of Chinese Steamed Bread
Five different extruded soy protein isolates (ESPIs) were obtained by extrusion and denoted by IVD1, IVD2, IVD3, IVD4, and IVD5. Then the SDS-PAGE results showed that the subunits of SPI decreased after extrusion, especially the subunits of 90.8, 32.8, and 31.3 kDa, whereas no isopeptide bond was formed. Although SPI improved both the development time (DT) and stability (S) of dough, ESPIs increased S but the DT decreased from 4.3min to 1.8–2.0min. Texture profile analysis (TPA) results showed that the hardness and chewiness of Chinese steamed bread (CSB) decreased in the order wheat flour+IVD2 (WF+IVD2), WF+SPI, WF+IVD4, WF+IVD1, WF+IVD3, WF, and WF+IVD5. As regards color, the total color ΔE decreased except for the WF+IVD1 (56.22); its positive and negative trends of L∗ and b∗ were invariant with the SPI or ESPIs mixture, whereas a∗ showed a positive trend. The sensory score increased from 82.7 to 83.4 with 3% of SPI addition and up to 87.8 when the substitution was IVD1. Therefore, SPI treated by extrusion may significantly improve the quality of CSB.
Introduction
Chinese steamed bread (CSB), a traditional fermented principal food of China, is gaining widely consumed by people in China and the emigrated Chinese people of many other Southeastern Asian countries [1].CSB is mainly composed of wheat flour (WF), water, and yeast, accounting for about 40% of wheat consumption for breakfast item in China every year [2].WF is the major ingredient of steamed bread and comprises mainly starch (about 70%-75%), water (about 14%), and proteins (about 10%-12%) [3,4].The quantities of protein and starch are important factors determining the gluten strength of dough; that is, CSB quality depends on dough strength.With the lack of lysine for WF, the amino acid balance of CSB was poor.What is worse, furosine is an indirect measurement of -N-formyl-(-N-deoxyglucose)-Lys, which is the major component of blocked Lys present after the early Maillard reaction [5].Bread may come into being a part of furosine when adding bran, rye, or maize to the wheat flour, respectively.Nevertheless a synergistic effect of suppressing furosine formation with some soybean flour mixture may occur simultaneously [6].
Soybean protein contains eight essential amino acids, especially Lys.It is mainly composed of 7S globulin or conglycinin and 11S globulin or glycinin.-conglycinin is a trimer comprising three subunits ( , , and ), whereas glycinin comprises six subunits, each making up an acidic polypeptide and a basic polypeptide [7].Soybean protein isolate (SPI) is widely used in the food industry because of its favorable water-holding capacity, oil-binding capacities, and other functional properties [8,9], and many researchers have also discovered the functions of soybean protein in alleviating osteoporosis and inhibiting hyperlipidemia and other physiological health functions [10,11].Therefore, adding some soybean protein to WF can not only improve the nutritional value of CSB but also be beneficial to human health.
WF mixed with some soy protein flour can provide high protein content and improve the balance of amino acids; it could also reduce the rate at which frozen storage attenuates quality of dough [12].Bread utilizing heat-treated (steamed and roasted) soy flour was likely to have a less beany odour and taste than those with non-heat-treated (raw and germinated) soy flour.Nevertheless, the bread with heattreated flour is perceived to have a better appearance and loaf volume than that with non-heat-treated flour [13].The sensory scores of bread increased from 40.2 to 52.2, when mixing WF with 6% of soy protein, and it does not have a negative effect on the body's absorption of iron, calcium, zinc, and other minerals [14].
Though oxidising improvers and surfactant improved dough strength of soy-wheat bread, they could not weaken soy-wheat bread beany flavors, especially at the high contents [15].Furthermore, the bioavailability of soy protein is limited by the trypsin inhibitors and hemagglutinin.These problems may be resolved by mild extrusion [16].Extrusion technology is widely used in industrialization because it has a large production capacity, high utilization of raw materials, product diversification, energy-conservation feature, and other advantages [17].Meanwhile, soy protein can endure complicated changes with the condition of high temperature, pressure, and shear forces in the extruder [16], whereas it has not been reported that extruded soy protein isolates (ESPIs) were applied to CSB.
The present work aimed to improve the in vitro digestibility of SPI and achieve large-scale production of CSB using the technology of extruded soy protein, which was explored with a DS32II double-screw extruder.The rheological properties and microstructure of dough were evaluated by farinograph and scanning electron microscope (SEM) analyses, and then CSB quality was assessed by sensory evaluation, colorimetry, and texture profile analysis (TPA).The ability of ESPIs to improve CSB properties, which can have significance in people's daily lives, was determined.
Preparation of ESPIs
. About 1000 g of SPI was mixed with 400 g of water by using a milling dough maker until no large boulders exist.Then appropriate feeding speed, discharging speed, and extrusion temperature were selected, to extrude SPI using a DS32II double-screw extruder (Jinan Saixin Machinery Co., Ltd.), as shown in Table 1.
In Vitro Digestibility (IVD).
In vitro digestibility of protein was determined using trypsin (250 U/mg; trypsin 1 : 250) and pepsin (250 U/mg protein; obtained from porcine gastric mucosa) enzyme system according to the reported method [18] with micromodification.About 1.0 g of protein was suspended in 20 mL of 0.1 M HCl and mixed with 0.1 g of pepsin in 1 mL of 0.01 M HCl.The mixture was incubated with slight shake at 37 ∘ C, for 6 h, and then added to 10 mL water; 5 mL of 0.50 M NaOH and 10 mL of 0.10 M phosphate buffer (pH 8.0) containing 20 mg trypsin were added.The digested mixture was slightly shaken for 12 h at 37 ∘ C and then 5 mL of aqueous solution of 20% (w/w) 5-sulfosalicylic acid dihydrate was added.Precipitated proteins were removed by filtration, and then the nitrogen content of supernatant was determined by the Kjeldahl nitrogen analysis.In vitro digestibility of protein was expressed as percentage of enzymatic digestion, as per the following formula: Enzymatic digestion% = Nitrogen (insoluble protein) released by enzyme Total nitrogen content of sample × 100%. (1) 2.4.SDS-PAGE.SDS-PAGE was applied to a discontinuous buffer system according to the slightly modified method of Tang et al. [19] using 12% separating gel and 5% stacking gel.The protein (20 g/mL, SPI or ESPIs mixture mixed with sample buffer, 1 : 1 v/v) was electrophoresed after heating for 4 min in boiling water.Every sample (10 L) was added to each lane.Before the sample exited the stacking gel, electrophoresis was performed at 18 mA and the other was at 35 mA.The gel was dyed with 0.1% Coomassie brilliant blue (R-250) in 25% ethanol and 8% acetic acid (ethanol : acetic acid : water, 250 : 80 : 670 v/v/v) and then destained in 25% ethanol and 8% acetic acid.
Dough Rheological
Properties.Dough rheological properties were determined with a farinograph (Brabender, Duisburg, Germany) according to the American Association of Cereal Chemists standard method 54-21 using about 300 g of composite flour containing 3% protein.Parameters, conducted at the average of double measurements, such as water absorption (WAC), dough development time (DT), stability time (S), degree of softening (DS), and farinograph quality number (FQN) were acquired from the software to evaluate the dough rheological properties.
Scanning Electron Microscopy (SEM) Analysis of Dough.
Before drying to the critical point, freeze-dried dough was broken into approximately 0.5 cm thick piece spot adjuncts and then coated with gold particles for 110 s.Manual smoothening of the fracture surface was difficult, so we acquired a relatively smooth area to observe the cross section of dough using SEM (JSM-6490LV, JEOL, Japan) analysis at magnifications of 900.
Preparation of CSB.
CSB was prepared according to the reported method [1,2] with slight modifications using composite flour (220 g), dry yeast (1.7 g), and water (78% of farinograph water absorption).After mixing for 4.0 min at low speed and then kneading to form dough, the dough was sheeted seven times and divided into three pieces (100 g per piece).These pieces were rounded and molded manually and then fermented for 45 min in a fermentation room at 38 ∘ C and 80% relative humidity.The fermented dough was steamed for 23 min using a steam cooker and boiling water.
2.8.Assessment of CSB Quality.CSB sensory scores were evaluated by a ten-person evaluation panel according to the method reported by [20,21] with slight modifications.CSB score includes specific volume (weighting, 20), exterior appearance (20), skin color (10), interior structure (15), taste (20), and flavor (20).CSB was cut into 15 mm thick slices with a bread knife for texture profile analysis (TPA) performed by a TA-XI2i PLUS Texture Analyzer (Stable Micro Systems, Ltd., Surrey, England) with the Pasta Firmness/Stickiness Rig probe (P36R).The test parameters were as follows: pretest speed, 5 mm/s; test speed, 1 mm/s; posttest speed, 1 mm/s; and compression, 50%.After TPA analysis, the color of CSB was tested by colorimetry which was based on a system that is very closely related to the perception of color difference to a human observer for most objects; that is, * (0 indicates black and 100 indicates white), * (+ * indicates redness and − * indicates greenness), * (+ * indicates yellowness and − * indicates blueness), and ΔE represented the total color of substance [22].Texture and color measurement of CSB were the average of three measurements at the same conditions: 2.9.Statistical Analysis.The analysis of variance (one-way ANOVA) was performed to analyze all measurements.Significant differences among the treatment group were analyzed by Duncan's test with SPSS software (Version 16.0, SPSS Inc., Chicago, IL, USA) and graphics were generated with Origin 8.5 software.1.
Results and Discussion
The subunits of soy 7S ( , , and ) and 11S (A and B) globulin and other subunits were all decreased, especially the subunits of 90.8 kDa, 32.8 kDa, and 31.3 kDa, whereas no isopeptide bond was formed.Extrusion processing exposed some hydrophobic group for SPI and resulted in complex changes with regard to the hydrogen bonds, disulfide bond, and hydrophobic interaction and their interactions were weakened, which may decrease protein solubility and parts of subunits content [23,24].Thus, the IVD of ESPIs improved, but no obvious variations in subunits occurred.
Properties of Dough.
Results of the composite dough farinograph indicated that the water absorption (WAC), dough stability (S), and farinograph quality number (FON) of dough were increased but the degree of softening (DS) decreased when mixed with 3% of SPI or ESPIs.Meanwhile, dough development time (DT) decreased in the order WF+SPI (5.60 min), WF (4.30 min), WF+IVD1 (2.00 min), WF+IVD2 (1.85 min), WF+IVD4 (1.85 min), WF+IVD3 (1.80 min), and WF+IVD5 (1.70 min).WAC increased and then decreased with ESPIs digestibility increase, whereas WAC ranged within WF+SPI (60.85%) and WF (58.85%).Although the S of WF+SPI (6.70 min), WF+IVD1 (6.60 min), WF+IVD2 (6.55 min), WF+IVD3 (7.00 min), WF+IVD4 (6.30 min), and WF+IVD5 (6.75 min) did not significantly differ, the DT decreased obviously as ESPIs were added.The reason may be that soy protein was amphiphilic, and extrusion caused the partially hydrophobic group to be exposed and promoted the gluten cross-linking and disulfide bond forming [16,23,24].Both SPI and ESPIs could improve the rheological properties of dough; above all ESPIs may save the cost of mixture time in the food industry (Table 2).SEM was used to observe the gluten network structure and distribution of starch granules.Dough had a continuity of gluten network structure and formed a closer gluten network structure than WF, when WF was mixed with 3% of SPI or ESPIs.The results showed that starch granules were embedded in the gluten network structure and they were basic and correlated with farinograph finding (Figure 2).The reason may be that SPI or ESPIs increased the S-S bonds of gluten network and contributed to the gluten protein crosslinking [16,24].The extrusion process enhanced gluten network tightness strength due to the denaturation that involved the intramolecular hydrogen bonds, van der Waals forces, weakened hydrophobic interaction, and exposed hydrophobic group and thiol [16,23].Thus, SPI treated by extrusion may significantly improve the rheological properties of dough.
Texture of CSB.
Results of TPA showed that CSB hardness and chewiness were decreased in the order WF+IVD2 (3564.60 g/2741.42g), WF+SPI (3277.48g/ 2626.27g), WF+IVD4 (3218.20 g/2464.96g), WF+IVD1 (3141.07g/2396.21g), WF+IVD3 (2992.88g/2334.40g), WF (2841.68 g/2229.66g), and WF+IVD5 (2773.03g/2183.33g).This result showed that SPI extruding can improve the tenacity of CSB in general, though there was no significant difference with regard to WF, WF+IVD1, WF+IVD3, and WF+IVD5.Similar to our data, bread utilizing soy protein isolate (SPI) may increase hardness and chewiness due to dilution of the gluten matrix, interchange of disulphide bonds between gluten proteins and SPI, and increasing the dough viscosity as the absorption of water increased by SPI [13,25,26].As the TPA of CSB was significantly associated with the sensory evaluation, particularly the hardness and chewiness had significant influence on its comprehensive score [27].High quality CSB should have an appropriate hardness and chewiness; being too large or too small may be bad for the quality (Figure 3).
Color of CSB.
With addition of 3% of SPI or ESPIs, red and yellow values increased and brightness decreased except for the WF+IVD1.Moreover, the * of WF (82.73),WF+IVD1 (82.78),WF+IVD3 (81.98), and WF+SPI (82.41) did not have significant difference, and the * value of CSB decreased compared with WF, except for WF+IVD1.Values of * indicated that color transformed from greenness to redness when 3% of SPI, IVD2, IVD3, IVD4, or IVD5 was added.Although there were no significant differences in * for WF compared with WF+SPI or WF+ESPIs except for WF+IVD1, the color was still yellow.Δ did not have significant difference when it came to WF, WF+SPI, WF+IVD1, WF+IVD3, and WF+IVD5.It may be attributed to the browning by polyphenol oxidase in processing and other natural pigments in WF with different colors such as B vitamins, flavonoid compounds, and carotenoid etcetera [28,29].Moreover, isoflavones and other color substances of SPI also have effect on CSB color [30,31].However, with addition of 3% of ESPIs, Δ did not have significant difference which may be due to the fact that the color substances and structure of SPI were destroyed after extrusion (Table 3) [23,24].Values within a column with different letters are significantly different ( < 0.05).
Sensory
Assessment of CSB Quality.The sensory scores and cross section of CSB were summarized in Table 4 and Figure 4, respectively.Specific volume and interior structure significantly affect CSB quality, and they were all almost improved in comparison with the control as 3% of SPI or ESPIs were used, especially WF+IVD1 and WF+IVD3.In terms of exterior appearance, no significant difference was observed when involving WF+SPI (17.6 scores), WF+IVD1 (18.0), WF+IVD2 (18.0), and WF+IVD3 (17.0) compared with the control.The sensory scores and cross section of CSB with regard to the WF+IVD1 (87.8 scores), WF+IVD2 (84.4), and WF+IVD3 (87.4) had higher sensory scores and were more exquisite, implying that CSB with appropriate hardness and chewiness, closer control group color, and exquisite cross section had a better sensory property.The reason may be that SPI and ESPIs could form a greater polymer network with the gluten protein and act as the nitrogen source of yeast and they could also increase the S-S bonds of gluten network [24,28], thereby improving the specific volume and interior structure of CSB.Moreover, the slight savoury released by SPI or ESPIs after cooking may improve the taste and flavor of CSB, especially by weakening the beany flavor by extrusion process.In the study by Shin et al. [13], similar to our data, the bread with heat-treated (steamed and roasted) soy flour has a lower beany odour and taste than those with non-heattread (raw and germinated) soy flour.
Conclusions
After extrusion, the IVD of ESPIs improved while the subunits of soy 7S ( , , and ) and 11S (A and B) globulin decreased, especially the subunits of 90.8 kDa, 32.8 kDa, and 31.3 kDa.Although SPI and ESPIs both improved the rheological properties of dough, ESPIs may save the cost of kneading time in the food industry.Compared to WF and WF+SPI CSB, the total score of WF+IVD1, WF+IVD2, and WF+IVD3 CSB had the higher scores and exquisite cross section.It demonstrated that SPI treated by extrusion may significantly improve the nutrition and quality of CSB.
Figure 3 :
Figure 3: Influence of SPI and ESPIs on hardness (a), chewiness (b), and springiness of CSB.Values within a column with different letters are significantly different ( < 0.05).
Table 1 :
The technological conditions of ESPIs.
Table 2 :
Effects of SPI and ESPIs on farinographic properties of dough.
Table 3 :
Effect of SPI and ESPIs on the color of Chinese steamed bread.
abcValues within a column with different letters are significantly different ( < 0.05).
Table 4 :
Effect of SPI and ESPIs on the sensory score of Chinese steamed bread. | v3-fos-license |
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} | pes2o/s2orc | Nanomaterials for Periodontal Tissue Engineering: Chitosan-Based Scaffolds. A Systematic Review.
Introduction. Several biomaterials are used in periodontal tissue engineering in order to obtain a three-dimensional scaffold, which could enhance the oral bone regeneration. These novel biomaterials, when placed in the affected area, activate a cascade of events, inducing regenerative cellular responses, and replacing the missing tissue. Natural and synthetic polymers can be used alone or in combination with other biomaterials, growth factors, and stem cells. Natural-based polymer chitosan is widely used in periodontal tissue engineering. It presents biodegradability, biocompatibility, and biological renewability properties. It is bacteriostatic and nontoxic and has hemostatic and mucoadhesive capacity. The aim of this systematic review is to obtain an updated overview of the utilization and effectiveness of chitosan-based scaffold (CS-bs) in the alveolar bone regeneration process. Materials and Methods. During database searching (using PubMed, Cochrane Library, and CINAHL), 72 items were found. The title, abstract, and full text of each study were carefully analyzed and only 22 articles were selected. Thirteen articles were excluded based on their title, five after reading the abstract, twenty-six after reading the full text, and six were not considered because of their publication date (prior to 2010). Quality assessment and data extraction were performed in the twelve included randomized controlled trials. Data concerning cell proliferation and viability (CPV), mineralization level (M), and alkaline phosphatase activity (ALPA) were recorded from each article Results. All the included trials tested CS-bs that were combined with other biomaterials (such as hydroxyapatite, alginate, polylactic-co-glycolic acid, polycaprolactone), growth factors (basic fibroblast growth factor, bone morphogenetic protein) and/or stem cells (periodontal ligament stem cells, human jaw bone marrow-derived mesenchymal stem cells). Values about the proliferation of cementoblasts (CB) and periodontal ligament cells (PDLCs), the activity of alkaline phosphatase, and the mineralization level determined by pure chitosan scaffolds resulted in lower than those caused by chitosan-based scaffolds combined with other molecules and biomaterials. Conclusions. A higher periodontal regenerative potential was recorded in the case of CS-based scaffolds combined with other polymeric biomaterials and bioceramics (bio compared to those provided by CS alone. Furthermore, literature demonstrated that the addition of growth factors and stem cells to CS-based scaffolds might improve the biological properties of chitosan.
Introduction
Periodontal disease is a chronic inflammatory disease caused by a bacterial infection, which leads to an inflammatory status, causing destruction of the tissue supporting the teeth (the gingival, bone, and periodontal ligament) [1]. Periodontal inflammation resolution and subgingival microbial biofilm removal could only require nonsurgical mechanical therapy, but in order to obtain restitution ad integrum of the periodontum, alveolar bone defects need to be regenerated [2]. For this purpose, several biomaterials have been introduced, giving a positive impact on oral tissue engineering. These biomaterials act as three-dimensional scaffolds, the surface of which promotes cellular adhesion, proliferation, and differentiation, creating a favorable environment for tissue regeneration. Moreover, biomaterials, which can be of natural or synthetic origin, are able to come in immediate contact with the living tissue, without developing any adverse immune reaction. The placement of these novel biomaterials in the affected area activates a cascade of events, inducing regenerative cellular responses, and replacing the missing tissue [3,4]. The scaffold needs to have the capacity to promote osteogenesis, osteoinduction, and osteoconduction processes, cellular components (preosteogenic cells) must be delivered or attracted to its surface, and they must be activated by osteoinductive growth factors. Scaffold biomaterials are responsible for maintaining the appropriate space, in order to allow the implanted cells to deposit the ECM and to proliferate [5]. The recent review by Rodríguez-Vázquez et al. (2015) [6] specified the main characteristics of scaffold biomaterials that are used in tissue engineering. Biomaterials must be biocompatible, absorbable and degradable, with similar resorption and degradation rate (in vitro and in vivo) to the rate of tissue regeneration; its surface must be chemically adequate and stable; resistance and mechanical properties must be proper and its degradation products shall not be toxic or carcinogenic. The scaffold design should perfectly adapt to the defected area. An interplay between porosity and density should be present, as density improves mechanical strength and porosity facilitates cell migration, growth factors delivery, and vascularization [7,8]. Furthermore, the combined use of these biomaterials with mesenchymal stem cells (MSCs) and bone morphogenetic proteins (BMPs) with osteoinductive properties represents a crucial component of bone tissue engineering. MCSs present several features, which improve the regeneration process: they have immunosuppressive capacity, act as endocrine secretors, and are able to differentiate into several cellular types, such as osteoblasts and adipose cells [9]. The BMPs stimulate osteogenesis and neovascularization [5]. Jafari et al. (2015) [10] subdivided the scaffold biomaterials in two main groups: natural-based polymers and synthetic-based polymers, with different biodegradation rates, mechanical and physicochemical characteristics. The latter include polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-co-glycolide (PLGA). Natural-based polymers are represented by chitosan, alginate, collagen, gelatin, elastin, and silk fibers. Natural polymers are perfectly biocompatibility and present several properties (pore size, porosity, fibrous structure), which guarantee positives results on living tissues. Chitosan (CS) is the fully or partially deacetylated form of chitin, which can be found in fungi and shells of sea crustaceans. It is the most abundant natural amino polysaccharide after cellulose and is formed by 2-acetamido-2-deoxy-β-d-glucopyranose and 2-amino-2-deoxy-β-d-glucopyranose groups, showing structural similarity to glycosaminoglycans of the extracellular matrix [11,12]. Chitosan is available in different forms, such as fibers, hydrogels, sponges, films and it is considered a valid biomaterial, capable of serving as two or three-dimensional scaffold in wound dressing or tissue engineering processes respectively. CS is a biodegradable, biocompatible, nontoxic, biologically renewable, and bacteriostatic biomaterial. The surface of chitosan is hydrophilic and it facilitates the adhesion, proliferation, and differentiation of the cellular component. Furthermore, it also has hemostatic and mucoadhesive capacity since its amino groups are positively-charged [13]. However, the studies by Alididi et al. and Marei et al. [14,15] demonstrated that chitosan was not osteoinductive or osteoconductive alone, but if combined with other molecules or biomaterials, such as growth factors, dental mesenchymal stem cells or hydroxyapatite, it could represent a valid help in bone regeneration.
Objectives
This research has the objective of reviewing the literature in order to obtain an updated overview of the utilization and effectiveness of chitosan-based scaffold (CS-bs) in the field of periodontal tissue engineering. This review focused, in particular, on CS-bs effectiveness in the alveolar bone regeneration process.
Clinical Question (PICO)
• P: Chitosan-based scaffold • I: utilization and efficacy of chitosan-based scaffold in periodontal tissue engineering, assessing, in particular, its contribution in alveolar bone regeneration • C: comparison between chitosan used alone and chitosan used in combination with other biomaterials, molecules or stem cells • O: general overview of the different chitosan scaffold forms and compositions and their application in periodontal tissue engineering. Evaluation of chitosan-based scaffold effectiveness in the alveolar bone regeneration process
Protocol and Registration
Methods and inclusion criteria of this systematic review were selected following the specific protocol provided by the PRISMA statement [16].
Inclusion and Exclusion Criteria
We have selected the most recent studies concerning periodontal tissue engineering, in which the alveolar bone regeneration was obtained by using a chitosan-based scaffold alone or in combination with other biomaterials, molecules or stem cells. The inclusion criteria were as follows: Available data about cell proliferation and viability, mineralization and alkaline phosphatase activity of the newly formed bone Study design: Randomized Controlled Trial Chitosan-based scaffold used in combination with other biomaterials, growth factors or stem cells Articles written in the English language Case reports and reviews were excluded from our study. Studies published before 2010 were not considered.
Search
Databases of PubMed, Cochrane Library, and CINAHL were used to conduct a systematic literature review, selecting the most recent studies about the utilization and effectiveness of chitosan-based scaffold, applied to periodontal tissue engineering. Only articles written in the English language and published within 2010 were included. No restrictions were imposed regarding the type of biomaterials, molecules, or cellular components that were combined with chitosan. During literature searching, we used the following keywords: "chitosan scaffold" "periodontal engineering", "oral bone regeneration" (combined with the Boolean term "AND").
Study Selection and Data Collection Process
Study selection was conducted by two reviewers (D.L., G.M.), who independently reviewed title, abstract, and full text of all the articles that were found during the literature search. Eligible articles were selected following inclusion and exclusion criteria. Data collection was provided by two researchers (E.T., D.C.), who extracted from each item several pieces of information: the design of the study (randomized controlled trial), in vivo or in vitro analysis, and scaffold biomaterials that were used for bone regeneration. Data about cell proliferation and viability (CPV), mineralization (M), and alkaline phosphatase activity (ALPA) were recorded from each article. Therefore, the principal outcome measures referred to these parameters (means). The flow chart used for the selection of studies is shown in Figure 1.
Quality Assessment
Quality assessment of the included studies was provided by the Newcastle Ottawa Scale [17]. The lowest score was 5, the highest was 7, and on average the quality of the articles was evaluated to be equal to 6.04 (Table 1). All the items compared their results with a control group, and most of them were conducted in vitro. The potential for bone regeneration of each chitosan-based scaffold was analyzed with reliable methods, such as measurements of cell proliferation and viability (CPV), mineralization (M), and alkaline phosphatase activity (ALPA).
Study Selection and Characteristics
Electronic research was conducted in PubMed, Cochrane Library, and CINAHL databases, and a total of 72 items were found. Title, abstract, and full text of each study were carefully analyzed and only 22 articles were selected. Thirteen articles were excluded based on title, five after reading the abstract, 26 after reading the full text, and six were not considered since they were published before 2010. After assessing the quality of the included articles using the Newcastle Ottawa Scale, they were submitted to data collection process. The twenty-two selected articles were randomized controlled trials, and all of them were written in the English language. Studies characteristics with reference to the author, in vivo/in vitro measurement, and type of biomaterial, are shown in Tables 2-6. In the selected studies, chitosan was used in combination with other biomaterials and molecules/cells. Biomaterials combined with CS were: hydroxyapatite (HA), alginate (AL), collagen, polylactic-co-glycolic acid (PLGA), pure polylactic acid (PLA), genipin, tricalcium phosphate, inorganic calcium phosphate, hyaluronic acid (Ha), dicarboxylic acid (DA), and polycaprolactone (PCL). Stem cells, growth factors, and proteins were also used in combination with CS: basic fibroblast growth factor (bFGF), periodontal ligament stem cells (PDLSCs), bone morphogenetic proteins (BMP), insulin-like growth factor-1 (IGF-1), osteoprotegerin (OPG), human jaw bone marrow-derived mesenchymal stem cells (hJBMMSCs) and human bone marrow stromal cells (hBMSCs). Ten of the included studies were performed in vitro, six of them were conducted in vivo and six both in vivo and in vitro. Studies in vivo used a sample of 48 mice (calvarial defects) and 12 beagles (alveolar bone). The included items analyzed several parameters, using different measurement methods. This study only reviewed data resulting from the evaluation of bone regeneration level, obtained thanks to the following measurement: (1) cellular proliferation and viability through MTT [40], Cell-Counting Kit-8®, AlamarBlue assays [41], and PrestoBlue assays (2) mineralization level using von Kossa, ARS (Alizarin Red S), Masson's trichrome staining and immunofluorescent staining for osteocalcin (OCN), and (3) alkaline phosphatase activity (ALP).
AlamarBlue assay of PDLCs and hBMSCs
Generally cell metabolic activity was higher in the CS/BG-NP group compared with the CS group for both PDLCs and hBMSCs.
Results of Individual Studies
The results of individual studies are presented in Tables 2-6. In order to evaluate the efficacy of chitosan-based scaffolds during bone regeneration, data about cell proliferation and viability were recorded from 10 of the included articles: five of them used the MTT assay of cementoblasts [18][19][20], periodontal ligament cells [18,28,37], one used the Cell-Counting Kit test (CCK-8) [23,26], four applied the AlamarBlue assay [25,29,30,32] and one the PrestoBlue test [22] (Tables 2-4). ALP activity of periodontal ligament cells [21], osteoblasts [34,35], and mesenchymal stem or stromal cells [24] was assessed in four studies (Table 5). Three items analyzed the mineralization level of the newly formed bone with the Masson's trichrome staining method [27,33,38], two assessed it with the ARS staining [31,36] and one with the immunofluorescent staining technique for osteocalcin (OCN) [39] ( Table 6). Twelve of the selected papers performed their experiment in vivo, creating bone defects, which were later covered by CS-based scaffolds: Ge et al. [21] created bilateral parietal bone defects (with a diameter of 5 mm) in eighteen eight-week-old rats (weight = 180-220 g), scoring the anesthetized cranial skin, exposing calvaria; parietal cranial 15 mm oval-shaped defects were obtained by Jayash et al. [25] using a bone trephine drill. A 5 mm diameter parietal defect was created in thirty-two five-month-old male rats by Li et al. [27] thanks to a trephine drill under copious saline irrigation. Guo et al. [23], after making an intraperitoneal injection with 10% chloral hydrate to 30 rats, performed a "V" type incision on the skull with a blade and drew with a drill, a 5 mm-diameter defect reaching the dura mater. Shah et al. [32] tested the CS-based scaffold on a subcutaneous pouch of eight-week healthy adult rats weighing 140-180 g. Xue et al. [36] anesthetize intramuscularly 3 white rabbits (4-6 months, weight 2-3 kg), exposing the lower edge of the mandible and creating a bone defect in the molar area of the mandibular body. Zang et al (2016) [38], used one-wall, box-shaped, infrabony defects (4 mm width, 7 mm depth) at the distal and mesial aspects of the third premolars and at the mesial aspects of the first molars. In the study of 2019 by the same author, bilateral class III furcation defects (4 mm wide and 5 mm high) were created on the third and fourth mandibular premolars [39]. The proliferation of cementoblasts (CB) and periodontal ligament cells (PDLCs) on pure chitosan scaffolds resulted in lower than those on the chitosan-based scaffolds combined with other molecules and biomaterials. The study by Akman et al. [18] compared the proliferation of these two cellular type on chitosan-based scaffold with the addition of HA and bFGF with those on pure chitosan one, showing that the absorbance values (at 570 nm) of the cells on day 7 and 8 were equal to 1.7 (CB) and 1 (PDLCs) and 0.6 (CB and PDLCs), respectively. Pure chitosan scaffolds were investigated in comparison with IGF-1, and BMP-6 added CS/AL/PLGA and β-tricalcium phosphate/CS scaffolds in the research by Duruel et al. [20] and Liao et al. [28] respectively, recording a higher absorbance value of CB and PDLCs in the second groups (2/2.6 and 0.9/1 on day 12 and 6, respectively). The MTT assay of PDLCs showed no significant differences between an autoclaved chitosan powder/β-glycerophosphate thermosensitive hydrogel (CS-PA/GP) and an autoclaved chitosan solution/GP hydrogel: absorbance values at 490 nm were equal to 0.7 and 0.6, respectively [37]. Dental pulp stem cells (DPSCs) were seeded on two scaffold types by Bakopoulou et al. [19]: CS combined with gelatine (CS/Gel) fabricated using 0.1% and 1% of the crosslinker glutaraldehyde (GTA). The OD values (545-630 mm) recorded in the MTT assay of DPSCs seeded on the two scaffolds after seven days were 1.6 for CS/Gel-0.1 and 1.3 for CS/Gel-1 (p < 0.01), but this statistical difference was compensated to non-significant at day 14. The trial by Miranda et al. [29] cultured osteoblast-and fibroblast-like cells on CS-Ha hydrogel, Ha hydrogel, and pure CS scaffolds; a quantitative evaluation of cell viability was conducted for 24, 48, and 72 h, using Alamar Blue, which was also added to a phosphate buffer solution without cells: the test showed increased cellular viability (20%) in both cellular groups compared with the control one; however, none of them were statistically different. The metabolic activity of hPDLCs and human bone marrow stromal cells (hBMSCs) recorded by the AlamarBlue assay in the trial by Mota et al. [30] presented higher values in the CS/bioactive glass nanoparticles (BG-NPs) membranes compared with CS one. The same test was performed by Jayash et al. [25] in order to assess the cell viability in a new osteoprotegerin-chitosan gel. After 24 h, the viability of the OPG-CS and CS gels (25 and 50 kDa) was significantly higher than those of the controls (OPG-CS and CS = 140%, controls = 110%). The cell proliferation and viability (assessed with AlamarBlue) of MC3T3-E1 cells on the trilayered functionally-graded CS membrane (FGM) with bioactive glass gradient (50%, 25%, 0% wt.) resulted in being higher than those in the control group: the relative percentage AB reduction after seven days was equal to 150% for tge FGM group and 90% for the control one [32]. In the in vitro study by Gümüşderelioglu et al. [22], CS-based multifunctional and double-faced barrier membrane was realized: hard tissue was put in contact with the porous side of the membrane coated with HA, in which BMP-6 was also embedded. The nonporous surface of the membrane was in contact with the inflammatory soft tissue, and it was coated with electrospun PCL fibers. PrestoBlue assay on day 21 assessed that mitochondrial activities of MC3T3-E1 cells seeded on different membranes showed no statistical differences (CS = 0.58, HA/CS = 0.63, HA/CS + BMP-6 = 0.64, HA/CS/PCL = 0.62). Data demonstrated that these cells grew on all CS-based scaffolds, recording higher cellular activity in HA/CS membrane. The CCK-8 test of PDLCs in the article by Li et al. [26] demonstrated higher OD values (0.7) in CS-based hydrogel/α β -GP scaffold loaded with BMP2 plasmid DNA (pDNA-BMP2) than in those without pDNA-BMP2 (0.6). The same test used by Guo et al. [23] highlighted better cell viability on the electrospun collage-chitosan composite membrane than in the electrospun collagen one (OD values were 0.7 and 0.4 respectively). Sundaram et al. [34] analyzed the ability of a bilayered construct composed by PCL multiscale electrospun membrane and a chitosan/2 wt% CaSO 4 scaffold to regenerate periodontal ligament and alveolar bone simultaneously. The authors of this study found a higher level of alkaline phosphatase activity of hDFCs on day 7 in the latter group (ALP protein concentration = 8 ng/mg) than in the control one (ALP protein concentration = 3.5 ng/mg). Multitissue simultaneous regeneration was also studied by Varoni et al. [35], who recorded no significant differences between the ALP activity of osteoblasts (OB) on day 7 provided by a CS-based genipin-cross-linked trilayered scaffold and the control group (460 and 480 pNpp/nmol min respectively). The RCT by Ge et al. [21] measured the ALP activity of PDLSCs up to 14 days in two different scaffold types: nanohydroxyapatite-coated -genipin-CS conjuction and genipin-CS-framework, showing higher values in the first group on day 7 (30 u/gprot and 25 u/gprot respectively), but registering similar values in both groups on day 14. The ALP activity of hMSCs seeded on HCG membrane recorded by Hunter et al. [24] showed a peak at 14 days of cultures, demonstrating that this type of membrane enhances hMSCs proliferation and osteogenic differentiation. Masson's trichrome staining performed by Sukpaita et al. [33] found an increased amount of collagen and bone matrix in CS/Dicarboxylic acid scaffold with and without PDLCs seeding. In the study by Zang et al. [38], the same test showed more dense and well-organized PDLCs in the chitosan scaffold with hJBMMSCSs than the chitosan/anorganic bovine bone and pure chitosan groups. ARS staining of hPDLCs performed by Xue et al. [36] recorded more mineralized nodules on the nPLGA/nCS/nAG complex than in negative control group, showing the that this type of membrane may promote cell mineralization. Human mesenchymal stem cells (MSCs) were cultured by Rammal et al. [31] on a bone-mimetic material (B-MM) made from inorganic calcium phosphate combined with CS and hyaluronic acid biopolymers, which acted as a framework for the osteogenic potential of MSCs. ARS staining detected the formation by MMSCs of the mineralized matrix on B-MM, contrary to the control glass coverslip, on which no morphological changes and no nodules were found. In the study by Zang et al. [39], the number of OCN-positive cells in beagles mandibular class III furcation defects resulted in being higher on CS/β-GP/BMP-7/ORN and on CS/β-GP/BMP-7 membranes (45 and 43, respectively) than those on CS/β-GP/ORN and control group (22 and 19, respectively). Finally, Li et al. [27] used the Masson's trichrome staining to compare the mineralization level of the newly formed bone (NB) in an injectable CS-based thermosensitive hydrogel scaffold with and without the incorporation of pDNA-BMP2 (CS/CSn(pDNA-BMP2)-GP). The study found out that the width of the NB was 500 µm for the first group and 300 µm for the second one, showing that CS/CSn-GP has greater capacity for alveolar bone regeneration when combined with pDNA-BMP2.
Discussion
This systematic review aimed to obtain an up-to-date overview of the usage and efficacy of chitosan-based scaffold used alone or combined with other biomaterials, (whose characteristics and properties are shown in Tables 7 and 8), molecules, and cellular components. Thanks to its multiple properties, CS has been used for years in periodontal regeneration techniques [42][43][44][45][46]; the analysis of the most recent literature conducted in our paper highlighted that this biomaterial might be combined with other natural-or synthetic-based polymers obtaining bi-and trilayered scaffolds, which allow the simultaneous regeneration of the different tissues of the periodontal apparatus [21,25]. This capacity clarifies the reason why CS-based scaffolds should be used in the field of periodontal tissue engineering. CS is obtained from chitin deacetylation, which can be performed both through chemical or enzymatic processes: the chemical method avails of acids or alkalis, while the enzymatic one is made it possible by the chitin deacetylase, which catalyzes the hydrolysis of N-acetamido bonds in chitin [47]. Pure CS exists in various forms, depending on the molecular weights (300-1000 kDa) and on the degree of deacetylation, which generally ranges between 50-95%. These two parameters determine many physicochemical properties of CS, such as its solubility, crystallinity, and degradation. When the degree of deacetylation is intermediate, CS presents a semi-crystalline structure, while high deacetylation leads to a maximum crystallinity. The degradation rate of CS must provide the time necessary for the formation of the new bone: high degrees of deacetylation guarantee low degradation rates (which is performed in vivo by lysozyme). The free amine groups on deacetylated subunits present cationic nature, giving CS hemostatic, mucoadhesion, and antimicrobial properties. This biomaterial is biocompatible, biodegradable, and osteoconductive, facilitating the adhesion and proliferation of cells on its surface. The CS-based scaffold can be combined with other polymers and molecules in order to improve its mechanical and biological properties [48,49]. As a confirmation of this, in all the selected studies the data obtained from the analysis of the mineralization level, cell proliferation/viability, and alkaline phosphatase activity demonstrated that pure chitosan scaffolds were less effective at regenerating the bone tissue than the chitosan-based scaffolds combined with other biomaterials, molecules, and stem cells. Previous studies recorded superior mechanical reliability and in vivo biomineralization of CS combined with hydroxyapatite compared to CS and HA used alone [50]. According to the study by Akman et al. [18], the addition HA created a novel scaffold structure, preserving the pore sizes and interconnectivity. As well as decreasing the swelling ratio, it has been shown that HA established a strong mechanical interface with CS, which forms a hydrophilic structure, interacting with the body fluids. It was also showed that the higher was the chitosan concentration, the lower was the scaffold's interconnectivity. In the same study, 100 ng of basic fibroblast growth factors were loaded to CS/HA scaffolds, demonstrating that the combination of these two biomaterials represents a superior carrier system for bFGF than CS alone. bFGF has the capacity to regulate periodontal wound healing, inducing the growth of immature PDL cells and also angiogenesis; it enhances the proliferation of osteoblasts, PDL cells, and cementoblasts. The addition of hydroxyapatite and the loading of basic fibroblast growth factor made possible for cementoblasts and periodontal ligament cells to increase their proliferation on the scaffold. The residual release of bFGF from the scaffold increased the proliferation of the cells, also thanks to its chemotactic effect [18,51]. Gümüşderelioglu et al. [22] state that the presence of HA coating in CS membranes may lead to an increase of osteoconductivity of the scaffold. In the study by Ge et al. [21], the alkaline phosphatase activity of PDLSCSs resulted in being increased in nanohydroxyapatite coated scaffold: this may be caused by the release of calcium phosphate ions during the partial dissolution of nanohydroxyapatite. The small pores of this biomaterial also enhanced the attachment and proliferation of osteoblasts. Dental tissues mesenchymal stem cells as those obtained from the periodontal ligament may amplify the regenerative effect when seeded on scaffolds with proper surface characteristics: biodegradable polymer-nanohydroxyapatite composites may stimulate the differentiation of stem cells into osteoblasts. Rammal et al. [31] demonstrated that a bone-mimetic material made of organic CS combined with hyaluronic acid and calcium phosphate, may promote pro-regenerative secretome from MSCs since it represents a versatile osteoinductive coating. Dental pulp stem cells represent a precious option in regenerative dentistry [52], since they may potentiate the reconstitution of mineralized tissues, such as bone and dentine/pulp complex. For this reason, Bakopoulou et al. [19] seeded DPSCs on two CS-based scaffolds, which were combined with gelatin, fabricated with 0.1 and 1% of GTA. The combination with a gelatin may improve CS mechanical strength and its initial cell attachment potential. As well as several natural biomaterials, gelatin represents an attractive solution in tissue engineering, thanks to its biocompatibility, cell viability/proliferation maintenance, osteogenic differentiation promotions, and antimicrobial activity. The results of this study showed that the first day's proliferation rate of DPSCs was lower in the scaffold with the highest concentration of GTA, a difference that disappeared at later time-points. Meanwhile, DPSCs seeded on CS/Gel-1 scaffold did not show upregulation of three differentiation markers, proving a long-term cytotoxic effect of GTA. It has been demonstrated that the combined action of bone morphogenetic proteins and scaffold polymers may enhance bone tissue regeneration. A study by Venkatesan et al. (2017) [53] proved that CS-based scaffolds have the capacity to systematically and sustainably release BMP-20 and Shu et al. [54] showed that the relationship between the osteogenic property of this protein and a 2-N,6-0-sulfated CS may enhance bone tissue development. Duruel et al. [20] highlighted the importance of growth factors in the regeneration process: IGF-1 promotes cell recruitment to the affected areas within a few hours, and BMPs are osteoinductive factors expressed in mature bone, which plays a crucial role in the regulation of bone metabolism. In this study, chitosan was combined with AL and PLGA microparticles. In order to analyze the effect of released growth factor on cellular functions, AL was used as a carrier for IGF-1 (which promotes OB proliferation and pre-osteoblasts differentiation), while PLGA as carrier for BMP-6 (that is an important biosignaling molecule in periodontal regeneration). AL was chosen because of its reversible swelling property, allowing growth factor release. PLGA is characterized by a low degradation rate, and it was demonstrated that it provided the growth factor release for a longer period than AL. Despite having many adequate properties, CS is nonbioactive but only biotolerable. This obstacle could be overcome, combining CS with calcium phosphates, which is bioactive and osteoconductive [28]. The association between CS and hyaluronic acid could be a valid option in periodontal tissue engineering: CS has better mechanical properties than Ha, but its bioresorption is longer than those of Ha [29]. Poor water solubility is one of the limitations of CS. Sukpaita et al. [33] prepared CS dissolving it in dicarboxylic acid, which also serves as crosslinking agents, giving higher mechanical properties to the chitosan scaffold. Periodontium is a complex structure, formed by different tissues (cementum, bone, periodontal ligament, and gingival). In order to obtain a multitissue simultaneous regeneration, the application of chitosan-based bylayered and trilayered scaffold seems to be a valid option [34,35]. The multilayered technique includes the use of a substrate, which is immersed in the CS solution, characterized by a cationic nature. In this way, the polymer deposits a thin film on the surface (layer). In order to realize the interaction between the positively charged groups of CS and the negative one, the system is immersed in a polyanionic solution. As a consequence, an upper layer is formed [55]. Bone marrow-derived mesenchymal stem cells may contribute to periodontal regeneration, as they can differentiate into cementum, bone, and periodontal ligament [38].
Conclusions
The efficacy of chitosan in periodontal tissue engineering has been widely demonstrated. Chitosan is a natural-based polymer with biodegradability, biocompatibility, and biological renewability properties. It is bacteriostatic and nontoxic and presents hemostatic and mucoadhesive capacity. In this systematic review, CS-based scaffolds combined with other polymeric biomaterials (AL, collagen, PLGA, PCL) and bioceramics (bioactive-glass, calcium phosphate, β-tricalcium phosphate, HA) have been analyzed, recording a higher periodontal regenerative potential compared to those given by CS alone. Data proved that natural biopolymers present high biocompatibility and antimicrobial potential; they have the capacity to recognize cell signals, to promote cell viability/proliferation and osteogenic differentiation. However, these biomaterials show weak mechanical characteristics. Synthetic polymers have poor potentiality in providing cell adhesion/migration and proliferation, but they show good mechanical properties, and their mechanical strength and degradation rate can be adjusted in order to reach the best performance. Bioceramics are bioactive, ensure excellent osteoconductivity and when combined with polymer matrix, they may improve the mechanical properties of the system. Furthermore, the addition of growth factors (bFGF, BMP, IGF-1) and stem cells (PDLSCs, hDFCs, hJBMMSCs) to CS-based scaffolds may improve the biological properties of chitosan, providing a better regenerative effect. Our analysis also highlighted that CS-based scaffolds accompanied by natural-or synthetic-based polymers might allow the simultaneous regeneration of the different periodontal tissues (gingival, cement, alveolar bone and periodontal ligament). | v3-fos-license |
2019-04-10T13:12:52.101Z | 2019-01-07T00:00:00.000 | 104400644 | {
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} | pes2o/s2orc | Towards homochiral supramolecular entities from achiral molecules by vortex mixing-accompanied self-assembly
By using a vortex mixing-accompanied self-assembly strategy, homochiral entities with controlled handedness were obtained from exclusively achiral molecules.
handed circularly polarized light, respectively. Experimentally, the value of g CD is defined as g CD = [ellipticity/32980]/absorbance at the CD extremum. The magnitude of CPL can be evaluated by the luminescence dissymmetry factor (g lum ), which is defined as g lum = 2 × (I L -I R )/(I L + I R ), where I L and I R refer to the intensity of left-and right-handed CPL, respectively. Experimentally, the value of g lum is defined as g lum = [elipticity/(32980/ln10)]/total fluorescence intensity at the CPL extremum. For the measurement of CD spectra, the cuvette was placed perpendicularly to the light path of CD spectrometer and rotated within the cuvette plane in or-der to rule out the possibility of the birefringency phenomena and eliminate the possible angle dependence of the CD signals. To estimate the contribution of LD effect on the true CD signal, 36 CD and LD spectra of the samples were measured in steps of 10° by rotating the sample which fixed in the homemade rotator.
Vortex mixing treatment 5 mg BTACA was first dissolved in 0.7 mL dimethylforma-mide (DMF) by sonication in a 5 mL vial, then 0.7 mL Mil-li-Q water was injected with a pipette and instant gels were formed in the mixed solvent. Upon heating (383 K), the gels became into transparent solution. Then vortex mixing at 2500 rpm was applied to the hot solution for 10 minutes. After that, white suspensions with good dispersion were obtained.
After that, these samples were placed in oil bathes at different temperature and stirred at 1000 rpm.
Synthesis and characterization of compounds
The BTA core with three functional cinnamic acid side chains (BTACA) was prepared by the hydrolysis of the ethyl cinnamate-substituted 1,3,5benzenetricarboxamide (BTACE).
The resultant powder was dried to give a white powder with a yield of 88%.
The obtained yellow solution was added 60 mL water and treated with 1 M dilute HCl until pH=1 and solid precipitate was obtained. After filtration, the solid was washed 5 / 47 with water and methanol to get the final product with a yield of 73%. 1 Volume ratio [a] Phase [b] 10-0 S
Supplementary Tables and Figures
The total volume of DMF/H 2 O is 1.4 mL and the concentration of BTACA is 5.54 mM.
[b] S: solution; PG: partial gel; G: stable gel; I: insoluble. The assemblies of BTACA in DMF/H 2 O through vortex mixing were characterized by means of X-ray diffraction (XRD), UV-Vis and fluorescence spectral measurements. Compared with its solution, UV-Vis spectra of BTACA assemblies broadened with a shoulder peak at 380 nm which suggested the existence of J-like aggregation (Fig. S3a). Meanwhile, the fluorescence intensity was significantly enhanced, accompanying with a drastic red-shift from 434 to 498 nm after assembly ( Fig. S3b). The XRD profile of BTACA xerogel showed a series of peaks at 2.29°, 4.68°, 5.21° and 6.82°, and corresponding distances were estimated to be 3.85, 2.70, 1.88 and 1.69 nm, respectively (Fig. S4a). This indicates that the molecular packing adopts a body-centred cubic structure pattern. Moreover, the in situ investigation of BTACA assemblies in DMF/H 2 O (1:1 v/v) showed identical peaks, which indicated the highly ordered molecular packing (Fig. S4b). Driving force of supramolecular assembly could be further confirmed by Fourier transform-infrared (FTIR) measurement. The peak at 1666 cm -1 for BTACA clearly indicated the formation of hydrogen bond from carboxylic acid groups (Fig. S4c) while the peaks at 1593 cm -1 (amide I band) and 1518 cm -1 (amid II band) suggested the existence of C=O and N-H in the hydrogen bond form. FTIR data demonstrated that the hydrogen bond between carboxylic acid groups played a crucial role in the supramolecular assembly of BTACA. with the mean spectrum of all 36 LD spectra (dash line).
In Fig. S7a, the CD difference from the maximum value (351 nm) to the minimum value (290 nm) fluctuated around 1600 mdeg. This nonzero value suggested that the observed CD signal represents the authenticity of helical chirality in the suspension system. On the contrary, the values of 36 individual LD spectra fluctuated with the testing angle, suggesting that the angle-dependent LD effect can be eliminated by averaging all of the LD spectra (Fig. S7b). Therefore, the true CD intensity can be obtained by averaging all of these 36 individual spectra as shown in Fig. S7c.
Moreover, the contribution of LD to the CD spectra could be quantitatively estimated by using the following semi-empirical equation (45):
Contamination of CD by LD = LD ×0.02 / CD observed
The contamination of CD by LD in the present case was estimated about 0.05%. The detection areas during CD measurement are also taken into consideration.
Since cuvette of 0.1 mm is used for measuring the CD spectra, 30 μL suspension is enough for each CD measurement. As shown in Fig. S8, we measured 20 times for one BTACA samples obtained by vortex mixing. Therefore, almost 600 μL suspension were measured for one sample. On the other hand, the effective detection areas for CD measurement (JASCO J-1500 spectrometer) is about 0.5 cm 2 (the diameter is 8 mm). Thus, about 10 cm 2 area was measured for one sample.
Clearly, there is no difference among them, indicating the well-dispersed and homochiral of BTACA assembles after vortex mixing process. SEM images in (a) shows the same SEM images as in (b) but without the markers.
Only P handed nanohelix were observed within the scope of 300 μm × 200 μm. The red boxes in (b) represented that the scopes were carefully analyzed, which were listed in the end of this file. By carefully looking the UV-vis absorbance spectra of the BTACA assemblies after vortex mixing (Fig. S3a), we could see a small shoulder peak at 380 nm besides the main peak at 328 nm. This shoulder peak can be attributed to the J-like aggregation (pi-pi stacking) of BTACA assembles. After the vortex mixing process, the assemblies were slender. With further ripening operation, the assemblies grew larger, which was reflected by a slight decrease of the main absorption peak and the entanglement of helical nanostrucutres (Fig. S15).
In the CD spectra, due to the formation of the chiral nanostructures, both of these species contributed to the CD signals. For the vortex mixing sample, the CD peak showed at 351 nm, while for the ripening processed assemblies, the CD maximum moved to 380 nm, which was the right position of shoulder absorption peak. In any cases, we saw that the crossover is in the same place at 313 nm (the position of the chromophore), indicating that the exciton coupling is based on the interactions between these chromophores after aggregation.
Considering that the ripening process needed much longer ripening time, these change in CD spectra might be caused by the enhanced J-like aggregation after thermodynamic equilibrium. In addition, tiny nanostructure difference of BTACA assemblies between these two process were observed from the SEM images ( Fig. 3 and S16), which might also lead to the different CD spectra. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2015-07-02T00:00:00.000 | 6468704 | {
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} | pes2o/s2orc | Controlled electromechanical cell stimulation on-a-chip
Stem cell research has yielded promising advances in regenerative medicine, but standard assays generally lack the ability to combine different cell stimulations with rapid sample processing and precise fluid control. In this work, we describe the design and fabrication of a micro-scale cell stimulator capable of simultaneously providing mechanical, electrical, and biochemical stimulation, and subsequently extracting detailed morphological and gene-expression analysis on the cellular response. This micro-device offers the opportunity to overcome previous limitations and recreate critical elements of the in vivo microenvironment in order to investigate cellular responses to three different stimulations. The platform was validated in experiments using human bone marrow mesenchymal stem cells. These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli. This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.
Stem cell biology has become a major research focus, but conventional culture systems are often limited in their ability to control local cellular microenvironments and spatiotemporal signaling. Recent studies have reported that mechanical stimulation influences the cell microenvironment and drives stem cell differentiation processes 1 . In parallel, electrical stimulation appears to be equally crucial for the development of conductive and contractile properties of cardiac tissue constructs, as extensively studied by Vunjak-Novakovic and colleagues 2 . Additionally, the simultaneous application of electrical, mechanical and chemical stimuli is required to fully reproduce in vitro the native microenvironment of striated muscle in vivo 3 . Specifically, for cardiac muscle, the in vitro system should be engineered with multiple stimulations to approach the in vivo condition in cardiac tissue where the electrical and mechanical signals are strongly coupled 2 .
Consequently, the capability to reproduce in vitro the complex native microenvironment combining these simulations, may offer the opportunity to investigate the role of each stimulation to delineate the individual or synergistic effects on the development, function, differentiation or regeneration of the tissue.
Previous studies combining multiple stimulations in a single platform mainly consist of bioreactors at the macroscale [4][5][6][7][8] . While these systems provided useful insights into electromechanical phenomena, they require large numbers of cells, large volumes of reagents, and are limited in their accessibility for high resolution and/or time-lapse imaging.
Therefore, the lack of advanced micro-tools to replicate fundamental aspects of the in vivo microenvironment (cardiac or skeletal muscle) in a highly controlled manner, including mechanical and electrical stimulation, represents a limiting factor in understanding the causal relationships between single or combined stimulations and their related electrophysiological and morphological consequences 9,10 . Specifically, we focused on mimicking the microenvironment of cardiac muscle tissue.
Results
Device design and fabrication. Polydimethylsiloxane (PDMS) was chosen as the main material for the production of the micro-bioreactor ( Fig. 1) due to its favorable features in cell culture applications (namely gas permeability and optical transparency) and the robustness of soft-lithography techniques. In addition, its elastic mechanical properties were exploited to apply controlled strains to cells, while the ability to embed electrical paths by locally doping the PDMS pre-polymer with carbon nanotubes (CNTs) 17 was used to deliver electrical stimulation.
The assembly process of the micro-bioreactor is schematically depicted in Fig. 2A(a-f). The device comprises three main layers, namely a pneumatic layer for mechanical stimulation, a conductive layer for electrical simulation, and a fluidic layer for cell culture. We produced the fluidic and pneumatic layers of the PDMS device by standard soft lithography ( Fig. 2A). The conductive layer was produced by casting a mixture of CNTs and PDMS on the silicon wafer mold.
When designing the microfluidic system, care was taken to enable precise control over multiple stimulation factors and to ensure compatibility with standard fluorescence and confocal microscopes, in terms of transparency and specimen thickness (to minimize focal plane distance). The PDMS device was designed with two identical chambers (1.2 mm wide, 15 mm long, and 250 μ m high), arranged vertically and separated by a thin (~100 μ m) membrane ( Fig. 1B-C). A design approach similar to the one adopted in the lung-on-a-chip device by Huh et al. 27 was used, in which the thin lateral walls deform when negative pressure is applied to the side channels, thus transmitting a highly uniform strain to the membrane where cells are adhered. Furthermore, both lateral walls of the top chamber are made of a conductive polymer, so that electrical stimulation can be provided to the cells, in addition to the mechanical stimulation. Lastly, biochemical factors can be introduced by pipetting solutions into the inlet of the central channel (Fig. 1A).
Each device that was produced was tested to verify its functionality by adding phosphate buffer saline (PBS) solution to the fluid channel and connecting the device to the vacuum line and electrical connectors. Two tubes were attached to the vacuum access ports by interference fit, and two gold-coated pins were inserted into the electrode access holes (Fig. 2C). The hydraulic tightness was assessed by visual inspection, and a leakage was evident when air bubbles formed in the PBS during mechanical actuation. Electrical connections were tested through measurements, within the solution, with an oscilloscope probe.
The stimulation setup, consisting of a vacuum pump, an electronic valve, an electrical stimulator, and a frequency generator, was located outside the incubator. Only one tube for the vacuum line was required, along with two metallic wires for insertion into the incubator, which was connected to a manifold in Scientific RepoRts | 5:11800 | DOi: 10.1038/srep11800 order to control multiple devices at the same time. This had the benefit of minimizing deviations from normal cell culture procedures.
Finite element modeling (FEM). Finite element simulations were conducted to test the actuation principle of the device and to determine the expected strain field profiles at different pressures. The objective of the simulation was to identify a configuration capable of attaining deformation values from 0% to 8% (to mimic physiological conditions 28 ), while minimizing delays that occurs by trial and error method. Efforts were concentrated into finding a simplified production strategy, in order to avoid the additional step required in the lung-on-a-chip microdevice 13 , i.e. the chemical etching of the side membranes resulting from the soft lithography procedure.
The overall system was considered as a 2-dimensional (2D) model, and the device model was comprised of PDMS, whose mechanical behavior was simulated differently depending on the expected deformation regime 29 ; thus, the PDMS was incompressible and linearly elastic for small deformations, and hyper-elastic when larger deformations were involved ( Supplementary Fig. 1). A static pressure of − 700 mmHg was applied to the inner surfaces of the vacuum compartment (orange in Supplementary Fig. 1).
The cell culture membrane attained the desired strain level, even near the chamber sidewalls, while the resulting curvature was minimal (corresponding to a maximum deflection of 14 μ m, perpendicular to the initial plane of the membrane). Therefore, we chose to produce the devices without etching the membranes in the side channels where the vacuum was applied, simplifying the production procedure and increasing the production efficiency of the chip. Device characterization. Membrane deformation-mediated mechanical stimulation of cells was achieved by controlling the time-dependent air pressure in the two lateral channels flanking the central region (Fig. 1C). The characterization of membrane strain in the device was conducted by tracking displacements of 1μ m diameter iron particles embedded in the spin-coated PDMS membranes (Fig. 3). Actuation pressures were produced using an eccentric diaphragm pump, and varied from 0 mmHg to − 700 mmHg in 50 mmHg incremental steps (Fig. 3C). By combining a custom-written MATLAB code and a particle tracker ImageJ plug-in 30 , a deformation heat map was constructed, which recorded the position of 25 speckles as they moved through time (Fig. 3A,B).
Color maps were obtained by plotting normal (ε YY , ε XX ) and shear strains (ε XY ) computed at the nodes of the matrix array (Fig. 3A,B). Measurements of deformation (Fig. 3B) revealed an increased normal strain perpendicular to the fluid channel (y direction) with increasing negative pressures. Conversely, there were no significant deviations in normal strain in the x direction (parallel to the cell channel) nor in shear strain, ε XY. These results were obtained with a spin-coated membrane of ~100 μ m thickness and, in this condition, an 8% maximum strain was measured at − 700 mmHg, consistent with the numerical characterization. A calibration curve obtained by measuring strain corresponding to vacuum pressure levels is shown in Fig. 3C.
The devices were operated for 7 days at 2 Hz to demonstrate their stable strain application. Performing this long-term run did not significantly affect the strain values, showing insignificant levels of material plasticity or fatigue, as supported by a previous long-term study of PDMS pneumatically-actuated elements 31 .
To evaluate the electrical stimulation, a stainless steel needle was inserted into the device through the top layer in order to position its tip at the center of the cell culture region. The needle was connected to a digital oscilloscope and used as a voltage probe. Ultimately, the efficacy of the electrical stimulator Cell orientation and immunofluorescence staining. Cell stretching experiments were performed using hMSCs (Lonza Group Ltd., Basel Switzerland), and the effects of the electromechanical stimulation on this cell line were analyzed. After a 24 h static culture, a 3% or 7% strain at 1 Hz was applied to the membrane to which the cells were adherent, as reported in the literature for similar applications 32,33 . For electrical stimulation, a biphasic signal was chosen, as used in previous studies 20,23 . Specifically, in order to stimulate the cells with an average electric field of 5 V/cm, biphasic square-wave pulses (+ 1.2 V for 1 ms, − 1.2 V for 1 ms, 1 Hz) were applied.
Cell orientation analysis was performed after 14 days of stimulation using the Fiji directionality plugin 34 , based on Fourier spectra analysis. To evaluate the orientation distribution of cytoskeleton fibers, results from five devices per configuration (namely control, mechanical, electrical and electromechanical) were analyzed by plotting means and standard deviations (SD). Orientation data were compared using a t-test with p values < 0.05 considered as statistically significant. Morphological changes in cells subjected to mechanical, electrical or electromechanical stimulation were compared to control cells that were not exposed to any stimulation (Fig. 4A). Interestingly, the actin fibers in mechanically-stimulated cells were oriented perpendicular to the strain direction and showed a more elongated spindle-like morphology compared to control cells. These findings are consistent with previous observation of cytoskeletal reorganization of MSCs and endothelial cells under shear stress stimulation 35,36 . Orientations were quantified (Fig. 4B) by setting 0° as the cell orientation perpendicular to the strain direction and 90° as that parallel to the strain direction. In the cases of mechanical or electromechanical stimulation with 3% strain, or electrical stimulation alone, the percentage of cytoskeletal fibers oriented in each defined sector was evenly distributed, and did not exceed 25% in any 30° sector, which was comparable to the unstimulated configuration (control). However, in devices that were mechanically stimulated with a 7% strain, cells oriented perpendicularly to the strain direction, namely in the 0-30° and 150-180° sectors, with ~30% of the cytoskeletal fibers in each of these sectors. Furthermore, this cellular reorientation was increased when mechanical stimulation at 7% strain was combined with electrical stimulation, resulting in more than 75% of the cytoskeletal fibers becoming aligned within ± 30° of the perpendicular direction.
In mature cardiac cells, the CX43 gap junction protein is essential for intercellular communication when single cells constitute a functional pluricellular complex. Therefore, CX43 serves as a marker of induced differentiation 37 , and its expression was evaluated here by fluorescence immunostaining (Fig. 5A). There were statistically significant differences in CX43 intensities between control samples and cells that underwent electrical stimulation (5 V/cm) coupled with low strain levels (3%) (Fig. 5B). No significant differences were observed between control cells and cells exposed to electromechanical stimulation with a higher strain level (7%). Moreover, no differences in cell density were observed between the stimulated (3% strain and 5 V/cm) and control samples after 14 days in culture (Fig. 5C). hMSC gene expression analysis. As a proof-of-principle, relative RNA expression analysis was performed using cells harvested from a single microfluidic device. RNA was extracted from five devices per configuration (control, mechanical and electrical), and prior to performing quantitative real time polymerase chain reactions (qRT-PCR), the quality and concentration of extracted RNA samples were evaluated with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA, USA) (Fig. 6A,B). To demonstrate the ability to measure changes in gene expression, we identified primers (see Supplementary Table 1) for the specific cardiac markers (GATA4, MEF2C, MYH7, NKX2.5, TUBB, CX43, TNNT2, and OCT4) using Primer Express 3.0 (Life Technologies), Carlsbad, CA) and optimized their measurement according to a previously reported protocol 38 . GAPDH was chosen as the housekeeping gene. Assays were performed in triplicate for each sample, and the data were analyzed and statistically evaluated using the delta-delta C T method, as previously described 39 .
After 14 days in culture, hMSC RNA concentrations were found to vary from ~800 pg/μ l to ~2 ng/μ l (Fig. 6A). The results exhibited intact RNA without partial degradation or contaminating genomic DNA, as well as typical 18 S and 28 S peaks, which had integrity numbers of 8.9 ± 0.1 (Fig. 6A). The relative fold-changes in gene expression for mechanically, electrically and electromechanically-stimulated cells were presented (Fig. 6C). Changes in gene expression for GATA4, MEF2C, and TUBB were marginally higher following mechanical stimulation compared to electrical stimulation, with no statistically significant differences observed. Differences between mechanical and electrical stimulation were statistically significant for MYH7, NKX2.5, and TNNT2 expression (p < 0.0001), where mechanical stimulation seems to play a key role in inducing their expression. Only CX43 exhibited a higher fold-change following electrical stimulation consistent with previous qPCR data on MSCs electrically stimulated in a tissue culture plate 22 . Moreover, fold-changes were significantly higher following electromechanical stimulation compared to each single stimulation for MEF2C (p < 0.005), MYH7 (p < 0.0001), NKX2.5 (p < 0.05), and TUBB (p < 0.0001), while TNNT2 was significantly lower (p < 0.0001) following electromechanical stimulation compared to mechanical stimulation but not statistically different compared to electrical stimulation.
Discussion
The novel micro-scale cell stimulator presented in this study is capable of providing controlled and simultaneous electrical, mechanical, and biochemical stimulations to cells cultured in a microfluidic system. The main advantage of our platform is the ability to apply each stimulation independently or to combine three different stimulations to study interactions of multiple stimuli, which more closely represents complex in vivo conditions. We designed the microfluidic device presented herein to accomplish these challenging tasks, and optimizing the geometrical parameters by FEM analysis before production. In addition, each stimulation can be appropriately fine-tuned to achieve specific experimental requirements, offering a wide range of practical bioengineering applications.
We achieved a high level of versatility in stimulating cells on a chip by designing a multilayer PDMS device (Fig. 1), where each layer performs a specific function. Specifically, the pneumatic layer performs mechanical stretching of the cell culture substrate, the conductive layer is used to apply a uniform electric field to cultured cells, and the fluidic layer provides the opportunity to deliver biochemical stimulation. The strain values applied fell within the range of those used in previous macro-scale mechanobiological experiments 32,33 . Prior calibration enables the operator to impose a specified strain by simply regulating the amplitude of vacuum pressure applied to the air channels. Values of electrical field gradients of 5 V/cm are easily attainable, and are comparable to those found in vivo 40 . The presence of easily-accessible channels for the addition of different media allow for time-varying administrations of factors, as needed, to optimize differentiation.
Our microfluidic device is compatible with fluorescence immunostaining and confocal imaging. Moreover, we demonstrated that our platform can be applied for monitoring spatiotemporal conditions and probing the effects of various stimuli on cells. Of notable interest, an immediate practical application of the device is the electromechanical stimulation of hMSCs, where we observed that mechanical stimulation induced morphological changes in cells and actin cytoskeletal rearrangements in the direction perpendicular to the applied strain (Fig. 4A). Most importantly, the stimulated cells can be easily harvested from the device to perform standard molecular biology analyses, such as qRT-PCR. As result, we observed changes in gene expression generally consistent with our expectation that either mechanical or electrical stimulation helped to induce activation of cardiac myocyte markers (Fig. 6C). When we compare the relative effects of different modes of stimulation, we note similar tendencies, although a stronger effect associated with mechanical strain as compared to electrical stimulation. These methods could be applied to ranges of conditions for both modes of stimulation in order to further investigate the relative impact and the potential for synergistic effects on cell differentiation. These findings intend to show the capabilities of our novel technology that, when combining multiple cell stimulations, offers an important tool for improving investigations of cellular responses in several biological areas, such as stem cell differentiation, cardiac tissue engineering, and regenerative medicine.
Methods
Device design and production. The device is comprised of three main layers of polydimethylsiloxane (PDMS) obtained from a base of pre-polymer (Sylgard 184, Dow Corning, Midland, MI, USA) at a ratio of 10:1 base to curing agent. The PDMS layers were obtained by replica molding from corresponding silicon molds produced in a clean room environment using standard photolithography techniques with SU-8 photoresist (MicroChem, Newton, MA, USA), starting from 4′ ′ silicon wafers. Each mold was initially treated with 50 μ l of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich, St. Louis, MO, USA) under a vacuum for 2 h to facilitate subsequent PDMS demolding. The assembly process of the micro-bioreactor is shown in Fig. 2A(a-f).
Mold A, containing the electrical paths (Fig. 2Aa), was used to cast a CNT-PDMS mixture (20%, w/w) prepared as described in our previous work 41 . The conductive CNT-PDMS mixture was added to fully cover the top of the mold and then carefully spread with a rubber spatula to fill the electrode features. Excess material was removed with a cell scraper (Fig. 2Ab), thus allowing the conductive polymer to occupy only the dedicated vertical features. The mixture was cured by placing the mold into an oven at 80 °C for 4 h, creating the conductive layer for electrical stimulations. Furthermore, a 100 μ m thick layer of PDMS was spin-coated on top of mold A (Fig. 2Ac) and allowed to cure in an oven at 80 °C for 4 h to produce a deformable membrane for mechanical stimulations.
Subsequently, the pneumatic actuation and fluidic layers were cast by pouring liquid PDMS on top of mold B and mold C, respectively. After complete polymerization (at 80 °C for 4 h), the pneumatic actuation was removed from the mold and bonded to mold A (Fig. 2Ad) by means of a standard plasma treatment of both layers. The binding is performed with the aid of a microscope to accurately align the pneumatic layer and mold A based on the corresponding features. Individual devices were trimmed from the wafer-size assemblies by cutting out rectangular sections (about 35 × 15 mm) with a razor blade (Fig. 2Ae), and holes were created with biopsy punches (Ted Pella Inc., Redding, CA, USA) to create access ports (1 mm and 3 mm in diameter for pneumatic actuation and cell conditioning, respectively). Each device was plasma bonded to the fluidic layer precisely aligning the features under the microscope (Fig. 2Af). Finally, another plasma treatment allows the bonding of the device onto a glass microscope slide as a supporting substrate. The final device featured two identical central chambers (1.2 mm wide, 15 mm long and 250 μ m high), arranged vertically and separated by a thin membrane where the cells were cultured (Fig. 1B,C).
Finite element modeling (FEM).
A computational model of the microfluidic platform was built to assess the magnitude of the strain imposed on cell monolayers when a negative pressure load was applied to the actuation (side) channels. The system was idealized as a 2-dimensional (2D) model, with all cell, air and media compartments represented, surrounded by a 500 μ m thick PDMS frame. The corresponding 2D plane strain finite element model (FEM) was implemented in the Abaqus/Standard 6.10-1 commercial software package (Dassault Systemes Simulia Corp., Providence, RI, USA), and discretized with 8-node biquadratic plane strain quadrilateral (CPE8) elements with a mapped meshing scheme. Mesh sensitivity studies were conducted to ensure consistency of results in terms of nodal displacement of the cell culture membranes. The resulting elements had an average edge size of ~25 μ m, thus generating a final mesh of ~10928 elements. As the mesh density increased, the displacement of the chosen nodes varied by less than 1%.
Different material properties were assigned to different parts of the geometry, depending on the corresponding load conditions 42 . In particular, the nonlinear behavior of PDMS was considered by implementing a hyperelastic model, where high deformations were present (i.e. side membranes). The stress-strain relationship of a hyperelastic material is represented by the following formula: where σ is the Cauchy stress, W is the strain energy function, B is the left Cauchy-Green deformation tensor, J is the volume ratio, and I 1 , I 2 , and I 3 are the first, second, and third invariants of B, respectively. For a Mooney-Rivlin material, such as PDMS, the strain energy function, W, can be described as: where C 1 and C 2 are material constants. The material constants were set as being equal to C 1 = 254 kPa and C 2 = 146 kPa 43 . Conversely, where small deformations were assumed, the PDMS was modeled as being incompressible and linearly elastic and the spin-coated membrane was modeled as being stiffer than the bulk material 44 . The material properties, Young's modulus, and Poisson ratio were thus set as being equal to E = 0.60 MPa and ν = 0.5 for the bulk material, and E = 1.2 MPa and ν = 0.5 for the membrane, respectively. The modeled geometry is depicted in Supplementary Fig. 1, highlighting the material properties based on the assigned sections.
The boundary conditions for the FEM were as follows: (i) the edges of the outer PDMS frame were fixed (encastre constrain) to consider their continuity with the bulk material of the device; (ii) self-contact was set, among all the internal surfaces of the compartments, with a no-slip tangential behavior once surfaces came into contact; (iii) a static pressure of − 700 mmHg was applied to the inner surfaces of the vacuum compartment (orange in Supplementary Fig. 1), through 20 incremental steps. Device characterization. Device characterization was focused on two aspects, namely membrane deformation as a function of negative pressure (vacuum) and electric signal recording within the cell culture channel.
For strain characterization, iron particles (Inframat Advanced Materials, Manchester, CT, USA) with an average diameter of 1 μ m were suspended in the PDMS mixture preparation and mixed for 1 min to break up aggregates. In this way, it was possible to embed the particles in the spin-coated membrane and track their displacement (Fig. 3A,B). A range of actuation pressures, ranging from 0 mmHg to − 700 mmHg (steps of − 50 mmHg), was applied to the pneumatic layer using an eccentric diaphragm vacuum pump (Trivac D&B, TX, USA). Pictures were taken at various actuation conditions using a 200X USB Microscope (AnMo Electronics Corporation, Taiwan) and stored for subsequent image processing.
A custom-written MATLAB code (MathWorks, Natick, MA, USA) was used to convert the images in binary format and track 25 bead locations at the vertices of a 5 × 5 matrix, using the particle-tracking algorithm of ImageJ 45 plugin (Speckle TrackerJ 30 ). As a result, the X and Y positions of each selected particle spot for each video frame were saved in a dataset file. The dataset files were then analyzed in order to visualize the strain map in different directions (XX, YY, and XY). This procedure was repeated for ten different devices and considering different membrane regions in the same device.
For the electrical characterization, a metal needle was injected into the PDMS from the top layer in the center of the cell seeding area, and the signal was recorded with an oscilloscope.
After production, each device was checked for mechanical and electrical functionality by controlling leakages during vacuum application and monitoring signals during electrical stimulation.
Control system. The system illustrated in Fig. 2B was assembled to perform cyclic-controlled vacuum and electrical stimulation procedures. A glass reservoir, connected to a diaphragm vacuum pump (Cole Parmer, Vernon Hills, IL, USA), was used as a vacuum source. An electronically-controlled valve (ITV Electronic Vacuum Regulator, SMC, Noblesville, IN, USA), controlled by a frequency generator, enabled tuning of the output pressure signal by imposing switching conditions between vacuum source and atmospheric pressure (both in terms of frequency and duty cycle). Furthermore, an electrical stimulator (STG4002, Multichannel Systems, Reutlingen, BW, Germany) was used to deliver a bipolar electrical signal, synchronized with the mechanical actuation, where negative-to-positive variation in the slope of the frequency generator signal was used as a trigger. Each device was equipped with tubing for pneumatic actuation and with gold pin wires for electrical signals (Fig. 2C). A manifold for pneumatic distribution and electrical connectors for signal distribution were placed inside the incubator and connected through the back port of the incubator to the output line of the valve and the output connectors of the electrical stimulator.
Cell culture and stimulation parameters. Before seeding cells into the microfluidic chambers, devices were sterilized by autoclaving and drying in an oven (80 °C) overnight, followed by incubation with 50 μ g/ml human fibronectin (Life Technologies) for 30 min (37 °C, 5% CO 2 ). Human bone marrow-derived MSCs (hMSC, Lonza, Basel, Switzerland) were seeded on fibronectin-coated T75 tissue culture flasks and cultured with growth medium (DMEM, 10% FBS, and 1% penicillin/streptomycin, Life Technologies). Upon reaching 80% confluence, cells were trypsinized (0.05% Trypsin-EDTA (1x), phenol red, Life Technologies) and resuspended in fresh growth medium at a density of 1.5 × 10 6 cells/ ml. Aliquots of 20 μ l cell suspensions were used for seeding microfluidic devices by injecting the suspensions into a fluidic well with conventional pipettes. To allow adequate adhesion of cells to the cell culture membrane, electrical and/or mechanical stimulation was applied at 24 h after seeding, and cells were maintained in culture in DMEM for an additional 14 days.
Mechanical stimulation was set as strain of 3% or 7% with 1 Hz frequency, following literature reporting a cyclic uniaxial stretch of 5-10% and 1-2 Hz for artificial cardiac tissue 3,46,47 . For the electrical stimulation we selected a pulsed stimulation to replicate the electrical environment of human heart tissue 21 . In particular, we chose biphasic square pulses of 1 ms at 1 Hz as biphasic stimulations are physiologically relevant and are known to avoid cell damage 48,49 . A field in the range of 5 V/cm was selected because it falls within the range of physiological values, 0.1-10 V/cm 18 , and is consistent with several previous studies 50-53 . Quantification of cell orientation. Cell orientation analysis was performed using Fiji software (Directionality plugin) 34 after 14 days of stimulation. The analysis was carried on cytoskeleton filaments, using images acquired after counterstaining. Five devices for each configuration (control, mechanical stimulation, or electrical stimulation, and combinations thereof) were analyzed. Statistical analysis of the means was performed by a two-tailed t-test, and differences were considered statistically significant in cases where the level of confidence exceeded 95%.
Immunofluorescence staining and intensity quantification.
To demonstrate the compatibility of the devices with fluorescent microscopy techniques, cells were stained following standard immunofluorescence protocols. After washing cells with 1x PBS, cells were fixed with 4% paraformaldehyde for 10 min and washed twice in 1x PBS. A solution of 0.1% Triton-X100 was used to permeabilize the cells for 15 min (Image-iT ® Fixation⁄Permeabilization Kit, Life Technologies). To prevent non-specific antibody binding, cells were treated for 2 h with a blocking buffer containing 1x PBS, 4% goat serum, and 5% bovine serum albumin. Samples were then incubated overnight at 4 °C with rabbit polyclonal antibody (Abcam, Cambridge, UK) against CX43 at a 1:200 dilution. After three washing steps with washing buffer, WB (1x PBS, 1% bovine serum albumin), cells were incubated with an Alexa Flour ® 488 secondary antibody (Life Technologies) at a 1:200 dilution for 1 h in the dark and washed again with WB. Finally, counterstaining was performed using DAPI (Sigma-Aldrich) for nuclei at a 1:200 dilution and ActinRed ™ 555 ReadyProbes ® Reagent (Life Technologies) for actin cytoskeleton staining. Images were captured using a LSM-780 confocal microscope (Zeiss, Oberkochen, Germany) and processed with Imaris software (Bitplane Scientific Software, Zurich, Switzerland). CX43 expression was quantified from immunofluorescence images through the ImageJ software, for at least three regions of interest (ROIs) for each device. Five devices were considered for each condition. The fluorescence intensities were normalized by the number of cells in the ROIs and plotted as mean ± SD. Cell number per mm 2 was assessed by counting the nuclei stained with DAPI and dividing by the area of the ROI. Statistical analysis was performed by a two-tailed t-test.
Scientific RepoRts | 5:11800 | DOi: 10.1038/srep11800 qRT-PCR. Quantitative real time polymerase chain reactions (qRT-PCR) were performed for a range of cardiac markers, using the StepOnePlus Real-Time PCR System (AB Applied Biosystems, Life Technologies). Cells were trypsinized within the device and collected in a 1 ml Eppendorf vial through three washing steps. RNA extraction was performed with the PicoPure ® RNA Isolation Kit (Life Technologies), following the manufacturer's recommendations, including the additional DNAse treatment step proposed by the manufacturer. qRT-PCR was carried out with RNA from cells harvested from a single microfluidic device. Five devices for each configuration (control, electrical stimulation, mechanical stimulation and electromechanical stimulation) were considered for the RNA extractions.
The qualities and concentrations of the extracted RNA samples were evaluated with the Agilent Bioanalyzer 2100 (Fig. 6A,B). To perform qRT-PCR, RNA (1 ng) was reverse transcribed into cDNA using the Sensiscript Kit (Qiagen, Venlo, Limburg, Netherlands). Polymerase chain reactions were performed using Power SYBR Green (Power SYBR ® Green PCR Master Mix, Life Technologies) following the manufacturer's recommended protocol in 20 μ l reaction volumes. Specific primers for amplifying GATA4, MEF2C, MYH7, NKX2.5, TUBB, CX43, TNNT2, and OCT4 mRNA are shown in Table 1 (Supplemental materials). Primers were designed using Primer Express 3.0 (Life Technologies) and optimized as described previously 38 . GAPDH was chosen as the housekeeping gene. The final reaction mixture for each well consisted of 10 μ l of Power SYBR Green, 0.5 μ l of cDNA, 8.5 μ l of water, and 1 μ l of forward and reverse primers. Reaction conditions were 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 3 s, annealing/extension for 30 s at 60 °C, and data collection for 3 s at 60 °C. The data obtained by qRT-PCR were then analyzed and statistically evaluated using the delta-delta Ct method 38 . | v3-fos-license |
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} | pes2o/s2orc | Photoswitchable basicity through the use of azoheteroarenes
Azoheteroarene photoswitches offer functional advantages over their more conventional azobenzene counterparts by virtue of their heteroaromatic ring(s). Here we report that azobis(2-imidazole) functions as a photoswitchable base due to the additional proton stabilisation that is possible in the protonated Z isomer, facilitated by the basic imidazole nitrogens. This thermodynamic difference in stability corresponds to a 1.3 unit difference in pKa values between the E and Z isomers. This pKa difference can be used to reversibly control solution pH.
Using light as a means of modulating a system is extremely attractive, as it is non-invasive and is capable of providing excellent spatial and temporal precision. As such, numerous attempts have been reported that aim to use light to control a huge range of processes, from photopharmacology and optochemical genetics to spin-state switching and optomechanical devices; all mediated by photoresponsive molecules. [1][2][3][4][5][6][7] One way in which light can be used to perturb a system is to modulate pH. This opportunity can, for example, lead to external control of the reaction rate of pH-dependent chemical/biochemical processes; 8-10 however thus far this is limited by the availability of suitable photoresponsive acids or bases. The change in acidity/basicity of a photoactive molecule could be an irreversible process, with examples reported for the release of a 'caged' proton upon irradiation with light. 10,11 Alternatively (and preferably), the process could be reversible, through use of a suitable photoswitchable acid or base. 8,9,[12][13][14][15][16] Perhaps unsurprisingly, given their privileged nature in this field (synthetic versatility, high extinction coefficients and quantum yields, high fatigue resistance, etc.), a number of the photoswitchable acids and bases reported are azobenzenes, 8,9,14 which can be reversibly switched between the thermally stable E and metastable Z isomers. Previously, differences in ground state acidity/basicity between azobenzene E and Z isomers has been demonstrated for 2-hydroxyazobenzene, where the E acid is stabilised by hydrogen bonding to the azo unit, whereas the Z isomer is not; translating to an increase in acidity of the Z isomer (or conversely an increase in basicity of the deprotonated E isomer). 13,14 Similarly, the azonium p-aminoazobenzenes reported by Woolley and co-workers, exhibit a pK a shift of approximately 1.5 units between isomers, due to hydrogen bonding between an ortho oxygen functional group on the azobenzene ring to the protonated azo unit in the E isomer (Fig. 1A). 15,17 Hydrogen bonding to the azo unit is not the only plausible strategy for modifying azobenzene acidity/basicity however. Hecht and co-workers, for example, have modulated the basicity of piperidine-containing azobenzenes through steric shielding in one isomer over the other (Fig. 1B). 8,18,19 We have previously reported novel azoheteroaryl photoswitches based on pyrroles and pyrazoles, 20 where the type, positioning, and substitution of the heteroaryl ring impacts key switching properties such as addressability, efficiency and thermal stability. However the presence of heteroatoms on the heteroaryl ring system presents other functional opportunities.
For example, Herges and co-workers have used the pyridine nitrogen of azopyridines as a ligating unit to access photodissociable ligands, 3,21,22 and have recently demonstrated an improvement in ligand affinity upon moving to arylazoimidazoles. 23 Herein we report the first photoswitchable Brønsted base of this class, utilising an azobisimidazole. Importantly with respect to the state of the art (Fig. 1), the protonation event occurs on one of the imidazole rings and is stabilised by the neighbouring ring, rather than through participation of the azo moiety. This results in a compound in which the Z isomer is over an order of magnitude more basic than the E isomer. Furthermore, by avoiding protonation of the azo linkage, the thermal half-life of the protonated Z isomer is increased relative to the neutral form.
Imidazole was chosen as the heteroaromatic ring for this study, due to its high basicity relative to other heteroarenes. Both azobis-(2-imidazole) 1 and azobis(4-imidazole) 2 (see Fig. 2) were considered to have the correct connectivity to enable proton stabilisation in the Z isomer. Energy minimised conformations and calculated UV/vis spectra were obtained for both these photoswitches in their neutral states using B3LYP/6-31G(d,p) and CAM-B3LYP/6-311G(2df,2p) respectively (Fig. 2). Azobisimidazole 1, which has the azo moiety attached at the 2-position of both imidazole moieties, was calculated to have a large n-p* absorbance in the Z isomer, and hence good separation of absorbances between the E and Z isomers, which should allow for excellent photoconversion between isomers at different wavelengths (good addressability 24 ). This large n-p* absorbance is due to the twisted conformation adopted by the Z isomer ( Fig. 2B), which minimises unfavourable steric interactions between a given N-methyl and the neighbouring azoaryl ring, and unfavourable lone pair interactions between the basic imidazole nitrogens and the azo nitrogens. Conversely, the energy minimised conformation of 2 predicts a planar conformation for the Z isomer and hence a negligible n-p* absorbance is predicted on symmetry grounds (Fig. 2B). These calculations are consistent with our findings on the conformational preferences and spectroscopic properties of the arylazopyrazoles and arylazopyrroles 20 -when there is a mirror plane through the molecule, as with the planar E isomers and Z-2, the n-p* transition is symmetry forbidden and no absorbance is predicted (although experimentally a weak absorbance is seen due to vibrational and rotational motion). The calculated spectrum for E-1 also had a significantly red-shifted p-p* absorbance compared to E-2, suggesting that visible light rather than UV light could be used for photoswitching. On the basis of these two features, azobisimidazole 1 was chosen for further study. Energy minimised conformations of protonated 1 confirmed that in the Z isomer, the proton would be stabilised by interacting with both basic nitrogens in the photoswitch (as shown in Fig. 4B) resulting in a planar conformation and, in turn, a decreased n-p* absorbance (Fig. 4A).
Azobisimidazole 1 was prepared in two steps from commercially available 2-nitroimidazole 3 (Scheme 1). First, N-methylation was performed using methyl iodide to give compound 4. In addition to impacting the conformation of these systems (vide supra), N-methylation of azole azoheteroaryl photoswitches is required to avoid very fast thermal isomerisation of the Z isomer through tautomerisation. 25,26 Reductive coupling of 4 using zinc in aqueous ammonium chloride, 27 gave the required photoswitch 1.
Irradiation was initially carried out on the neutral compound 1 in aqueous buffer at pH 9 (where 1 was assumed to be not-significantly protonated; this was later shown to be the case, vide infra) using a range of wavelengths (Fig. 3A). The best E-Z photoconversion was achieved using a 408 nm laser diode, with an estimated photostationary state (PSS) of (90 AE 3)% Z (see ESI, † for estimation); however 415 nm irradation (generated by a dye laser), with an PSS of (79 AE 5)% Z, was chosen to carry out further photoswitching, due to the tunability of the power using this light source. ‡ 532 nm irradiation of the n-p* absorbance of the Z isomer resulted in quantitative switching of the azobisimidazole back to the E isomer. § Notably, Z-1 has an intense n-p* absorbance (l max = 495 nm, e = 8000 M À1 cm À1 ), much stronger than that seen for Z azobenzenes (l max = B450 nm, e = 1500 M À1 cm À1 ). 28 This is in line with the DFT calculations that predict a twisted conformation (vide supra). The thermal isomerisation rate of neutral 1 was measured at 25 1C using UV/vis spectrometry in aqueous buffer (at pH 9) and the half-life was found to be 16 seconds. It is interesting that the Z isomer of the unsymmetrical azole photoswitch phenylazo-2-imidazole has been previously reported to have a thermal half-life of 9 hours (albeit in a different solvent). 29 Clearly, the move to a symmetric bisimidazole azo compound has a large impact on the energetics of thermal stability in imidazole-based photoswitches. The origins of thermal stability in such heteroaromatic azo compounds is under further study and will be reported in due course.
In order to determine the pK a of both the E isomer and the 415 nm PSS (79% Z), a pH titration was carried out on a buffered aqueous sample of 1. At each pH increment the UV/vis spectra of the E isomer and PSS were measured. Due to the reasonably fast thermal isomerisation rate, the irradiated sample was allowed to thermally isomerise back to the E isomer before adjusting the pH. The p-p* absorption maximum of the E isomer at 440 nm ( Fig. 3A) was plotted against pH in order to obtain the corresponding titration curves (Fig. 3B). The pH titration confirmed that at pH 9 azobisimidazole 1 was indeed neutral. The pK a was found to be 4.7 for the E isomer and the apparent pK a was 5.9 for the E/Z mixture present in the 415 nm PSS. By considering the PSS and the equilibria present in the system (Fig. 3C), the pK a of the Z isomer was estimated as 6.0 (see ESI †). A rough titration of the E isomer showed a second protonation event (assigned to protonation of the second imidazole ring) with an approximate pK a of 1.6 (see ESI †). No second protonation event was observed for the Z isomer above pH 3.
Given this data, the UV/vis spectrum for E-1 was recorded in acidic aqueous buffer at pH 3, to measure the photoswitch parameters of the protonated compound 1H + . Irradiation at 415 nm led to a PSS with a very similar spectrum to that of the E isomer (Fig. 4A). Although at first this could be considered to be a result of a PSS containing predominantly the E isomer, we instead attribute it to the fact that the protonated E and Z isomers E-1H + and Z-1H + have very similar UV/vis spectra. This assignment is supported by TDDFT calculated spectra for the protonated E and Z isomers (Fig. 4A); the spectra of both isomers is highly similar due to their planar conformation (Fig. 4B), as opposed to the neutral compound 1 (vide supra). As was the case for the neutral compound 1, the protonated compound 1H + could be switched between two PSS upon irradiation with 415 and 532 nm light, however due to the similarity in the E and Z spectra, the PSS compositions could not be obtained. ¶ The thermal isomerisation rate of protonated 1H + at 25 1C was found to be approximately 20-fold slower than for the neutral compound in aqueous buffer (t 1/2 = 352 s for protonated vs. 16 s for neutral). This increased half-life of the Z isomer on protonation is attributed to the requirement to (partially) break the intramolecular hydrogen bond present in Z-1H + in either the rotation or inversion transition states for thermal Z-E isomerisation. This trend is opposite to that observed by Woolley and co-workers for their tetra-o-methoxy substituted aminoazobenzene photoswitches upon protonation (Fig. 1A); where the protonated Z isomer has a much shorter half life than the neutral species. 15,17 This difference is explained by the site of protonation for the different photoswitches; protonation of the azo linkage in the substituted aminoazobenzenes (Fig. 1A) increases the thermal Z-E isomerisation rate, whereas in our case, protonation of 1 does not involve participation of the azo linkage.
Energy minimised conformations of E-1H + support our assumption that protonation occurs on the imidazole nitrogen rather than the azo. The global minimum for the imidazolium is significantly lower energy than for the azonium (DG(azonium À imidazolium) = 44.4 kJ mol À1 -see ESI †). In order to further characterise the protonated compounds, the carbon NMR chemical shifts (d exp ) for deuteronated E-1 were compared to calculated chemical shifts (d calc ) for E-1H + protonated on either the imidazole or the azo (see ESI †). The difference in chemical shift between d exp and d calc was much smaller for the imidazolium calculated structure than the azonium. Experimentally, pK a values for azonium cations are generally several units lower than imidazolium cations, [30][31][32][33] with the exception being when the azonium proton can be additionally stabilised by hydrogen bonding as in the systems reported by Woolley and co-workers (e.g. Fig. 1A). 15,17 Together the computational and experimental data supports our assumption that the protonation occurs on the imidazole rather than the azo nitrogen.
Given the significant pK a difference between the E and Z isomers of azobisimidazole 1, we sought to achieve a reversible pH 'jump' upon photoswitching. In principle, switching solution pH could have many uses, such as controlling the rate of a specific base catalysed reaction, or modulating enzyme activity. 10 In order to measure the pH of the solution a pH indicator was used. 34 Bromocresol purple (BCP) was chosen as it has a similar pK a value (vide infra) to that of the photoswitch, and it has an absorption maximum at a wavelength where the azobisimidazole has a negligible absorbance. A pH titration was carried out on BCP (Fig. S5, ESI †) and the pK a value obtained (6.8) was similar to the literature value of 6.2-6.4. 35 Prior to attempting the pH 'jump', pH titrations of both E-1 and the 405 nm PSS with equimolar BCP were carried out (Fig. 5A). From this a graph of A max (1)/A max (BCP) against pH was plotted (for both E isomer and PSS) and the exponential fit obtained (Fig. S6, ESI †). Using this graph the pH value could be extracted from the UV/vis spectrum of a given sample of 1 and equimolar BCP. Irradiation of a 2.7 Â 10 À4 M aqueous solution of 1 at 405 nm led to an immediate change in pH (Fig. 5B). A change of 0.29 pH units was observed, equating to a two-fold reduction in proton concentration in the PSS. In theory, using a 1 : 1 mixture of E-1 and E-1H + should enable a pH jump equal to the DpK a of the photoswitch (approximately 1.3 in this case). However, limitations on the concentrations used in this experiment and buffering by the indicator and potentially by carbonate is likely to decrease the range that can be obtained. Decreasing the concentration from 10 À4 to 10 À5 M resulted in a decrease in the pH 'jump' observed. While an increase in concentration would be expected to result in an increase in the pH jump, we were unable to assess this due to limitations in the method used for pH detection and the fast thermal isomerisation rates of the photoswitch. Nonetheless, several repeated cycles of photoswitching were carried out to ensure that the pH jump was reproducible over multiple irradiation cycles (Fig. S7, ESI †). The pH jump was consistent each time, within 3% of the mean value, suggesting that no irreversible photochemistry involving the indicator was occurring.
In conclusion, we have designed an azoheteroaryl photoswitch that bears basic nitrogens on the two heteroaryl rings, correctly positioned to stabilise a proton in the Z isomer. This results in a significant and reversible shift in pK a values between the E and Z isomers. We believe extension of the concept developed in this paper may lead to related photochromic molecules with an increased difference in pK a between the isomers, and that such systems may lead to a wide range of applications. For example, photoswitchable bases have already been shown to have potential as switchable general base catalysts, 8 but significant basicity differences between isomers may open up the possibility of switchable specific base catalysis and enzyme catalysis. Furthermore, light-driven proton concentration oscillations 14 may be used to extract kinetic data on proton transfer events in a range of chemical and biological systems.
We thank the Engineering and Physical Sciences Research Council and the Leverhulme Trust (RPG-2012-441 to R. D. R.) for funding. We also thank Dr Kuimova for equipment access.
Notes and references ‡ When Z-E thermal isomerisation is fast, higher power is needed to reach the PSS. § This occurred at a faster rate than that of the Z-E thermal isomerisation. ¶ The thermal isomerisation was too fast to allow quantification of the PSS by other spectroscopic methods (for example, by NMR). | v3-fos-license |
2019-12-11T14:01:57.047Z | 2019-12-10T00:00:00.000 | 209162618 | {
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} | pes2o/s2orc | Aconitase 2 inhibits the proliferation of MCF-7 cells promoting mitochondrial oxidative metabolism and ROS/FoxO1-mediated autophagic response
Background Deregulation of the tricarboxylic acid cycle (TCA) due to mutations in specific enzymes or defective aerobic metabolism is associated with tumour growth. Aconitase 2 (ACO2) participates in the TCA cycle by converting citrate to isocitrate, but no evident demonstrations of its involvement in cancer metabolism have been provided so far. Methods Biochemical assays coupled with molecular biology, in silico, and cellular tools were applied to circumstantiate the impact of ACO2 in the breast cancer cell line MCF-7 metabolism. Fluorescence lifetime imaging microscopy (FLIM) of NADH was used to corroborate the changes in bioenergetics. Results We showed that ACO2 levels are decreased in breast cancer cell lines and human tumour biopsies. We generated ACO2- overexpressing MCF-7 cells and employed comparative analyses to identify metabolic adaptations. We found that increased ACO2 expression impairs cell proliferation and commits cells to redirect pyruvate to mitochondria, which weakens Warburg-like bioenergetic features. We also demonstrated that the enhancement of oxidative metabolism was supported by mitochondrial biogenesis and FoxO1-mediated autophagy/mitophagy that sustains the increased ROS burst. Conclusions This work identifies ACO2 as a relevant gene in cancer metabolic rewiring of MCF-7 cells, promoting a different utilisation of pyruvate and revealing the potential metabolic vulnerability of ACO2-associated malignancies.
BACKGROUND
The TCA cycle represents the core pathway for aerobic oxidation of carbohydrates, lipids and proteins in mitochondria, by supplying the reduced coenzymes NADH and FADH 2 necessary for ATP production through the oxidative phosphorylation (OXPHOS). 1,2 However, many cancer cells prefer to enhance the glycolytic rate for energetic purposes, by favouring glucose transporters and glycolytic enzymes rather than the TCA cycle machinery, and this results in increased lactate production even in normoxia, a phenomenon known as aerobic glycolysis or the 'Warburg effect'. 3 This peculiarity is significant as the intermediates of the TCA cycle can diverge towards anabolic reactions, leading to amino acids, lipids and nucleotide synthesis necessary for supporting the high rate of proliferation. 1,2 Based on this, the manipulation of glycolysis and of the TCA cycle reactions by cancer cells represents a core strategy for their metabolic demands, such as energy production, biomass assimilation and redox control. In this effort, many cancer cells avidly catabolise several metabolites among which is glutamine that, by providing alfa-ketoglutarate (α-KG), replenishes the anabolic reactions of the TCA cycle for lipid and nucleotide synthesis, as well as for redox homoeostasis and protein O-GlcNAcylation. 4,5 Other wellestablished metabolites used to sustain cancer growth and adaptation include, for instance, acetate and fatty acids, and this metabolic plasticity expands the concept beyond the oncogenedriven metabolic rewiring. 3,6,7 The classical idea that the establishment of the Warburg effect is solely a consequence of dysfunctional mitochondria, as suggested by initial associations with mutations affecting TCA cycle or electron transport chain (ETC) proteins, is completely changed in a more realistic view of a cancer cell that constantly readapts to tumour microenvironment fluctuations, including nutrient and oxygen availability. 1, 3 In fact, many studies have clearly demonstrated that cancer cells relying on aerobic glycolysis retain intact mitochondrial pathways, 8,9 including biogenesis and mitophagy that greatly improve metabolic networks in response to nutrient availability. Moreover, the enhancement of oxidative capacities by reorientation of metabolites towards mitochondria 10,11 or by the disposal of damaged mitochondria via mitophagy 12 was demonstrated to be harmful to cancer cells addicted to the Warburg effect. Consistently, interfering with tumour metabolic phenotype is a novel therapeutic strategy that can be exploited to preferentially kill cancer cells. 13 Considering the advantages, in terms of energetic and anabolic precursors, that a cancer cell can acquire by deregulating the TCA cycle, in this paper, we have assessed whether the modulation of aconitase 2 (ACO2), the second enzyme involved in the TCA cycle, could induce metabolic rearrangements and proliferation defects in cancer cells. This enzyme is also known as mitochondrial aconitase as it catalyses the reversible isomerisation of citrate to isocitrate in mitochondria, a reaction that can be also performed in the cytosol by aconitase 1 (ACO1). We have focused the attention on ACO2 because it belongs to a branch of the TCA cycle particularly important for cancer metabolic features as it is interposed between citrate, which plays a role in lipid anabolism, and α-KG that can be replenished by glutamine anaplerosis. 1,2 Moreover, other relevant cues in support of a putative involvement of ACO2 in cancer are that (i) its reduced levels in pluripotent stem cells make glutamine a key fuel for the TCA cycle, 14 (ii) it was found inactivated in fumarate hydratase (FH)deficient tumours 15 and (iii) its expression was found downregulated in cancer cells. 16,17 Here we show that ACO2 expression is reduced in breast cancer, and increasing the levels of the enzyme in MCF-7 cells can inhibit cell proliferation. By generating cells with stable overexpression of ACO2 gene, we demonstrated that proliferation inhibition is associated with enhanced oxidative metabolism mainly due to redirection of pyruvate to mitochondrial oxidation. The metabolic phenotype of ACO2-expressing cell was favoured by autophagic/ mitophagic response to increased production of reactive oxygen species (ROS) by the activation of Forkhead box protein O1 (FoxO1) signalling.
Western blot analyses, mitochondrial and nuclear fractionation Protein lysates were obtained by incubation of total, nuclear and mitochondrial fractions on ice for 20 min in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 5 mM Na 4 P 2 O 7 and 2 mM Na 3 VO 4 ) supplemented with protease inhibitor cocktail (AMRESCO) and followed by sonication. Lowry's method was used for protein concentration before performing electrophoresis by SDS-PAGE and blotting onto a nitrocellulose membrane (Bio-Rad). The following primary antibodies were used: ACO2 (Novus Biologicals), IRP1/ACO1, GRP75, p-CREB, CREB, LAMIN B1, TOM20, HSP60, HSP75/TRAP1, AMPK, p-AMPK (Santa Cruz Biotechnology), IDH2, NRF1, PDHB (Abnova), β-ACTIN, PARKIN, FoxO1, p-FoxO1, ACTININ, ALBUMIN, DRP1, p-p70S6K, mTOR, p-mTOR, PARP-1 (Cell Signaling Technology), PGC-1α (Calbiochem), H3, UBIQUITIN (Merck Millipore), α-tubulin, LC3B (Sigma-Aldrich), FH (GeneTex) and total OXPHOS Human WB Antibody Cocktail (Abcam). Nuclear extraction was performed as previously described in Ciccarone et al. 18 Mitochondrial purification was obtained by resuspending cells in mitochondria isolation buffer (MIB) composed of 1 mM EGTA, pH 7.4, 5 mM MOPS, 5 mM KH 2 PO 4 , 0.1% BSA and 0.3 M sucrose. Cells were broken mechanically with dounce (10 strokes) and with a tight pestle (30 strokes) in ice. The homogenate was centrifuged at 2600×g for 5 min at 4°C, and the supernatant containing mitochondria was collected while the pellet was processed as before three times to enrich mitochondrial fraction. Supernatants were then centrifuged at 15,000×g for 10 min at 4°C . The pellet was washed three times with MIB and then centrifuged at 15,000×g for 10 min at 4°C. Quantitative real-time PCR (RT-qPCR) RNA extraction was obtained by using TRItidy G (PanReact AppliChem) according to the manufacturer's instructions. Synthesis of cDNA was obtained from 1 µg of total RNA by using PrimeScript™ RT Reagent Kit (Perfect Real Time) (Takara), and RT-qPCR reaction was performed by using the SYBR® Premix Ex Taq (Tli RNase H Plus) (Takara) on a StepOne real-time PCR System (Applied Biosystems). All reactions were run as triplicates, and relative quantification obtained by the comparative cycle threshold method by using ACTB for normalisation. Primers are listed in Supplementary Table 1. Cell proliferation assays Cell proliferation was evaluated by Trypan blue exclusion test procedure and by bromodeoxyuridine (BrdU) incorporation assay. For the latter, cells were incubated with 10 µM BrdU for 4 h and then fixed for 30 min in ethanol:acetic acid:water solution (18:1:1). DNA denaturation was obtained by incubation on ice for 10 min with 1 N HCl and then 10 min with 2 N HCl. Treatments with PBS/0.4% Triton X-100 solution for 10 min and then PBS/3% BSA for 1 h were performed before incubation for 16-24 h with an anti-BrdU antibody (Santa Cruz Biotechnology) followed by 1 h of incubation with an Alexa Fluor™ 568 donkey secondary antibody; nuclei were stained with 1 µg/ml of Hoechst 33342 for 5 min. Images of cells were obtained with a Delta Vision Restoration Microscopy System (Applied Precision, Issaquah, WA) equipped with an Olympus IX70 fluorescence microscope (Olympus Italia, Segrate, Milano, Italy).
Fluorescence lifetime microscopy (FLIM) of NAD(P)H autofluorescence FLIM data were acquired with a Nikon A1-MP confocal microscope equipped with a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA) by producing 80-fs pulses at a repetition rate of 80 MHz. A PML-SPEC 16 GaAsP (B&H, Germany) multi-wavelength detector coupled to a SPC-830 TCSPC/FLIM device (B&H, Germany) was used to collect the decay data. A 60 × oil-immersion objective, 1.2 NA, was used for all experiments. Samples were excited at 750 nm. Signals were integrated into the wavelength region of 408-496 nm. For image acquisition, the pixel frame size was set to 512 × 512 and the pixel dwell time was 60 µs. The average laser power at the sample was maintained at the milliwatt level. In the FLIM images, mitochondrial NAD(P)H responses were separated by the rest of the autofluorescence (cytoplasm, nuclei) by means of fluorescence intensity analysis. 19 Colorimetric assays The aconitase 2 activity was determined by using the Aconitase Activity Assay Kit (Sigma-Aldrich) following the manufacturer's Aconitase 2 inhibits the proliferation of MCF-7 cells promoting. . . F Ciccarone et al. instructions. Citrate Assay Kit (Sigma-Aldrich), α-Ketoglutarate Assay Kit (Sigma-Aldrich) and Fumarate Assay Kit (Abnova) were used to measure the levels of citrate, α-ketoglutarate and fumarate, respectively. Values were normalised on total protein amount.
Citrate synthase activity Citrate synthase activity was determined spectrophotometrically according to Oexle et al. 20 About 2 × 10 6 cells were harvested and washed in ice-cold PBS. Pellets were lysed in 100 mM Tris-HCl, pH 8.1 and 0.25% Triton X-100 supplemented with protease inhibitor cocktail (AMRESCO) for 30 min in ice. A total of 25 µg of proteins were used for each enzymatic reaction performed in 250 µl of reaction buffer (100 mM Tris-HCl, pH 8.1, 0.25% Triton X-100, 0.1 5,5-dithiobis(2-nitrobenzoate) (DTNB), 0.5 mM oxaloacetate and 0.31 mM acetyl-CoA). The principle of the assay is based on the reaction between DTNB and CoA-SH to form TNB with a maximum absorbance at 412 nm. The citrate synthase activity is proportional to the intensity of the absorbance. After a delay of 5 s, the reaction proceeds at 37°C for a period of 4 min and absorbance recorded at 10-s intervals by an Eppendorf BioSpectrometer®. Enzyme activity was represented as a change in absorbance per minute (U), normalised on total protein amount.
Oxygen consumption and ATP measurement Oxygen consumption was determined by using a Clark-type oxygen electrode as described in Di Leo et al. 11 ATP levels were determined by the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science) according to the manufacturer's instructions. Values were normalised on the protein amount.
Extracellular lactate assay
The level of extracellular lactate was measured as previously described with minor modifications. 11 Following 24 h from cell plating, the medium was replaced with a fresh one, and after 3 h, 500 µl of cell medium was precipitated with 250 µl of 30% trichloroacetic acid. Media were frozen after centrifugation at 14,000×g for 20 min at 4°C, 10 µl of supernatant was incubated for 30 min at 37°C in 290 µl of a buffer containing 0.2 M glycine/ hydrazine buffer, pH 9.2, 0.6 mg/ml NAD + and 17 U/ml LDH. NADH formation was followed at 340 nm by using an Eppendorf BioSpectrometer®.
Cytofluorimetric analysis Thirty minutes before the end of the experimental time, cells were incubated with 2-NBDG (100 µM) for measurement of glucose uptake, MitoTracker Green (200 nM) or Nonyl Acridine Orange (NAO) (50 nM), MitoTracker Red CMXRos (200 nM) for mitochondrial membrane potential, dihydroethidium (DHE) (50 µM) and MitoSOX Red (5 µM) for ROS determination and Bodipy 493/503 (1 µM). Cells were washed and then collected in PBS, and the fluorescence intensity immediately analysed by means of a FACScalibur instrument. In total, 10,000 events were counted, and mean fluorescence intensity expressed as arbitrary units.
Metabolite determination by HPLC Cell cultures were collected after 24 h from plating, washed with ice-cold PBS and subsequently centrifuged at 1890×g for 10 min at 4°C. Cell pellets were deproteinised as described in detail elsewhere. 21 Briefly, cell pellets were treated with a precipitating solution composed of CH 3 CN 75% and KH 2 PO 4 25% (10 mM) at pH 7.4 and then centrifuged at 20,690×g for 15 min at 4°C. Supernatants were collected and subjected to two chloroform washings in order to obtain an upper aqueous phase that was directly injected onto the HPLC, and analysed to determine concentrations of uridine diphosphate (UDP), uridine-diphosphate galactose (UDP-Gal), cytosine, uridine-diphosphate-n-acetylgalactosamine (UDP-GalNAc), uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), glutamate (Glu), glutamine (Gln) and taurine (Tau). Amino acids were analysed as ortophtalaldehyde (OPA) derivatives by using a method with precolumn derivatisation, 22 whilst cytosine and UDP derivatives were separated and quantified according to an ionpairing HPLC method previously set up. 21 For both analyses, the HPLC apparatus consisted of a SpectraSystem P4000 pump and a highly sensitive UV6000LP diode array detector (ThermoElectron Italia), equipped with a 5-cm light-path flow cell, set up between 200-and 400-nm wavelength for acquisition of chromatographic runs. Data were acquired and analysed by Chrom-Quest® software package provided by the HPLC manufacturer. Separation of the various compounds was carried out by using a Hypersil 250 3 4.6mm, 5 mM particle-size column, which was provided with its own guard column (ThermoElectron Italia). Species identification and quantification in deproteinised cell extracts were determined by matching retention times, peak areas and absorption spectra of those of freshly prepared ultrapure standards. If needed, cochromatograms were performed by adding proper standards with known concentration to the medium samples. Concentrations of UDP, UDP-Gal, cytosine, UDP-GalNAc and UDP-GlcNAc were determined at 260-nm wavelength and those of NO 2 and NO 3 were calculated at 206-nm wavelength. Differently, concentrations of OPA-derivatised amino acids were calculated at 338-nm wavelength. Intracellular glutathione levels were determined as previously described. 23 Determination of protein carbonylation Carbonylated proteins were detected by the OxyBlot Kit (Millipore, S7150) as previously described, 24 by using 15 μg of total proteins that were resolved in 12% SDS-polyacrylamide gels.
Chromatin immunoprecipitation (ChIP) analysis ChIP assays were performed on nuclear lysates as previously described. 25 Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature of 37%, and the reaction was quenched by 5 min of incubation in 0.125 M glycine. Cell monolayer was harvested in ice-cold PBS containing protease inhibitors, and nuclei isolation performed as previously described. 18 Chromatin sonication was achieved by using Bioruptor NextGen (Diagenode) to high power. Sonicated DNA of~500-1000 bp was pre-cleared with Protein A-coupled Sepharose beads pre-saturated with Salmon Sperm DNA and then immunoprecipitated with anti-FoxO1 antibody (Cell Signaling Technology) or normal rabbit IgGs (Santa Cruz Biotechnology) for 16 h at 4°C. Immunoprecipitated DNA amplification was performed by using SYBR® Premix Ex Taq (Tli RNase H Plus) (Takara) on a StepOne real-time PCR System (Applied Biosystems). All reactions were run as triplicates. The results are expressed as fold enrichment with respect to IgG control. Primers used are listed in Supplementary Table 2. Bioinformatic analyses ACO2 expression was assessed by Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) with accession numbers GSE15852 (n = 43 breast tumours vs normal counterpart) and GSE294318 (n = 54 breast tumours vs n = 12 healthy breast samples) through an Affymetrix Human Genome Array. Relapsefree survival (RFS) analysis referred to breast cancer was obtained from the Pan-cancer RNA-seq present in the Kaplan-Meier Plotter database. Mutational analysis of the ACO2 gene was performed by consulting the TGCA database.
Statistical analysis
The results are shown as means ± SEM and derive from at least three independent experiments. Statistical evaluation was conducted by using the unpaired two-tailed Student's t test for metabolic analysis and enzymatic activity assays, paired Student's t test for cytofluorimetric analysis and RT-qPCR analysis Aconitase 2 inhibits the proliferation of MCF-7 cells promoting. . . F Ciccarone et al.
and one-way ANOVA with post hoc Tukey test for cell counts after treatments.
ACO2 expression is reduced in breast cancer and increasing its levels dampens cell proliferation of MCF-7 cells
To assess the expression of ACO2 in breast cancer cells, we compared the non-tumorigenic human breast epithelial cell line MCF10A with a panel of breast cancer cell lines, most of which exhibit a dramatic reduction of ACO2 protein levels (Fig. 1a). The decreasing trend observed for ACO2 in these cell lines was not a common feature for all the aconitase isoforms or TCA cycle enzymes, as stated by the levels of ACO1 and IDH2 proteins, respectively. Moreover, such changes were not ascribed to relevant fluctuations in mitochondrial mass, as demonstrated by the expression of GRP75 (Fig. 1a). RT-qPCR demonstrated that the transcriptional downregulation of ACO2 was also effective along with decreased protein levels ( Supplementary Fig. 1a). The analysis of publicly available gene expression datasets deposited in the Gene Expression Omnibus (GEO) highlighted that ACO2 expression levels were reduced in human breast cancer biopsies (T) when either compared with non-tumoural adjacent counterpart (NT) or with normal breast tissue from other individuals (H) (Fig. 1b, c). The Kaplan-Meier Plotter demonstrated that the high expression of ACO2 in human tumour specimens is associated with good clinical outcomes in terms of relapse-free survival (RFS) 26 ( Supplementary Fig. 1b-d). Less than 1% of genetic alterations were instead identified for the ACO2 gene in breast cancer according to the TCGA data set ( Supplementary Fig. 1e).
Prompted by the evidence that ACO2 levels are downregulated in breast cancer cells and in human breast cancer specimens, we transiently overexpressed myc-tagged ACO2 in MCF-7 cells ( Supplementary Fig. 1f), demonstrating that high levels of ACO2 reduced cell proliferation ( Supplementary Fig. 1g). To corroborate this result, we generated MCF-7 cells with stable overexpression of ACO2, and we choose to perform our analyses on two independent clones (#2 and #4) with different degrees of ACO2 overexpression as well as enzymatic activity (Fig. 1d, e). Trypan blue exclusion test (Fig. 1f), bromodeoxyuridine (BrdU) incorporation ( Fig. 1g; Supplementary Fig. 1h) and analysis of cytosine levels ( Supplementary Fig. 1i) demonstrated dose-dependent inhibition e ACO2 activity determination obtained by colorimetric assay (n = 3; *p < 0.05, **p < 0.01 vs EV). f Cell proliferation assayed by Trypan blue direct cell-counting procedure (n = 3; **p < 0.01, ***p < 0.001 vs EV). g Cell proliferation assayed by BrdU incorporation assay (n = 3; *p < 0.05, **p < 0.01 vs EV). Representative pictures of BrdU incorporation are shown in Supplementary Fig. 1h of cell proliferation in ACO2-overexpressing clones with respect to control MCF-7 cells bearing the empty vector (EV).
ACO2 overexpression boosts mitochondrial metabolism
Mitochondrial metabolism was then monitored to reveal any influence due to the stable increase of ACO2 expression in MCF-7 cells. We observed an increase in the citrate synthase (CS) activity ( Fig. 2a) and consequently of the levels of the TCA cycle intermediates citrate, α-KG and fumarate in ACO2overexpressing cells (Fig. 2b). To investigate whether increased TCA cycle flux could result in activation of ETC, we performed lifetime imaging of NADH, which is extensively used to monitor changes in metabolism. 27 free or protein-bound state, respectively. 28 Since metabolic states can change the extent of NADH enzymatic binding, fluorescence lifetime imaging microscopy (FLIM) permits us to investigate, with submicrometric resolution and organelle specificity, whether the induced alteration in the TCA cycle was associated with increased binding of the reduced cofactor NADH. Figure 2c shows a significant increase in the average mitochondrial NAD(P)H lifetime, retrieved from the NAD(P)H FLIM images reported in Fig. 2d, in ACO2-overexpressing cells, thus indicating an increase in the enzyme-bound state of NADH species corresponding to higher mitochondrial NADH levels used by ETC. The elevated mitochondrial membrane potential (Fig. 2e), oxygen respiration (Fig. 2f) and ATP production (Fig. 2g) corroborated that ACO2 overexpression boosts OXPHOS in MCF-7 cells in a dose-dependent fashion. To test whether these changes were associated with an increase in mitochondrial mass, we assessed the relative amount of the mitochondrial translocase TOM20 with respect to the cytosolic protein ACTB (Fig. 2h) and the fluorescence intensity of mitochondrial-selective labels ( Fig. 2i; Supplementary Fig. 2a). These assays demonstrated that ACO2-overexpressing cells have an increased mitochondrial content along with expression of TCA cycle enzymes ( Supplementary Fig. 2b, c) and of both mitochondrial-and nuclear-encoded genes of the OXPHOS complexes ( Supplementary Fig. 2d-f). Consistently, the analysis of nuclear factors involved in mitochondrial biogenesis, including PGC-1α and ser129-phosphorylated CREB (p-CREB), were more highly detectable in ACO2-overexpressing clones (Fig. 2j). The unaltered expression of the mitochondrial chaperones HSP60 and HSP75 suggested a preserved mitochondrial homoeostasis in ACO2-overexpressing cells (Supplementary Fig. 2c).
ACO2 overexpression affects metabolic features of cancer cells and differentiated mammary epithelial cells
To assess whether improved mitochondrial metabolism promoted changes in typical features of cancer metabolism, we focused our attention on aerobic glycolysis and glutamine addiction in MCF-7 cells. After ACO2 overexpression, we observed the reduction of extracellular lactate release (Fig. 3a) that was not ascribable to altered glucose uptake, as demonstrated by the unaffected incorporation of the glucose fluorescent analogue 2-NBDG (Fig. 3b). Since this pattern was paralleled by an upregulation of the pyruvate dehydrogenase complex (i.e., β-subunit of the E1 component, PDHB) (Fig. 3c) and augmented acetyl-CoA levels (Fig. 3d), a more efficient way to funnel pyruvate towards mitochondria seemed to occur in ACO2-overexpressing cells. To demonstrate that the enhancement of oxidative metabolism is the cause of MCF-7 decreased proliferation observed after ACO2 overexpression, we replaced glucose with galactose to force cells to use OXPHOS for ATP production. 29 In this condition, control MCF-7 cells decreased their proliferation (Fig. 3e), whereas ACO2overexpressing cells underwent cell death (Fig. 3f).
Concerning glutamine metabolism, no alteration of glutamine and of glutamate levels was evidenced in our clones (Supplementary Fig. 3a). However, when cells were subjected to glutamine deprivation, we observed a significant decrease of cell proliferation in control cells with respect to ACO2-overexpressing cells (Fig. 3g) and no sign of apoptosis (data not shown). Treatments with the allosteric inhibitor of glutaminase, BPTES, had similar effects, thus highlighting that MCF-7 cells are less dependent on glutamine metabolism after ACO2 overexpression (Fig. 3h).
In parallel to metabolic aspects of cancer cells, we also monitored a typical metabolic trait of differentiated epithelial mammary cells, consisting of the accumulation of milk components or precursors. In fact, a decreased proliferation of breast cancer cells can be accompanied by cell differentiation, including MCF-7. 30 The increased levels of UDP-galactose, combined with UTP decline, indicated the activation of the lactose biosynthetic pathway after ACO2 overexpression (Supplementary Fig. 3b). In support of a different utilisation of UDP galactose, we found an impairment of glucosamine pathways as shown by the reduction of UDP-GlcNAc and UDP-GalNac ( Supplementary Fig. 3c). In this scenario, the increase in the human milk components taurine ( Supplementary Fig. 3d) and albumin (Supplementary Fig. 3e) together with the accumulation of neutral lipids ( Supplementary Fig. 3f) demonstrated that ACO2-overexpressing clones have a more differentiated phenotype.
ACO2 overexpression induces oxidative stress-mediated autophagic/mitophagic flux The enhancement of mitochondrial metabolism in ACO2overexpressing cells may be associated with augmented oxidative stress. Consistently, we observed an increase in intracellular (Fig. 4a) and mitochondrial ROS (Fig. 4b), as well as carbonylated proteins ( Supplementary Fig. 4a) after ACO2 overexpression. This pattern was accompanied by variations in the antioxidant system as demonstrated by the upregulation of GCLC and SOD2 genes ( Supplementary Fig. 4b), by decreased levels of reduced glutathione (Supplementary Fig. 4c) and by increased NADP + / NADPH ratio (Supplementary Fig. 4d). No sign of nitrosative stress was instead identifiable as suggested by the unchanged levels of intracellular nitrite and nitrate ( Supplementary Fig. 4e, f). We then tested the functional role of oxidative stress in cancer-related processes by incubating MCF-7 cells with the ROS scavenger Nacetylcysteine (NAC). We observed that ROS abrogation is associated with increased cell growth and with molecular features of epithelial-mesenchymal transition phenotype (i.e. upregulation of VIM and downregulation of CDH1) in ACO2-overexpressing cells ( Supplementary Fig. 4g, h).
ROS production by mitochondria is known to promote autophagy, which is necessary for the removal of damaged/ exhausted mitochondria via mitophagy. 31 Therefore, we measured the levels of lipidated LC3B protein (LC3B-II) as a marker of autophagy activation. The increased levels of LC3B-II in our clones and the further accumulation observed in the presence of the autophagy inhibitor chloroquine (CQ) demonstrated that ACO2 overexpression triggers an active autophagic flux (Fig. 4c). We further revealed an increase in mitophagy, as shown by the high levels of ubiquitinated proteins (Fig. 4d) and of PARKIN recruitment (Fig. 4e) in mitochondrial fractions. These results were corroborated by the co-localisation of mitochondrial matrixtargeted mitoDsRed with GFP-LC3 ( Supplementary Fig. 5a) and by the high expression of the mitochondria fission protein DRP1 32 in ACO2 clones ( Supplementary Fig. 5b). We definitely demonstrated that autophagic induction in ACO2-overexpressing cells was a consequence of ROS production by the treatment with the ROS scavengers NAC, MitoTEMPO and ascorbate that were able to inhibit the accumulation of LC3B-II protein ( Fig. 4f; Supplementary Fig. 5c). To define the relevance of autophagy induction in our cells, we inhibited it by preventing the acidification of lysosomes with bafilomycin A1 (BafA1). The reduction in the number of ACO2-overexpressing cells (Supplementary Fig. 5d) and the appearance of the cleaved fragment of PARP-1 protein, a typical marker of apoptosis, after BafA1 ( Supplementary Fig. 5e) suggested an important contribution of autophagy/mitophagy in the maintenance of cell homoeostasis buffering ROS.
Autophagy induction downstream of ACO2 overexpression is associated with ROS/FoxO1 signalling To identify the molecular pathways behind autophagic response in ACO2-overexpressing cells, we firstly focused on the key autophagy inhibitor mTOR. 33 The unchanged levels of Ser2448phosphorylated mTOR and its target Thr389-phosphorylated p70S6 kinase excluded nutrient-limiting conditions as determinants of ROS-mediated autophagic flux in our clones (Fig. 5a). Such evidence is corroborated by the low levels of Thr172 phosphorylation of the energy sensor AMPK (Fig. 5b). In support of Aconitase 2 inhibits the proliferation of MCF-7 cells promoting. . . F Ciccarone et al. this, we showed that our cells retain the ability to trigger autophagy when challenged with amino acid starvation (achieved by EBSS medium incubation), which is a canonical stimulus impinging on mTOR activity (Supplementary Fig. 5f). Then, we demonstrated that the activation of autophagic flux was associated with transcriptional upregulation of the key autophagic genes LC3B and ATG4B as well as of the mitophagic gene PINK1 (Fig. 5c). Considering that all autophagic genes here analysed are targets of FoxO proteins and that we have previously demonstrated that FoxO1 is a key transcription factor known to connect metabolism-associated oxidative stress with autophagy/ mitophagy induction, 24 we assessed the levels of FoxO1 in our experimental conditions. We firstly revealed that FoxO1 is highly abundant in the nuclear fractions of ACO2-overexpressing cells with respect to those of control (Fig. 5d). To determine whether FoxO1 nuclear localisation was a consequence of ROS buildup, we performed NAC treatment, but we did not evidence any specific change in its subcellular distribution (Fig. 5e). We thus analysed Fig. 3 ACO2 overexpression affects aerobic glycolysis and glutamine addiction. a Extracellular lactate content measured by an enzymatic/ spectrophotometric combined technique (n = 6, **p < 0.01, ***p < 0.001 vs EV). b Glucose uptake assessed by cytofluorimetric analysis in FL-2 channel assessed by using the fluorescent analogue of glucose 2-NBDG. MFI mean fluorescence intensity, a.u. arbitrary unit (n = 4). c Representative western blot (n = 3) showing the level of a subunit of the mitochondrial PDH complex. ACTB was used as loading control. d Determination of acetyl-CoA levels by HPLC (n = 3; *p < 0.05, **p < 0.01 vs EV). e Cell proliferation assayed by Trypan blue direct cellcounting procedure after incubation with galactose (GAL) or glucose (GLU) for 48 h (n = 3; *p < 0.05, ***p < 0.01 as indicated). f Percentage of Trypan blue-positive cells determined after incubation with galactose (GAL) or glucose (GLU) for 48 h (n = 3; ***p < 0.001 as indicated). g Cell proliferation assayed by Trypan blue direct cell-counting procedure after glutamine (GLN) depletion for 24 h (n = 3; *p < 0.05 as indicated). h Cell proliferation assayed by Trypan blue direct cell-counting procedure after glutaminase inhibitor (BPTES) treatment for 24 h (n = 3; **p < 0.01, ***p < 0.001 as indicated) the levels of Ser256-phosphorylated FoxO1 demonstrating that this modification is less marked in nuclear fractions of untreated ACO2-overexpressing cells while it increases after NAC treatment (Fig. 5e). To evidence whether this scenario was associated with changes in FoxO1 recruitment at autophagic gene promoters, we tested sequences known to be targeted by FoxO1 performing ChIP analysis after NAC treatment. This experiment demonstrated that FoxO1 is highly enriched at LC3B, ATG4B and PINK1 promoters (Fig. 5f) in untreated ACO2-overexpressing cells, whereas its binding is largely disrupted after ROS removal by NAC.
DISCUSSION
The specialised metabolic landscape that is established in each cancer cell derives from the activation of oncogenic pathways or abrogation of tumour-suppressor signalling and is continuously shaped by the interaction with tumour microenvironment comprising stromal/immune cells and nutrient/oxygen availability. 3 In some cases, cancer metabolic reprogramming can be a direct consequence of oncogenic driver mutations affecting metabolic genes as shown for half of the enzymes belonging to the TCA cycle: fumarate hydratase, succinate dehydrogenase, isocitrate dehydrogenase 2 and malate dehydrogenase 2. 1,2 All these mutations are responsible for cellular transformation due to accumulation of TCA cycle intermediates or aberrant production of oncometabolites. Changes in the expression levels of several TCA cycle enzymes, such as CS and IDH3α, 34,35 have also been shown to affect tumour phenotype suggesting that alteration of any step of TCA cycle can be detrimental for cell metabolic homoeostasis. Although no mutation in ACO2 sequence has been associated with tumour susceptibility, in this paper, we have demonstrated that expression levels of ACO2 are deregulated in breast cancer and that its overexpression in MCF-7 cell line is able to dampen cell proliferation. On the contrary, the modulation of the cytosolic ACO1 was shown to have no impact on breast cancer proliferation. 36 Besides our present work, a direct contribution of ACO2 in tumorigenesis was exclusively demonstrated in prostate cancer, and this is mainly justified by the importance of citrate metabolism in non-malignant prostate epithelial cells. In fact, prostate tissue physiologically accumulates high amount of citrate, thanks to a zinc-mediated inhibition of ACO2. 37,38 Even though it is well known that ACO2 is not a rate-limiting enzyme of the TCA OE ACO2 cycle, our data pointed out the importance of its reaction in promoting cancer metabolic rewiring. In fact, we demonstrated that increased levels of ACO2 in MCF-7 cells are able to affect aerobic glycolytic rate as demonstrated by the reduction of extracellular lactate efflux. This evidence could give effort to the MCF-7 glycolytic shift observed under defective assembly of iron-sulfur clusters, which are essential for aconitase activity. 39 The unaffected glucose assimilation along with increased level of acetyl-CoA and PDH complex demonstrated a different fate for pyruvate, prevalently directed to mitochondria, in cells with increased ACO2 levels. The same changes were observed during treatment of colorectal cancer cells with dichloroacetate (DCA). This compound induces cell growth arrest by inhibiting pyruvate dehydrogenase kinase (PDK) with consequent activation of PDH, thus promoting the entry of pyruvate into the TCA cycle. 40 We also demonstrated that ACO2 expression affects glutamine addiction of MCF-7 cells, indicating that feeding TCA cycle with pyruvate and channelling it for aerobic oxidation in the presence of adequate ACO2 levels dislodge cancer cells from using glutamine for anaplerosis. The re-routing of glucose-derived pyruvate towards mitochondria can be considered as the guiding force involved in the proliferation inhibition of ACO2-overexpressing cells. Consistently, we observed a decrease of cell proliferation in control cells forced to use OXPHOS by galactose treatment. 29 The enhancement of mitochondrial metabolism and the reduction of the Warburg effect also underlie the anti-proliferative effects observed after alanine aminotransferase inhibition, 41 improved triacylglycerol catabolism in hepatocellular carcinoma 11 and DCA treatment in colorectal cancer. 40 Moreover, the increase in mitochondrial biogenesis due to PGC-1α activation was shown to be another efficient strategy against the Warburg effect. [42][43][44] It has to be noticed that after ACO2 overexpression, we also observed an increase in mitochondrial mass associated with high levels of PGC-1α and Ser129-phosphorylated CREB. This would imply that ACO2mediated metabolism is able to trigger a mitonuclear retrograde response culminating in mitochondrial biogenesis that needs further investigations. Whatever the causes, pyruvate redirection, mitochondrial biogenesis or both, our data support that ACO2 can boost OXPHOS weakening the Warburg effect. Mitochondria are the major physiological source of ROS and can cause an increase in oxidative stress following enhanced metabolic rate or electron leakage to oxygen when they are dysfunctional. 45,46 The contribution of ROS in breast cancer aetiology and progression is dual when they act as signal molecules in metabolic adaptations and proliferation pathways, as well as when they act as harmful factors, eliciting pro-apoptotic or mutagenic effects by damaging macromolecules, primarily nucleic acids. 47 In general, low concentrations of ROS are believed to promote cancer cell survival as demonstrated for the maintenance of breast cancer stem cells and resistance to radiotherapy. 48 High concentrations of ROS instead can promote oxidative damage and eradication of tumour cells via programmed cell death, a mechanism frequently underpinning the action of chemotherapeutic drugs. Along with dosage, also type, duration and site of generation dictate ROS functional outcome. 47 In the context of ACO2-overexpressing cells, a deleterious effect of ROS can be envisaged after the enhancement of mitochondrial oxidative metabolism by galactose administration that commits them to cell death. Under standard glucose conditions, the viability of ACO2overexpressing cells was assured by the induction of protective antioxidant systems and autophagy as survival mechanisms. This result is in agreement with ROS-mediated activation of autophagy occurring after DCA-dependent reorientation of pyruvate towards the TCA cycle. 40 The role of autophagy in breast cancer as much as in the majority of tumour types is complex for the deep integration into metabolism, stress response and cell-death pathways. 49,50 The loss of the essential autophagic protein Beclin1 in mammary epithelial cells induces tumorigenesis as a consequence of genome instability. 51 Another tumour-suppressor role of autophagy in breast cancer entails the removal of damaged mitochondria as exemplified by the augmented proliferation and aggressiveness resulting from the downregulation of PARKIN or BNIP3, two key pro-mitophagic proteins. [52][53][54] Nevertheless, autophagy induction promotes cell survival against stressful conditions (e.g. nutrient limitations, hypoxia) as described for dormant breast cancer cells responsible for tumour recurrence 50,55 or against chemotherapeutic interventions contributing to apoptosis resistance. 56,57 In the absence of any causal change in the nutrient-sensing pathways of mTOR and AMPK, the recruitment of the ubiquitin E3 ligase PARKIN at mitochondria with consequent increase in ubiquitin-conjugated proteins indicated that ACO2 overexpression leads to the activation of a mitophagic process. This result is also supported by the increased levels of DRP1 protein, which promotes mitochondrial fission, a mechanism generally coupled to mitophagy following oxidative stress. 58 The fact that mitophagy occurs in parallel with mitochondrial biogenesis suggests an active turnover of mitochondria rather than a definitive disposal. 32 Therefore, the activation of mitophagy caused by ACO2 overexpression likely accounts for the removal of damaged/exhausted mitochondria due to the enhanced oxidative metabolism in the context of a mitochondrial quality control process. It is interesting to notice that many other papers have demonstrated how modulation of autophagic/mitophagic process impacts on cancer metabolism. In particular, the Warburg effect was shown to be promoted by loss of the pro-mitophagic proteins PINK1 and PARKIN, [59][60][61] while it was dampened after autophagy stimulation achieved by mTOR inhibition, serum/amino acid starvation or ATG7 overexpression. 62,63 The transcriptional activation of ATG4B, LC3B and PINK1 in ACO2-overexpressing cells is consistent with a lasting autophagic/ mitophagic process necessary for the adaptation of cells to stable ACO2 overexpression. In our context, ROS seem to act also as signalling molecules activating the redox-sensitive transcription factor FoxO1. In fact, FoxO1 is able to couple oxidative stress to autophagic response 24,64,65 and to participate in the retrograde response triggered by ROS following starvation or mitochondrial dysfunction. [66][67][68] The molecular mechanism driving FoxO1 action upon oxidative stress mainly consists of its post-translational modification and nuclear accumulation. [69][70][71] Although we observed no evident change in nuclear levels of FoxO1, the high level of FoxO1 Ser256 phosphorylation after NAC is noteworthy because this modification is present in the DNA-binding domain restraining FoxO1 transcriptional activity. 72 Consistently, we observed a reduced FoxO1 occupancy at autophagic/mitophagic promoters after ROS abrogation. Therefore, FoxO1 may be a key player in the communication between mitochondria and the nucleus in cancer cells for the maintenance of oxidative metabolism through mitochondrial quality control. Ongoing research is devoted to the evaluation of other redox-sensitive transcription factors in the metabolic adaptation induced by ACO2 overexpression.
Overall, this work has evidenced that ACO2 expression modifies metabolic features of MCF-7 cells imposing repression of the Warburg effect and repurposing pyruvate in the aerobic route speeding OXPHOS. This scenario is accompanied by a decrease in cell proliferation associated with ROS/FoxO1 signalling necessary for preserving cellular homoeostasis. These data suggest that ACO2 can be included in metabolic reprogramming exploited by cancer cells, considering that it can be found deregulated in breast, gastric and prostate tumour biopsies. 16,37 Future studies are necessary to enlarge this evidence on other cell systems or in vivo models with the final aim to evaluate whether ACO2 can be a suitable therapeutic target or a biomarker of metabolic vulnerability. In fact, it is worth to highlight that this neglected enzyme of the TCA cycle seems to interfere with different aspects of cancer metabolism from aerobic glycolysis to glutamine addiction and autophagy, without excluding the relevant implication it may have in cancer immunometabolism by supplying cisaconitate for the production of the anti-inflammatory metabolite itaconate. 73
AUTHOR CONTRIBUTIONS
F.C. conceived the study, designed and performed the experiments, interpreted the data and wrote the paper; L.D.L. performed activity assays and TCA cycle/ATP/oxygen measurements; G.L. and B.T. performed HPLC analysis; G.M. and F.D.G. performed fluorescence lifetime imaging of NADH; M.R.C. conceived and supervised the study, interpreted the data and revised the paper. All authors reviewed and approved the paper.
Competing interests: The authors declare no competing interests. Aconitase 2 inhibits the proliferation of MCF-7 cells promoting. . . F Ciccarone et al. | v3-fos-license |
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} | pes2o/s2orc | Partially spatially coherent digital holographic microscopy and machine learning for quantitative analysis of human spermatozoa under oxidative stress condition
Semen quality assessed by sperm count and sperm cell characteristics such as morphology and motility, is considered to be the main determinant of men’s reproductive health. Therefore, sperm cell selection is vital in assisted reproductive technology (ART) used for the treatment of infertility. Conventional bright field optical microscopy is widely utilized for the imaging and selection of sperm cells based on the qualitative analysis by experienced clinicians. In this study, we report the development of a highly sensitive quantitative phase microscopy (QPM) using partially spatially coherent light source, which is a label-free, non-invasive and high-resolution technique to quantify various biophysical parameters. The partial spatial coherence nature of light source provides a significant improvement in spatial phase sensitivity and hence reconstruction of the phase of the entire sperm cell is demonstrated, which was otherwise not possible using highly spatially coherent light source. High sensitivity of the system enables quantitative phase imaging of the specimens having very low refractive index contrast with respect to the medium like tail of the sperm cells. Further, it also benefits with accurate quantification of 3D-morphological parameters of sperm cells which might be helpful in the infertility treatment. The quantitative analysis of more than 2500 sperm cells under hydrogen peroxide (H2O2) induced oxidative stress condition is demonstrated. It is further correlated with motility of sperm cell to study the effect of oxidative stress on healthy sperm cells. The results exhibit a decrease in the maximum phase values of the sperm head as well as decrease in the sperm cell’s motility with increasing oxidative stress, i.e., H2O2 concentration. Various morphological and texture parameters were extracted from the phase maps and subsequently support vector machine (SVM) based machine learning algorithm is employed for the classification of the control and the stressed sperms cells. The algorithm achieves an area under the receiver operator characteristic (ROC) curve of 89.93% based on the all morphological and texture parameters with a sensitivity of 91.18%. The proposed approach can be implemented for live sperm cells selection in ART procedure for the treatment of infertility.
pathways required for human sperm activation, whereas high level impairs sperm function, leading to infertility. Specifically, oxidative stress is known to affect the integrity of the sperm genome, result in lipid peroxidation, loss in membrane fluidity, and decrease in sperm motility 6,7 .
Adverse effects of oxidative stress might not be explored with light microscopy. For that reason, sperm cells with impaired fertilizing potential can be picked by embryologists for intracytoplasmic sperm injection (ICSI). At the same time, routine oxidative stress screening is not performed in IVF laboratories because of high cost and complexity of standard tests 8 . Moreover, implementation of rapid diagnostics could replace long and cumbersome multi-step analytic procedures that require complex experimental equipment.
The human sperm cells are relatively transparent in nature and have almost similar optical properties as surroundings leading to low refractive index (RI) contrast. Therefore, it is difficult to obtain a good contrast image by using bright field microscope. Several optical techniques have been developed for the contrast enhancement of sample images [9][10][11] , however, they do not provide any quantitative information of the specimen 12,13 . When the light passes through the specimen, the optical path delay (OPD) is generated in light field due to the RI difference between the cell and the surrounding medium. The OPD is measured using quantitative phase microscopy (QPM) techniques, which are based on the principle of interferometry. It can be further utilized to measure several optical properties of specimen. The QPM techniques have been employed for the visualization and the evaluation of specimens that are particularly useful in cell biology [12][13][14] . The key advantage of these techniques is that they provide high resolution 3-D quantitative information of the specimen without any labelling.
In this study, we have investigated the effects of externally induced oxidative stress by treating healthy sperm cells with hydrogen peroxide (H 2 O 2 ) using spatially low coherent QPM and further the findings are correlated with clinically relevant motility parameter of the sperm cells. A number of studies have been implemented for the quantitative assessment of normal and immotile sperm cells utilizing QPM [15][16][17][18][19] , however the effect of oxidative stress on the morphology and motility of the sperm cells is not done previously. In addition, existing QPM either utilized a narrowband (i.e., lasers) or broadband (i.e., light emitting diodes and halogen lamp) light sources for phase imaging of sperm cells [17][18][19][20] . The use of highly temporal and spatial coherent light sources, like lasers, degrades the interferogram's quality due to speckle and spurious fringe formation, which eventually reduces the spatial sensitivity of the system [21][22][23] . This makes difficult to perform quantitative phase imaging (QPI) of the tail of sperm cells as it offers minute OPD 18,19 . The phase sensitivity can be improved by utilizing broadband light sources like white light, light emitting diodes and super-luminescent diodes [24][25][26] . However, such light sources require chromatic aberration corrected optics and dispersion compensation mechanism. In addition, single shot phase recovery over the whole camera field of view (FOV) is not possible due to low fringe density with low temporal coherent light sources 25 . Thus, a monochromatic extended (i.e., pseudo-thermal) light source can be implemented in QPM technique, which carries advantages of both narrow-band and broad-band light sources. Several methods have been proposed to synthesize pseudo-thermal light sources using rotating diffuser and vibrating multiple mode fiber bundle (MMFB), previously 22,23 .
A monochromatic laser beam is passed through a rotating diffuse to synthesize a pseudo-thermal light source which carries high temporal coherence (helps to obtain high fringe density over whole camera FOV) and low spatial coherence (generate speckle and spurious fringe free interfergrams) properties. Such light source is employed with Linnik-type interference microscopy system to record off-axis holograms of sperm cells. The phase maps of sperm cells are then recovered with improved spatial phase sensitivity of the order of 20 ± 1.5 mrad. It is exhibited that phase map of the tail of sperm cell is nicely recovered with pseudo-thermal light source, which is otherwise not possible in laser based phase imaging. Most of the QPM techniques are, therefore, implemented only on dried sperm cells to recover phase map of the tail of sperm cells, previously 17,19 . Further, diagnosis of both the head and the tail of sperm cells are important for the procedures of artificial reproductive technologies. According to the WHO criteria, healthy tail having principle piece should be uniform along its length, be thinner than the midpiece, with a length of about 45 µm and without any sharp angle 27 . Thus, quantitative assessment of sperm tail can help to choose a healthy sperm in the clinical practice. QPM may also provide a better visualization to detect the abnormalities like defects of head neck attachment, primary ciliary dyskinesia (PCD), or dysplasia of fibrosis sheath (DFS).
It was observed that the optical thickness of the sperm's head decreases as a function of increase in the H 2 O 2 concentration. Several morphological and texture parameters were extracted from the phase maps to measure the changes during oxidative stress. A support vector machine (SVM) based classifier is developed for the classification of normal and stressed sperm cells. The morphological and texture parameters extracted from phase maps were used to train the algorithm for better classification. For the training of the classifier, 60% of total samples were used and rest 40% were used as test specimen. We have achieved an accuracy of 89.93% for the classification of control and test sperm cells with SVM model. The observations support the hypothesis that changes caused by the oxidative stress could result in the decrease of maximum phase value of the sperm cell as compared to the normal one. The findings of QPM were correlated with a dose-dependent decrease in progressive motility of the sperm cells. The decrease in sperm motility with an increase in the H 2 O 2 concentration was observed as compared to the controlled samples.
Methods and Materials
Principle of DHM. DHM is based on the principle of interferometry, in which a full or partially coherent light is divided into two beams, one as reference and other illuminates the specimen called object wave. Further, the scattered wave from object and reference waves interfere to generate the hologram and the 2D intensity distribution can be expressed as: x y www.nature.com/scientificreports www.nature.com/scientificreports/ where a(x, y) and b(x, y) represent the background (DC) and the modulation terms, respectively, Δφ(x, y) is the phase difference between the object and reference fields, f x and f y are the spatial frequencies of the interference pattern along x and y directions, respectively.
For the convenience, the above intensity pattern of hologram can be rewritten in the following form The hologram reconstruction allows the retrieval of the complex object field. To retrieve the phase information, Fourier transform of the hologram is taken and one of the twin image peaks is filtered with numeric band pass filter in the frequency domain. Further, inverse Fourier transform is performed to reconstruct the hologram (h filt ) as a 2D array of complex numbers. The phase profile of the specimen is then simply measured as: The phase Δφ depends on the thickness of the specimen and the RI difference of the specimen and the media containing the object itself. This phase variation having information of the morphology of specimen under investigation thus holography provides a 3D topographic profile of the specimen. The phase is related to the optical path difference (OPD) by the relation 12 : where λ is the wavelength of incident light, h is the geometrical thickness of the specimen; n s and n o are the refractive indices of the specimen and surrounding medium, respectively and there is an extra factor of 2 appears because the reflection configuration is utilized to record the hologram.
Morphological and Statistical Analysis. The analysis of recovered phase is very important for the image based computer-aided diagnosis (CAD), which provides excellent accuracy in early stage disease detection 28,29 . Machine learning is a subfield of computer science having a range of applications in biomedical imaging, which uses the extracted morphological and texture features of the image to make predictions 28,29 . For the classification of sperm cells under control and oxidative stress conditions, the phase map of the head of sperm cells are utilized for the calculation of the various texture parameters, which were further utilized in SVM algorithm.
Once the phase maps of the sperm cells were extracted from the hologram, the head of the sperm cell isolated to extract the phase map based morphological and texture features. The optical thickness (OT) is related to the phase of the specimen by the relation φ λ π = * OT x y ( , ) /4 (for reflection geometry), where λ is the wavelength of the light. The measured OT is utilized to measure the volume of sperm head and can be calculated by integrating OT over projected area as 30,31 where dx and dy are the calibrated pixel width along x and y directions, respectively. The area element dS of the cell surface is calculated by Monge parameterization defined as 30,32 where G x and G y are the gradients along the x and y directions, respectively. Further, the surface area 'S' is defined as the sum of all the area elements and the projected area 32 . Next, sphericity 'Ψ ' of the sperm head was determined, whose values lie between 0 and 1 (for laminar disk and perfect sphere, respectively). It is defined as the ratio between the surface area (S) of a cell with the volume of the same cell and calculated as 30,31,33 semen preparation. Semen samples were obtained from men who attended the IVF clinic for the investigation and/treatment of infertility. The Regional Committee for Medical and Health Research Ethics of Norway (REK_nord) approved the project. An informed consent was obtained from all participants. The semen sample was collected according to the guidelines of the World Health Organization with an abstinence period of 3-5 days. After collection, the sample was allowed to liquefy for 30-40 min. Sperm counts were evaluated using the Neubauer-improved counting chambers. All ejaculates used in the experiments had an original sperm concentration more than 60 million of cells per milliliter, progressive motility more than 50% and with normal morphology >14% following strict criteria. The sperm fraction with high motility was isolated by density gradient centrifugation method (Vitrolife, Sweeden). One milliliter of semen was carefully placed on the gradient layers (90% and 45% layers) and centrifuged at 500 g for 20 min. The pellet from the centrifuge tube was washed Scientific RepoRts | (2019) 9:3564 | https://doi.org/10.1038/s41598-019-39523-5 www.nature.com/scientificreports/ twice with human Quinn's sperm washing medium (SM; Origio, Denmark) at 300 g for 10 min. The supernatant was discarded, and the pellet was re-suspended in QA fert-medium supplemented with 5 mg/ml HSA and was used for following procedures.
To perform oxidative stress experiment, sperm sample was diluted to a concentration of 5.0 × 10 6 cells/ml using culture medium. Further, 96 well tissue culture plates were filled with sperm in medium (Quinn's Advantage Protein plus Fertilization medium, SAGE, Denmark) with different concentrations of H 2 O 2 (10 µM, 40 µM, 70 µM, 100 µM) and the reference chamber was filled with the same concentration of semen without H 2 O 2 . The samples were incubated for 1 hour at 37 °C, 5-6% CO 2 . After incubation motility of each sperm sample was graded in two clusters: progressive motility (PR) and non-progressive motility (NP), which were reported as in percentages.
For QPM, the cells of each concentration were placed in a PDMS chamber on reflecting silicon (Si) chip after 1 hour of incubation. To immobilize the sperm cells, samples were fixed with 4% PFA for 30 min at RT and washed in phosphate-buffered saline (PBS) for 5 min. Finally, 50 μL of PBS were added in the PDMS chamber with fixed cells and the samples were covered by cover glass. experimental Details. The schematic of the partial spatial coherence gated QPM/DHM system based on Linnik interferometer is shown in Fig. 1. To reduce the phase noise of the system, the spatial coherence of the laser light source is reduced and the resulting light beam illuminates the specimen. It is demonstrated that when a coherent light incident on a rotating diffuser (RD) and the diffused light is coupled into the multiple multi-mode fiber bundle (MMFB) then its output acts as a pseudo thermal light source having partial spatial and highly temporal coherence properties. The detailed study of the speckle reduction can be found elsewhere [21][22][23] .
A highly coherent laser light (He-Ne @632.8 nm) beam is expanded using microscope objective MO 1 and passed through a RD. The beam spot size of 4.5 mm is made onto the diffuser plane to match the diameter of MMFB. The scattered photons are collected by lens L 1 (focal length f 1 = 50 mm) and pumped into the MMFB. The light from MMFB output is first collimated and then focused at the back-focal plane of the MO 3 by utilizing the lenses L 2 (f 2 = 75 mm), L 3 (f 3 = 150 mm) and beam splitter (BS). Thus the samples are illuminated by a nearly collimated beam for their accurate phase imaging. In the reference arm an optically flat mirror (of the order of λ/10) is used. The reflected light from the reference mirror and the specimen are re-combined at BS to form interference pattern. The interferograms are then projected on the CMOS image sensor (Hamamatsu ORCA-Flash4.0 LT, C11440-42U) using tube lens L 4 (f 4 = 200 mm). The camera exposure time is kept 50 ms.
Comparison of coherent laser and pseudo-thermal light source based phase imaging. In the proposed geometry, a pseudo thermal light source is used to reduce the spatial phase noise of the system which further enhances the measurement accuracy of the system. First, we have compared the spatial phase sensitivity of the system by imaging the sperm cell with fully coherent and partially coherent (pseudo-thermal) light sources. Figure 2 shows interferogram, reconstructed phase map of a sperm cell and the spatial phase noise of the system for fully and partially spatially coherence light sources. The spatial phase sensitivity of the system is enhanced when the test specimen is illuminated by the partially spatially coherent light source. Figure 2(a,d) show the interferograms of the sperm cell utilizing direct laser and synthesized pseudo-thermal light sources, respectively. Highly coherent nature of light source leads to speckle and non-uniform illumination of the specimen as shown in Fig. 2(a), while pseudo-thermal light source provides a speckle free uniform illumination (Fig. 2(d)). The object is clearly visible in Fig. 2(d) with illumination of pseudo-thermal light source which is otherwise not visible with direct laser source ( Fig. 2(a)). Figure 2(b,e) show their corresponding reconstructed phase maps of the interferograms depicted in Figs 2(a) and 2(d). It can be observed from the phase images that the finer features of the sperm cells i.e. neck and tail is not resolved in phase map of hologram recorded by the direct laser, while whole sperm cell is clearly reconstructed in case of pseudo-thermal light source. In case of direct laser, the generation of speckle www.nature.com/scientificreports www.nature.com/scientificreports/ and non-uniform illumination reduces the over-all spatial phase sensitivity of the system which results in poor resolution. The spatial phase sensitivity of the system for both kind of light sources were measured and compared. High spatial phase sensitivity is essential where minute phase variations in the target are needed to be quantified. Here, we utilized pseudo-thermal light source to enhance the spatial phase sensitivity of the phase microscopy system. The difference in phase values of the controlled and the 10 μM sperm cells is only 8%, which would be difficult to differentiate with direct laser based QPM technique due to high spatial phase noise. Figure 2(c,f) show the spatial phase noise of the system for the direct laser and pseudo-thermal light sources, respectively, where the color bars having different scale values. By measuring the standard deviation of the phase distribution, one can estimate the spatial phase sensitivity of the system. In our case, the phase sensitivity is observed to be 300 ± 11.9 mrad and 20 ± 1.5 mrad for direct laser and pseudo-thermal light source, respectively.
The effect of oxidative stress on sperm motility has been demonstrated in number of studies [34][35][36] . H 2 O 2 is externally supplemented agent to induce oxidative stress on sperm cells. Our results support the previous studies that the extent of motility decrease depends on the concentration of H 2 O 2 . The underlying mechanism of H 2 O 2 influence to sperm motility is described previously 37,38 . Membrane lipids of sperm cells contain unsaturated fatty acids which are vulnerable to peroxidation. Sperm incubation with H 2 O 2 triggers lipid peroxidation cascade results in membrane loss of flexibility and plasticity which determines disrupted tail motion 3,34,39 . Moreover, motility may be decreased because of restriction of energy production by damaged mitochondria after oxidation 40,41 . Quantitative phase imaging of sperm cells. The quantitative morphological analysis of the sperm cells provides a better understanding of the behaviour of sperm cells under control and oxidative stress conditions. Figure 4 shows the recorded hologram and pseudo colour unwrapped phase map of sperm cell. Figure 4(a) shows a typical low spatial coherence hologram of the sperm cell and 2D view of the recovered phase map is shown in Fig. 4(b). The basic structure of the sperm cell composed of the head, mid piece, tail and end piece, the head is partially covered with nucleus and acrosome. Figure 4(c) shows the pseudo 3D phase map of the same sperm cell where maximum optical path delay is generated by the head of the sperm cell having value approximately 4 rad.
The low spatial coherence QPM/DHM is further used for the evaluation of the effects of oxidative stress on the morphology of the human sperm cells. Figure 5 shows the recovered phase maps of sperm cells treated with different concentration of H 2 O 2 . Figure 5(a-e) show the reconstructed 3D phase maps for the control, 10 μM, 40 μM, 70 μM and 100 μM concentration of H 2 O 2 , respectively. It is observed from the phase images that with increasing concentration of H 2 O 2 , the maximum value of the phase of sperm head decreases which indicates that there is a change in the morphology of the sperm head. For the study of morphological changes in the sperm head during www.nature.com/scientificreports www.nature.com/scientificreports/ oxidative stress, several morphological parameters have been extracted from the phase maps. In total, more than 2500 sperm cells were analysed to measure the optical and morphological parameters. Figure 6(a) shows the whisker box plots of maximum phase of the sperm head at different concentrations of H 2 O 2 . Figure 6(b) shows the whisker box plot for the optical thickness of the sperm head for control and 10 μM concentration of the H 2 O 2 . The optical thickness decreases after oxidative stress which further changes the morphology of the sperm cells. The structure of sperm suggest that the nucleus is tallest part sperm followed by acrosome and mid-piece which allows observer to distinguish nucleus from acrosome 17,18 . The reconstructed phase map show a clearly detectable edge of cell boundary and maximum phase in the nucleus region. The identification and quantification of the optical thickness of nucleus may provide the deformation of nucleus during oxidative stress as shown in Fig. 5. The acrosome having significant low OT due to lesser thickness as compared to nucleus. Hence, the quantification of the change in the OT of nucleus during deformation can be a good marker for the quantification of oxidative stress. Here, we have chosen control and 10 μM concentration of the H 2 O 2 only for the comparative study because there is almost linear decrease in the maximum phase with increasing concentration of the H 2 O 2 (Fig. 6a).
Characterization of morphological and texture parameters during oxidative stress. In order to determine the effects of oxidative stress on the morphology of the sperm cell head, the morphology of head is quantified from the phase maps using calculations describes in materials and method section. Surface area (S), volume (V), surface to volume ratio (S/V) and sphericity (Ψ) parameters were analysed for classification of control and 10 μM concentration of the H 2 O 2 . Figure 7(a-d) show the whisker box plots of these parameters for sperm head under control and oxidative stress conditions. The results show that the surface area increases in www.nature.com/scientificreports/ sperm cell head after the externally induced oxidative stress (Fig. 7a), while the volume is approximately constant during this process as can be seen in Fig. 7(b). There is an increase in the surface to volume ratio while sphericity decreases after oxidative stress. The increase in the S and S/V with decrease in ψ indicates that the flattening of the cells under stress assuming constant RI of the sperm head during whole process (Fig. 7(c,d)).
For the statistical analysis, selection and extraction of texture features are also important for the classification of any disease. Here, we have extracted various texture features from the phase maps of the sperm heads such as: mean, variance, entropy, kurtosis, skewness and energy. All the parameters were extracted by choosing a region of interest (ROI) of the sperm cells and listed in Table 1. There is a decrease in the mean value of the phase distribution over entire sperm head reflects the flattening of the sperm head i.e. decrease in the optical thickness of the sperm head after introducing 10 μM of H 2 O 2 concentration. The decrease in the variance shows the less spread of data points around its mean value, while there is an increase in entropy predicts the increase in the randomness of phase distribution over entire sperm head. The increase in kurtosis and skewness show the more flatness and asymmetricity in phase distribution of sperm head. The decrease in the energy value shows the increase of heterogeneity in phase distribution of sperm cell head.
Once all the morphological and texture parameters were extracted from the phase maps of sperm cells, a support vector machine (SVM) classifier has been developed for the classification of the control and oxidative stress induced sperm cells 28,29,42 . Eleven parameters: OT, S, V, S/V, ψ, mean, variance, entropy, kurtosis, skewness and energy were utilized as input predictor variables and the genuine state of the sperm as a response variable i.e. 0 for control and 1 for 10 μM H 2 O 2 concentration treated sperm cells. Sensitivity, specificity and area under receiver operating characteristic (ROC) curve were calculated to check the accuracy of the model. Total data points are divided into two sets, 60% for the training of the model and 40% for the testing purpose. Figure 8 shows the ROC curve for the testing data points with a specificity and sensitivity of 88.61% and 91.18%, respectively with an accuracy of 89.93% for the classification of control and test sperm cells.
Conclusion
In present study, the capability of DHM using low spatial coherence light source alongwith SVM classifier exploited to measure change in morphology of sperm head after oxidative stress. It is exhibited that pseudo-thermal light source based phase imaging provides reconstruction of the biological structure having minute optical thickness (i.e., tail of the sperm cells), which is otherwise not possible under coherent illumination. www.nature.com/scientificreports www.nature.com/scientificreports/ It is known that fertilization capability of sperm cell is impaired under biological oxidative stress. The oxidative stress was induced using H 2 O 2 treatment. Concentrations of H 2 O 2 exceeding physiological threshold trigger the changes in semen leading to sperm cell dysfunction 7,35,38 . The evidence from previous studies suggests decrease of sperm cell motility due to membrane translocations of phospholipids 3,43 . In addition to membrane peroxidation, H 2 O 2 initiates concentration-dependent increase of DNA fragmentation because of DNA strand breaks 3,36,44,45 . Using conventional microscopy, sperm cells with this type of anomalies might be amongst selected cells for intracytoplasmic sperm cell injection (ICSI) procedure leading to treatment failure. Therefore, it is of great significance to develop noninvasive methods for sperm cells selection. DHM appears to be one of the most promising noninvasive technique for the quantification of optical parameters of sperm cells 13,15,17 .
We found that H 2 O 2 induces oxidative stress to the sperm cells which leads to the sperm cell dysfunction by decreasing its motility. The result of our study suggests the association between gradual progressive motility loss (Fig. 3) and the shift of optical properties of the sperm head (Figs 6, 7) after exposure to various concentrations of H 2 O 2 . The head morphology changes resulting from peroxidation might be due to de-condensation of genetic material because of DNA fragmentation. Quantitative evaluation of the phase shift by DHM provides an opportunity to use SVM to obtain new information on the exact structure and better distinguish sperm cells that are normal from those under oxidative stress (Fig. 8). Development of such machine learning algorithms could play an important role in automatic classification of the healthy and stressed sperm cells. The origin of decrease in the maximum phase of sperm head could be due to various reasons such as: deformation in nucleus, structural organization of sperm DNA, condensation of chromatin etc 19 . The morphometric values obtained in our study can provide the volumetric estimation for the quantitative comparison between control and H 2 O 2 treated sperm cells. The correlation of decrease in the phase and deformation in the nucleus can be quantify by multimodal imaging in future where the boundary can be located by fluorescence imaging and QPM can provide the changes in the maximum phase of nucleus. QPM may have capability to quantify the changes due to fragmentation in DNA after introducing oxidative stress in human sperm which can be the motivation for this kind of analysis on the fertilization capacitance of sperm cell 46,47 .
One of the obstacles in IVF treatments is to recognize the sperm cells morphology by observing them under optical microscope whether it is under oxidative stress or not. However, by utilizing low spatial coherence DHM together with machine learning algorithms might provide better sperm selection during ICSI procedure. Moreover, as mentioned above, "hand-picked" spermatozoon for ICSI procedure might contain fragmented DNA, which can be detected indirectly by measuring sperm optical features using noninvasive, label-free QPM/ DHM technique. We believe that our approach with DHM and machine learning based algorithm for sperm analysis at the cellular level has a strong potential for improving IVF procedures and their outcomes. | v3-fos-license |
2018-04-03T05:29:54.556Z | 2013-09-23T00:00:00.000 | 17053673 | {
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} | pes2o/s2orc | Skeletal Muscle Regeneration on Protein-Grafted and Microchannel-Patterned Scaffold for Hypopharyngeal Tissue Engineering
In the field of tissue engineering, polymeric materials with high biocompatibility like polylactic acid and polyglycolic acid have been widely used for fabricating living constructs. For hypopharynx tissue engineering, skeletal muscle is one important functional part of the whole organ, which assembles the unidirectionally aligned myotubes. In this study, a polyurethane (PU) scaffold with microchannel patterns was used to provide aligning guidance for the seeded human myoblasts. Due to the low hydrophilicity of PU, the scaffold was grafted with silk fibroin (PU-SF) or gelatin (PU-Gel) to improve its cell adhesion properties. Scaffolds were observed to degrade slowly over time, and their mechanical properties and hydrophilicities were improved through the surface grafting. Also, the myoblasts seeded on PU-SF had the higher proliferative rate and better differentiation compared with those on the control or PU-Gel. Our results demonstrate that polyurethane scaffolds seeded with myoblasts hold promise to guide hypopharynx muscle regeneration.
Introduction
Hypopharynx carcinoma is one of the common head and neck cancers; approximately 2500 new cases are diagnosed in the United States each year with the peak incidence in males and females aged 50 to 60 years [1,2]. Laryngopharyngectomy and reconstruction followed by chemoradiotherapy has been the traditional and relatively practicable treatment presently. However, surgical interventions are inevitably associated with quality-of-life impairments including severe speech and swallowing disability [3]. Also, physical deformities and emotional trauma persist long after the conclusion of surgical intervention. For the severe and large area hypopharynx defects, visceral or myocutaneous flaps, such as an ileocolic flap, a radial forearm flap, and an anterolateral thigh flap, a submental island flap, and an infrahyoid myocutaneous flap, are common methods used for surgical repair [4][5][6][7][8]. However, these grafts cannot fully restore the function of hypopharynx tissue, in particular the reconstruction of the complex structures surrounding the larynx and hypopharynx, while postoperative dyspnea may be inevitable when hypopharynx pharyngeal constrictors were sectioned [9,10].
Skeletal muscle is the most abundant tissue type in the human body. Muscle fiber consists of a longitudinal arrangement of myofilaments with actin and myosin as major components [11]. Huang and colleagues cultured murine myoblasts (ATCC C2C12) on elastic poly(dimethylsiloxane) (PDMS) films topographically micropatterned with 10 m wide microgrooves. The myoblasts formed long and unbranched myotubes that had uniform diameter and were aligned parallel to the microgroove direction to suggest that microgrooves promote end to end fusion of myoblasts [12]. In another study by Ma and coworkers, ATCC were seeded onto a collagen composite scaffold and cultured in a roller bottle cell culture system to create a 3-dimensional (3D) tissue graft in vitro. The 3D graft was then used for in vivo muscle tissue repair by implanting it into defect sites created in mice models. The scaffolds were found to degrade slowly over time, and muscle healing was improved as shown by an increased quantity of innervated and vascularized muscle fibers. These results suggest that 3D muscle grafts created in vitro from collagen composite scaffolds seeded with myoblasts can be used for defect muscle tissue repair in vivo [13].
In our experiment, scaffolds with linearly aligned microchannels were fabricated to align the seeded myoblasts through contact guidance. Protein grafted surfaces promote human hypopharyngeal cell proliferation and differentiation. Furthermore, we will offer a new perspective on the implications of ex vivo niche system to advance the scientific understanding of stem cell functions as well as supporting clinical applications.
Materials.
Poly(ester urethane) (PU, 58213 NAT 022) was purchased from Estane Co., China. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotic antimycotic solution (AAS, 100 U/mL penicillin, 100 g/mL streptomycin, and 100 g/mL amphotericin B), and Trypsin-EDTA solution were purchased from Gibco (Invitrogen, Co., New York). Anti-MyoD1 antibody was supplied by Abcam (Hong Kong) Ltd. FITC-conjugated AffiniPure goat anti-mouse IgG (H+L) was purchased from Jackson ImmunoResearch. Rabbit anti-beta-actin (loading control), HRP-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-rat IgG (H+L), were supplied by Beijing Biosynthesis Biotechnology Co., Ltd. All chemical reagents used for Western blotting were purchased from Beyotime Institute of Biotech (JiangSu, China). Other chemical reagents used in this experiment were from Sinopharm Chemical Reagent. All water used in the experiment was doubly distilled. Phosphate buffer saline (PBS, pH = 7.4) used in cell culture was sterilized.
Extraction of Silk Fibroin (SF).
Silk fibroin was extracted from the natural silkworm cocoon (Zhejiang Province, China) using a method reported previously [14]. The cocoons were heated to boil for 1 h in an aqueous Na 2 CO 3 solution (0.5 wt%) and rinsed with water to remove the sericin. The extracted fibers were subsequently dissolved in calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ⋅4H 2 O) at 80 ∘ C to yield a homogeneous solution (5%, w/w). The solution was then dialyzed using a tubular cellulose membrane (MW cutoff, 12,000-14,000, Sigma) in water for 3 days at room temperature and thereafter replenished with water every 4 hours (a total about 12 to 16 times) to remove the residual salts. The dialyzed solution was subsequently collected, and the SF concentration was measured to be 0.018 g/mL.
Scaffold Preparation.
Based on our previous work, a soft polydimethylsiloxane (PDMS) mould was fabricated from a silica wafer patterned with unidirectional microchannels of 200 m in width and separated by walls with 30 m wide and 30 m high. PU was dissolved in 1,4-dioxane to get a concentration of 12% (w/v). PU/1,4-dioxane solution was casted onto the PDMS mould and dried at 37 ∘ C for 12 h, yielding a transparent PU membrane with parallel microchannels of 200 m width and 30 m depth. The membrane was then immersed in alcohol solution (95 v%) for 3 h to remove any dirt and rinsed repeatedly with large amounts of water and dried in air.
To immobilize proteins onto the PU surface, the patterned PU membrane was immersed in 1,6-hexanediamine/ propyl alcohol solution (0.06 g/mL) for 10 min at 37 ∘ C and rinsed with water for 24 h at room temperature to remove the unreacted 1,6-hexanediamine. It was subsequently vacuumdried at 30 ∘ C for 12 h to remove residual propyl alcohol. This aminolyzed PU membrane was soaked in aqueous glutaraldehyde solution (1.0 wt%) for 3 h at room temperature to transform amino groups into the aldehyde groups and was further rinsed with abundant water to remove superfluous glutaraldehyde. The membrane was then incubated in a gelatin/PBS solution (2 mg/mL) or a SF/PBS solution for 24 h at 4 ∘ C, respectively. The gelatin-immobilized (PU-Gel) and SF-immobilized membranes (PU-SF) were rinsed with water for at least 12 h to remove all free gelatin or SF.
For cell culture tests, scaffolds were sterilized in 75% alcohol for 4 h and rinsed with sterilized PBS to remove residual alcohol. Prior to cell seeding, the scaffolds are immersed in DMEM for 30 min and placed into 96-well tissue culture polystyrene (TCPS) plates.
Mechanical Property Test.
Mechanical testing was performed by a linear tensile tester (Instron 3366, USA) at the stretch rate of 60 mm/min. Dumbbell-shaped scaffolds with a gauge length of 22 mm and cross-sectional area of 0.2-0.3 mm × 3.8 mm were used. The mechanical properties of PU, PU-Gel, and PU-SF scaffolds were separately tested.
For testing the alteration of scaffolds' mechanical properties after in vitro degradation, the scaffolds were firstly sterilized in aqueous alcohol solution (75 v%) for 2 h and rinsed in water to remove residual alcohol. They were then incubated at 37 ∘ C in PBS (pH = 7.4) supplemented with penicillin (100 U/mL)-streptomycin (100 g/mL). The mechanical testing was performed in the same way after different degradation periods (20, 105, 145, 185, 220, and 260 days). Three repeats were conducted for each scaffold type.
Contact Angle Measurement.
The static contact angle of each PU scaffold (planar face) was measured using a surface tension-contact angle meter (DIGIDROP, GBX, France) at ambient humidity and temperature. Drops of deionized water about 1.0 L in volume were applied.
Cell
Culture. Skeletal muscle biopsies were obtained from hypopharyngeal constrictors of 10 patients, who underwent surgery for laryngeal or hypopharyngeal carcinoma at the Department of Otolaryngology, Head and Neck surgery, Lihuili Hospital of Medical School, Ningbo University (Ningbo, China). Only specimens which were determined to BioMed Research International 3 be free of cancer by the pathologist were used. Collection of biopsy samples was approved by the Ethics Review Boards of Ningbo University, and an informed consent was obtained from every patient.
The biopsy specimens stored in PBS (pH = 7.4, ice-cool) supplemented with AAS upon collection were immediately transported to our laboratory for further processing. The muscle samples were first rinsed with PBS, and adherent fat and tendons tissues were removed with ophthalmic scissors. The muscle tissues were then minced into small tissue pieces of approximately 1 × 1 × 1 mm 3 and transferred into culture flasks (Corning, USA). Tissues were adhered to the tissue culture plate before 2 mL of culture medium containing 15% FBS and AAS was added. The medium was subsequently renewed every three days. Cells from the tissue pieces were allowed to migrate outwards and grow to be confluent in the culture plate. Cells were passaged by enzymatic treatment with 0.25% Trypsin-EDTA solution.
Cells from 2nd to 5th passages were seeded on the scaffolds at a density of 8 × 10 4 cells/mL.
Mitochondrial Activity Assay.
Mitochondrial activity of the cells seeded on each scaffold was assayed using the MTT method at 2, 5, and 10 days, respectively. 20 L of MTT solution (0.5 mg/mL) was added to each culture well and incubated with cultured cells for 4 h at 37 ∘ C in the dark. 150 L of dimethylsulphoxide (DMSO) was subsequently added to each well to dissolve the purple formazan crystal. Absorbance was measured at 490 nm using an ELISA reader (MaxM5, Spectra). The absorbance of DMSO without formazan crystal was used as blank reference. Data were compared between cells grown on PU, PU-Gel, and PU-SF scaffolds and TCPS. Triplicates of each sample were averaged.
Immunofluorescence
Staining. Cells cultured on scaffolds for 10 days were fixed in 4 wt% paraformaldehyde (Sigma, USA) for 30 min and rinsed three times with PBS for 10 min each time. The samples were immersed in 0.2% Triton X-100 for 10 min, rinsed three times for 10 min each time with PBS, and then blocked in 4% goat serum for 1 h in order to minimize nonspecific binding. The entire process was carried out at room temperature. The blocking solution was drained from the samples (without washing) and incubated over night in anti-MyoD1 mouse monoclonal antibody (1 : 200 dilution in PBS) at 4 ∘ C. After rinsing with PBS three times for 10 min each time, the samples were subsequently incubated in anti-mouse IgG (H+L) secondary antibody conjugated with FITC (1 : 100 dilution in PBS) for 1 h in the dark. For nuclei observation, the samples were dipped in 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Sigma, 3 g/mL in PBS) and immediately rinsed with PBS. In the stained image, the MyoD1 (myogenic differentiation antigen) displayed green fluorescence while the nuclei displayed blue fluorescence.
Scanning Electron Microscope (SEM)
Imaging. Samples were fixed in 2.5 wt% glutaraldehyde solution for 30 min at room temperature after cells were seeded for 2, 5, and 10 days, respectively. After rinsing three times for 10 min each with water, the scaffolds were mounted on an aluminum stub and coated with gold. The cell morphology was observed using a Philips CM100 electron microscope (Philips Electronics Nederland BV, Eindhoven, Netherlands) with an accelerating voltage of 10 kV.
Western
Blotting. Cells grown on scaffolds (24-well plates) for 2, 5 and 14 days were washed with PBS three times, 5 min for each time. 200 L of Membrane and Cytosol Protein Extraction Kit (Beyotime, China) was added to each sample for 30 min. The lysate was then collected and transferred to a microcentrifuge tube. The whole process was performed on ice. Subsequently, the lysate was centrifuged at 12000 rpm for 5 min at 4 ∘ C. The supernatant was transferred to a new tube for further analysis. About 30 g of total protein (as measured by Coomassie blue staining), each with 5 L of loading buffer (5×), was then loaded into a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel. Electrophoresis was performed in electrophoretic buffer solution at 100 V for 2 h. The separated proteins on the gel were then electrically transferred onto a polyvinylidene fluoride membrane (PVDF, Roche) in transfer buffer at 4 ∘ C. After being blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature, the membrane was incubated overnight in anti-MyoD1 mouse monoclonal antibody (1 : 500 dilution in BSA blocking solution) at 4 ∘ C. After three rinses of 10 min each with 0.05 v% Tween-20 in Tris-buffered saline (TBST), the membrane was incubated with horse radish peroxidase-conjugated anti-mouse antibody (1 : 5000 dilution in blocking solution; Bioss, China) for 1 h at room temperature. Enhanced chemiluminescence (ECL) Western blotting detection reagents were subsequently added to the membrane to allow detection. Membranes were then exposed in a gel imaging system (Tanon-4200SF, China) where the intensity of the detected proteins was captured. The relative levels of detected proteins were assessed by the ratio between the intensity of MyoD1 and beta-actin. Beta-actin was used to normalize the cellular protein content. The results presented were from at least three separate experiments.
Statistics.
Data are expressed as mean ± standard deviation (SD). Statistical comparisons were made by analysis of variance (ANOVA). -test was used for evaluations of differences between groups. values less than 0.05 were considered to be significant.
Properties of the Scaffolds.
Poly(ester-urethane) is widely used as substrates in biomedical engineering due to its high mechanical elasticity, favorable hemocompatibility, and biodegradability [15]. However, its poor hydrophilicity and intrinsically inert surface result in poor cell-material interaction. In contrast, SF, a fibrous protein found in natural silkworm cocoon, has been used in a variety of biomedical applications due to its favorable mechanical properties, remarkable biocompatibility, and controllable degradation rate [16]. SF grafting is hypothesized to have potential in promoting the cytocompatibility of the scaffold to skeletal muscles. Hence, in this experiment, biomacromolecules like SF and gelatin were grafted on the surface of PU scaffolds by aminolysis and glutaraldehyde (GA) crosslinking, a method previously developed by our group [17,18]. The grafting reaction was illustrated as Scheme 1.
The scaffolds were tested for their wettability using static contact angle measurements (Figure 1). Following grafting, both PU-SF (76.03 ∘ ± 2.03 ∘ ; < 0.01) and PU-Gel (82.27 ∘ ± 2.24 ∘ ; < 0.01) scaffolds were significantly more hydrophilic than the unmodified PU scaffold (101.93 ∘ ± 1.59 ∘ ). The improvement in the wettability of the surfacemodified scaffold also confirmed the success of the grafting of biomacromolecules on the PU surface. Figure 2 shows the stress-strain behaviors of the three micropatterned scaffolds under uniaxial elongation. The PU-SF scaffold demonstrated the highest ultimate tensile strength (UTS) and maximum strain while the unmodified PU had the lowest UTS. The UTS of the PU-Gel scaffold was between that of the PU-SF and PU scaffolds. However, the moduli of all three samples were similar. As SF possesses favorable mechanical properties, the improvements in mechanical properties following the grafting of SF were expected [19], while the modulus of the surface-modified samples was predominantly determined by the intrinsic chemistry of the bulk material, that is, PU. In tissue engineering applications, degradation is an important consideration as it is essential that the scaffold degradation characteristics match the rate of cell growth and tissue regeneration. The data obtained by in vitro degradation will allow us to better understand and predict the in vivo degradation characteristics of the scaffolds [20,21]. We evaluated the degradation properties of PU scaffold via the measurements of weight loss and UTS over a period of 260 days. Degradation usually occurs when soluble oligomeric components diffuse and dissolve in incubation medium due to the hydrolysis of the polymeric chains [22]. The weight loss observed for all scaffolds at day 20, 105, 145, 185, 220, and 260 was very minimal, and the difference of weight loss between each sample type was minimal too. That is because the surface grafting performance shall not greatly affect the intrinsic chemistry of PU matrix. On the other hand, the ultimate tensile strength (UTS) initially rises before gradually declining over time (Figure 3). Polyurethane (58213) is an aromatic polyester-based thermoplastic polymer, which is formed from the reaction of methylene diphenyl diisocyanate (MDI) and polyester polyol. This variation in mechanical properties over the course of degradation can be expected since the relative proportion of the hard segments (MDI) in PU increases due to the hydrolysis of soft segments (polyol). While the relative increase in the proportion of hard segment can enhance the UTS of PU, further increases beyond the optimal point can also adversely affect the properties of PU.
Aligned Microchannels Promote Linear Alignment of Myoblasts.
As the alignment of skeletal muscle cells is an important prerequisite for functional muscle tissue, a polymeric scaffold with unidirectional microchannels was designed to provide environmental cues to mimic the organization of myotubes in hypopharynx skeletal muscle. After 10 days of in vitro culture, the seeded myoblasts were found to be aligned parallel to the channels (Figure 4(b)). In contrast, cells cultured on smooth PU surface were found to be in random state (Figure 4(a)). Apart from being an important requirement for functional skeletal muscle tissue, such alignment may also potentially enhance myotube striation by restricting cell spreading to suppress myoblast proliferation and to promote cell fusion and skeletal muscle differentiation.
Effects of Surface Modification on Cell Growth and Differentiation.
Previous reports have demonstrated that surface wettability is primarily influenced by the outermost layer of the polymer, provided that the surface is uniformly flat and can be quantified by measuring the static contact angle of deionized water. Thus, changes in surface wettability resulting from aminolysis and SF or Gel grafting can also be determined by static contact angle measurements. PU surfaces can be modified by techniques including cholesterol [23], plasma [24], photooxidization and ultra-violet (UV) irradiation [25], so that methacrylic acid (MMA), hydroxyethyl acrylate (HEA), acrylic acid, or cholesterol can be grafted. In our work, SF and gelatin were separately grafted onto the PU scaffold surface using the aminolysis and glutaraldehyde cross-linking method [18] because SF and gelatin are nontoxic and have favorable biocompatibility [26,27]. The grafting of these biomacromolecules resulted in significant increases in wettability of the scaffold surface, compared with that of nonmodified PU surface. The increase in hydrophilicity of both PU-SF and PU-Gel scaffolds enhanced the attachment of myoblasts on scaffolds more than the control PU did ( Figure 5, day 2). Thus, both PU-Gel and PU-SF have the higher cell counts (higher Abs) than the unmodified PU, as assessed by the MTT method at day 5 and day 10 ( Figure 5), though both of them have still much lower cell counts than TCPS has. It may be caused by the lower attachment area of PU-Gel and PU-SF than smooth TCPS due to the channelwall patterns. The grafting of biomacromolecules like gelatin and SF greatly improved cell compatibility whereas the intrinsic inert property of PU was comparatively inferior for cell attachment and growth. As myoblasts grow and subsequently reach confluence, they become slender and spindle-like (Figures 6(a) at day 2 and 6(b) at day 10) [28]. To ascertain the phenotypic state of the myoblasts, immunohistochemistry analysis ( Figure 7) and Western blotting ( Figure 8) were used for investigation. MyoD1 (myogenic differentiation antigen) is a wellrecognized muscle-specific transcription factor expressed in quiescent satellite cells early in the activation process. It also acts as a "master switch" for skeletal muscle differentiation [29]. In response to muscle injury, damage, or degeneration, satellite cells become activated and start to proliferate, giving rise to a population of myogenic progenitor cells known as myoblasts. Myoblasts induce the expression of MyoD1 which is necessary for differentiation into fusion-competent cells. Further fusion into myofibers is associated with an induction of factors essential to myofiber function, including MyoD1, myogenin, and myosin heavy chain (MHC) [29]. The immunofluorescence with anti-MyoD1 as the primary antibody ( Figure 7) exhibited that all cells originated from human Hypopharynx grew along the microchannels; the green fluorescence generated from MyoD1 antibody demonstrated that the cells maintained their progenitor capability after they were cultured on scaffolds for 10 days. Figure 8 showed that the MyoD1 expression of cells was related to the surface profiles; cells on PU-SF expressed the highest MyoD1 while the cells on PU expressed the lowest. Thus, PU-SF was considered to be the most favorable choice for PU substrate in the point of skeletal muscle specification.
Conclusions
In this study, we have fabricated microchannel patterned scaffold and immobilized proteins like SF and gelatin onto the scaffold surface using aminolysis and glutaraldehyde crosslinking technique. The resulting scaffolds showed the appropriate bulk mechanical properties as well as suitable surface chemistry for cell proliferation and growth. The results about cell culture demonstrated the successful isolation of primary hypopharynx myoblasts, in vitro cell expansion, and differentiation into human myofibers using unidirectionally aligned microchannels on PU matrix. We thus concluded the potential of this surface-modified PU scaffolds with aligned microchannels for future applications in skeletal muscle tissue engineering.
Conflict of Interests
There is no conflict of interests associated with this paper. | v3-fos-license |
2017-08-27T13:09:01.014Z | 2004-01-01T00:00:00.000 | 43434038 | {
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} | pes2o/s2orc | A molecular dynamics study of Al 3 + in water : hydrolysis effects
A molecular dynamics study of Al in water was performed. A flexible non-constrained central force model for water molecules was used. This permitted one to take into account a tendency of cation hydrolysis effects. We observed strict octahedral arrangement of neighbours in aluminium cation hydration shell, two of six neighbours being water molecules, the rest four being OH groups that appeared as a consequence of cation hydrolysis. Four created protons leave the first hydration shell and transfer to the second one. Those protons are strongly bonded to cation hydration shell, which implies that total charge of hydration shell does not change. Structural and dynamical features of the obtained hydration shell of Al are reported.
Introduction
A concept of cation hydrolysis was introduced about a hundred years ago by Bjerrum [1].In accordance with this concept the initial steps, which are involved in the hydrolysis of metal ions in aqueous solutions, are considered as a series of removals of protons from water molecules in ion hydration shell.At the next stage there appear polynuclear ions [2,3] caused by condensation reaction between hydrated aqua-ions.However, a mechanism of cation hydrolysis, including the initial steps, is not quite understandable so far.For example, the primary evidence of the existence of such hydrated aqua-ions as Al(OH) 2+ , Al(OH) + 2 or Al(OH) 3 in aqueous solutions of aluminium salts appear to be just indirect; they are indispensable to fit solubility data over a broad pH range [4].
For the last three decades an impressive progress has been reached in computer modelling of the structure of hydration shells of monovalent alkaline cations and divalent alkaline earth cations [5].It was shown that for divalent cations the electrostatic repulsion between cation and protons of water molecules can cause notable modifications of an intramolecular geometry of water molecules in hydration shell.One can suppose that it can be sufficiently strong to repel protons of water molecules from hydration shell causing the hydrolysis effect.However the computer studies of hydration of cations with the hydrolysis effects have not been practically performed.This can be explained by inapplicability of standard water models in treating the hydrolysis effects and by difficulties of quantum-mechanical calculations of hydrated-hydrolyzed complexes with a sufficient degree of reliability in order to be used in further computer simulations [6].In computer simulations, in order to treat cation hydrolysis effects explicitly, water should be considered in the framework of a non-constrained flexible model.The modelling of cation-water interaction is more complex.It should contain numerous details connected with the treatment of many-body cation-water interactions, the covalent bond effects caused by the specific features of cation electronic configuration etc.
Since the intensity of cation hydrolysis increases with an increase of ion charge and/or decrease of ion size [7], the electrostatic interaction between cation and water molecules plays a prevalent role in this phenomenon.Due to this we recently proposed a model of a primitive cation M Z+ [8][9][10] for the treatment of hydratedhydrolyzed structure of cation.In this model we use a non-rigid model of water CF1 [11,12] in order to describe the effect of a cation charge Ze (e is the elementary electric charge) on intramolecular structure of water molecules.The interaction of primitive cation M Z+ with water molecules is similar to an interaction of the potential of cation Na + [13] with water but we also assume that an ion can possess different valency Z.A molecular dynamics study for this model has shown that the increase of cation charge stabilizes an octahedral hydration structure of this cation and significantly modifies the intramolecular geometry of water molecules in hydration shell.For highly charged cations such as M 4+ and M 5+ , we observed the effect of some proton loss by water molecules in hydration shell, which was treated as a cation hydrolysis.
In this paper we report a molecular dynamics modelling of hydration structure and dynamical properties of one of the simplest realistic trivalent cations -Al 3+ .Due to a comparatively small size and high valency of Al 3+ the electrostatic cationwater interaction is strong enough for hydrolysis initiation.The aqueous speciation of Al 3+ is very important for many industrial processes such as waste water treatment, pharmaceutical design, catalysis optimization, remediation of wastes from plutonium production etc. [4,14].For example, the speciation of Al 3+ hydrolysis products, especially the polynuclear complexes, appear to be a key aspect of aluminum rhizotoxicity [15].However, in spite of large efforts to describe the cation-water interaction correctly no hydrolysis effects are taken into account directly in previous computer simulations of Al 3+ in water [16,17].
In present investigation water is considered in the framework of flexible CF1 model [11,12].The description of cation-water interaction is taken in the form given in [18], which was drawn from quantum-chemical calculations.The results of our investigations demonstrate a strict octahedral configuration of Al 3+ shell with the tendency for cation hydrolysis.The obtained results for hydration structure and selfdiffusion coefficient of Al 3+ are in good agreement with the results of other computer simulations and experimental data.
Model
This study of Al 3+ aqueous solution was performed in the framework of a mixed model.The interaction between Al 3+ and water was taken from [18] in the form (energies are given in kJ/mol, distances are in Å): (1) V Al−H (r) = 1375.26/r+ 160.657/r 2 + 287.456 • exp(−0.35461r). ( The water-water interaction in [18] is described by Bopp-Jancso-Heinzinger model (BJH) [19].In this study we used the CF1 model for water-water interaction.Both models are identical in the sense of description of intermolecular interaction, but differ for intramolecular part of interaction.The intramolecular terms in BJH are described by anharmonic expansion, which are unfavorable in the sense of water molecule dissociation.
As a first step of our investigation for simplification we neglect the role of threebody cation-water interactions, which was discussed in [18].
Simulation details
Molecular dynamics simulations were carried out using the standard DL POLY package [20].A simulation unit cell (L x =L y =33.645Å, L z =46.263Å) with periodic boundary conditions in three directions contained 1727 water molecules and one Al 3+ ion in the center.The production run over 4 • 10 5 steps was performed in isotropic NPT ensemble.The density of the solution at temperature 298 K and pressure 1 bar was 1 g/cm 3 .Equations of motion were integrated according to Verlet algorithm with time step equal to 10 −16 s.
For a treatment of Coulombic interactions the Ewald summation procedure (Ewald convergence parameter was 0.285 Å−1 , maximal summation parameters for reciprocal lattice were |n x | = |n y | = 9, |n z | = 12) was used.We also made a pilot simulation with the use of shifted force procedure to manage the long-range interactions.The comparison between those two approaches has shown that shifted force procedure neglects in a special way the long-range part of interaction, which appears in stronger hydrolysis of water molecules in Al 3+ hydration shell.For short range parts of potentials, a cut-off distance was chosen to be 10 Å.
Results
It is convenient to explore the hydration shell of aluminium cation by means of radial distribution functions (RDF) g Al−O (r), g Al−H (r) and corresponding running coordination numbers: where β=O, H of water molecules.A function n αβ (r) indicates the number of species β in the sphere of radius r with species α in the center; ρ β is the number density of species β.In figure 1 we present Al 3+ -water RDFs and coordination numbers.The main peak of g Al−O (r) distribution is located at 1.78 Å.This result slightly underestimates the value range 1.8-1.97Å yielded from calculations at various levels of theory in [16,17,22], while the experimental results collected in [21] are within the range 1.87-1.9Å. However the number of oxygens in the first hydration shell in our calculations coincides with Al-O coordination numbers reported from all mentioned investigations and is equal to 6.A well defined plateau on the n Al−O (r) indicates that no exchange processes between the first and the second hydration shells were observed during simulation time.
Al-H distribution is characterized by g Al−H (r), n Al−H (r) functions.Position of a first peak of Al-H RDF is 2.71 Å. Different theoretical approaches [16,17] yielded a range of values 2.47-2.59Å for r max .The deviation of our result can be easily explained if one takes into account that our water model is non-rigid (in spite of the ones used in the above references).The strong electrostatic repulsion between Al 3+ and hydrogens stretches the O-H bonds of the aluminium cation hydration shell neighbours.In [16] a stretching of O-H bonds in water molecules in the first hydration shell was noticed too.Such a deformation was also observed in simulations of the effect of cation charge on intramolecular structure of water in hydration shell of highly charged model cation [9].The corresponding running coordination number n Al−H (r min ) indicates that the number of protons in the first hydration shell is equal to 8, instead of 12.We treat this result as a cation hydrolysis: strong electrostatic repulsion between Al 3+ and protons pushes four of them outside the first 2.One can clearly see two water molecules, four OH − groups and four protons that appeared due to hydrolysis.Four protons are located in the second hydration shell and are strongly bonded to the first one.All the neighbours are octahedrally arranged around the aluminium ion.As it was mentioned before the protons in OH − groups are oriented outside the hydration shell.The strong electrostatic interactions cause strict octahedral arrangement of Al 3+ hydration shell.For example, in Na + hydration shell there are six neighbours too.However, the charge of sodium ion being lower, the octahedral arrangement is feebly pronounced [9].A bond-angle distribution O-Al-O in hydration shell is presented in figure 3.This di-stribution demonstrates two peaks at 0 and -1, which correspond to angles 90 0 and 180 0 .This confirms the conclusion that during the simulation time the octahedral arrangement of Al 3+ neighbours was predominant keeping the coordination number equal to 6. Another aspect of our investigation is connected with dynamical properties of Al 3+ and oxygens in the first hydration shell.We calculated the normalized velocity autocorrelation functions (VACF) for aluminium ion and for oxygens of hydration shell.The spectral densities f α (ω) of hindered translational motions were drawn by Fourier transformations of VACFs where v α (t) is the velocity of particle α =M,O at time t.The functions f M (ω), f O (ω) are presented in figure 4. The Al 3+ ion distribution of frequencies is characterized by three main peaks approximately at: 25, 80 and 170 ps −1 ; one additional peak is located near 120 ps −1 .On the spectrum of oxygens one can see four peaks approximately at: 20, 70, 120 and 165 ps −1 .Obviously there is correlation between the spectra of central cation and oxygens.Furthermore, the characteristic frequencies coincide, which allows us to conclude that the motion of Al ion is strongly coupled with the motion of water molecules and OH − groups in hydration shell.The self-diffusion coefficient of aluminium ion, in our study is equal to 0.46 • 10 −5 cm 2 /s.In [17] a set of results is reported for the self-diffusion coefficient of aluminium ion for different models.It varies within the range 0.17 − 0.47 • 10 −5 cm 2 /s, while the experimental value drawn from [23] is 0.6 • 10 −5 cm 2 /s.
Conclusions
In the current investigation, a molecular dynamics simulation of Al 3+ aqueous solution was performed.We made an attempt to take into account a non-rigid origin of water molecules; this permitted us to observe a tendency of a cation hydrolysis effect and to explore how this effects the structural and dynamical properties of aluminium ion first hydration shell.CF1 model makes it possible for the water molecules to dissociate in spite of rigid models, for which the hydrolysis effect is impossible.
The aluminium cation hydration shell demonstrated a stable octahedral arrangement of neighbours.Coordination numbers n Al−O (r) and n Al−H (r) indicate the presence of 6 oxygens and 8 hydrogens respectively in the first hydration shell.A snapshot of this configuration shows that aluminium cation neighbourhood consists of two water molecules while four OH − groups appeared as a consequence of hydrolysis of water molecules due to strong electrostatic repulsion between aluminium cation and hydrogens.4 protons, which transferred from the first hydration shell to the second one, are still strongly bonded to the cation, which implies that the whole hydrated complex does not change its total charge.The octahedral strict arrangement of neighbours around the cation is confirmed by the O-Al-O bond-angle distribution with two peaks at 90 0 and 180 0 .The calculated self-diffusion coefficient of aluminium cation agrees well with other theoretical and experimental results.The characteristic frequencies of both spectra of translational motions of alumunium cation and oxygens in its hydration shell coincide.This fact agrees with the conclusion about the leading role of electrostatic interactions in complexation processes in "highly charged cation+hydration shell" clusters.
Acknowledgements
M. Holovko thanks to STCU for partial support of this research (Grant No. 1706).
Figure 1 .
Figure 1.Radial distribution functions (solid lines) g Al−O (r), g Al−H (r) and corresponding running coordination numbers (dashed lines) n Al−O (r), n Al−H (r).
Figure 3 .Figure 4 .
Figure 3. Bond angle distribution O-Al-O in the first hydration shell. | v3-fos-license |
2018-01-12T07:41:12.884Z | 2011-05-11T00:00:00.000 | 15281509 | {
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} | pes2o/s2orc | Diesel Exhaust Activates and Primes Microglia: Air Pollution, Neuroinflammation, and Regulation of Dopaminergic Neurotoxicity
Background: Air pollution is linked to central nervous system disease, but the mechanisms responsible are poorly understood. Objectives: Here, we sought to address the brain-region–specific effects of diesel exhaust (DE) and key cellular mechanisms underlying DE-induced microglia activation, neuroinflammation, and dopaminergic (DA) neurotoxicity. Methods: Rats were exposed to DE (2.0, 0.5, and 0 mg/m3) by inhalation over 4 weeks or as a single intratracheal administration of DE particles (DEP; 20 mg/kg). Primary neuron–glia cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were used to explore cellular mechanisms. Results: Rats exposed to DE by inhalation demonstrated elevated levels of whole-brain IL-6 (interleukin-6) protein, nitrated proteins, and IBA-1 (ionized calcium-binding adaptor molecule 1) protein (microglial marker), indicating generalized neuroinflammation. Analysis by brain region revealed that DE increased TNFα (tumor necrosis factor-α), IL-1β, IL-6, MIP-1α (macrophage inflammatory protein-1α) RAGE (receptor for advanced glycation end products), fractalkine, and the IBA-1 microglial marker in most regions tested, with the midbrain showing the greatest DE response. Intratracheal administration of DEP increased microglial IBA-1 staining in the substantia nigra and elevated both serum and whole-brain TNFα at 6 hr posttreatment. Although DEP alone failed to cause the production of cytokines and chemokines, DEP (5 μg/mL) pretreatment followed by lipopolysaccharide (2.5 ng/mL) in vitro synergistically amplified nitric oxide production, TNFα release, and DA neurotoxicity. Pretreatment with fractalkine (50 pg/mL) in vitro ameliorated DEP (50 μg/mL)-induced microglial hydrogen peroxide production and DA neurotoxicity. Conclusions: Together, these findings reveal complex, interacting mechanisms responsible for how air pollution may cause neuroinflammation and DA neurotoxicity.
Research
Air pollution is a prevalent source of environ mentally induced inflammation/ oxidative stress, and each year millions of people are exposed to levels of air pollution above promulgated safety standards (Block and Calderón-Garcidueñas 2009). Importantly, air pollution has been strongly associated with deleterious central nervous system (CNS) effects, including increased stroke incidence (Chen 2010), decreased cognitive function (Calderón-Garcidueñas et al. 2008a), and Alzheimer's disease (AD)-like or Parkinson's disease (PD)-like neuro pathology (Block and Calderón-Garcidueñas 2009). Although prospective epidemiology studies of PD and air pollution are unavailable at this time, elevated levels of manganese in the air have been associated with enhanced PD risk (Finkelstein and Jerrett 2007). Consistent with findings from human populations, animal studies in dogs, mice, and rats show that air pollution components cause neuro inflammation, oxidative stress, and DNA damage and up-regulate markers of neuro degenerative disease (Block and Calderón-Garcidueñas 2009). However, although evidence supports an effect of air pollution on CNS pathology and disease, underlying mechanisms of such effects are unknown (Block and Calderón-Garcidueñas 2009).
Diesel exhaust (DE) is a major constituent of near-road and urban air pollution and is commonly used as a surrogate model of air pollution in health effects studies (Hesterberg et al. 2010;Ma and Ma 2002). CNS responses to DE have been documented: Exposure has been shown to affect electroencephalogram parameters in human subjects (Cruts et al. 2008). Animal studies in rats using a month-long inhalation model ) and a model using 2-hr-long exposure by nose-only inhalation ) have demonstrated that DE elevates pro inflammatory factors in select brain regions. Recent studies have also shown gene expression changes in the rat cerebrum after peri natal exposure to DE (Tsukue et al. 2009). In addition, in utero exposure to DE has been shown to affect dopamine (DA) neuro chemistry and cause motor deficits in mice (Suzuki et al. 2010;Yokota et al. 2009). Our previous in vitro work has shown that microglia are activated by DE particles (DEP) to produce extracellular superoxide through NADPH oxidase, which is selectively toxic to DA neurons ). Further exploring cellu lar mechanisms of DE's CNS effects, we have also shown that DEP impair the bloodbrain barrier and cause capillaries to release tumor necrosis factor-α (TNFα) in vitro, contributing to inflammation (Hartz et al. 2008). However, although DE causes neuroinflammation, perturbs DA neuro chemistry, and impairs motor behavior, the cellular and molecular mechanisms driving these effects are poorly understood. In the present study, we used DE as a model of air pollution to further define the deleterious CNS effects and to begin to address the complex mechanisms that mediate pathology.
Background: Air pollution is linked to central nervous system disease, but the mechanisms responsible are poorly understood. oBjectives: Here, we sought to address the brain-region-specific effects of diesel exhaust (DE) and key cellular mechanisms underlying DE-induced microglia activation, neuroinflammation, and dopaminergic (DA) neurotoxicity. Methods: Rats were exposed to DE (2.0, 0.5, and 0 mg/m 3 ) by inhalation over 4 weeks or as a single intratra cheal adminis tration of DE particles (DEP; 20 mg/kg). Primary neuron-glia cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were used to explore cellular mechanisms. results: Rats exposed to DE by inhalation demonstrated elevated levels of whole-brain IL-6 (inter leukin-6) protein, nitrated proteins, and IBA-1 (ionized calcium-binding adaptor molecule 1) protein (microglial marker), indicating generalized neuro inflammation. Analysis by brain region revealed that DE increased TNFα (tumor necrosis factor-α), IL-1β, IL-6, MIP-1α (macrophage inflammatory protein-1α) RAGE (receptor for advanced glycation end products), fractalkine, and the IBA-1 microglial marker in most regions tested, with the midbrain showing the greatest DE response. Intratracheal administration of DEP increased microglial IBA-1 staining in the substantia nigra and elevated both serum and whole-brain TNFα at 6 hr post treatment. Although DEP alone failed to cause the production of cytokines and chemokines, DEP (5 μg/mL) pre treatment followed by lipopolysaccharide (2.5 ng/mL) in vitro synergistically amplified nitric oxide production, TNFα release, and DA neurotoxicity. Pretreatment with fractalkine (50 pg/mL) in vitro ameliorated DEP (50 μg/mL)-induced microglial hydrogen peroxide production and DA neurotoxicity. conclusions: Together, these findings reveal complex, interacting mechanisms responsible for how air pollution may cause neuroinflammation and DA neurotoxicity. key words: air pollution, brain, microglia, neuroinflammation, oxidative stress, Parkinson's disease. (Boston, MA). We purchased the tyrosine hydroxylase (TH) anti body from Millipore (Billerica, MA); the ionized calciumbinding adaptor molecule 1 (IBA-1) anti body from Wako (Richmond, VA); the α-synuclein anti body from Millipore; and the biotinylated horse anti-mouse and goat anti-rabbit secondary anti bodies from Vector Laboratories (Burlingame, CA). All other reagents were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Animals. For the in vivo studies, 12-weekold male Sprague-Dawley rats and 12-to 14-week-old male Wistar Kyoto (WKY) rats were purchased from Charles River Laboratories (Raleigh, NC). Animals were acclimated to the housing facility for 1 week before studies began. For the primary cell culture studies, timed-pregnant (gestational day 14) adult female Fisher 344 rats were purchased from Charles River Laboratories. Housing, breeding, and experimental use of the animals were performed in strict accordance with National Institutes of Heath guidelines. All animals were treated humanely and with regard for alleviation of suffering.
Animal treatment. Inhalation. DE was generated by a 30-kW (40 hp) fourcylinder indirect injection Duetz diesel engine (BF4M1008), as previously described (Gottipolu et al. 2009;Stevens et al. 2008), and animals were exposed to DE in exposure chambers [for details, see Supplemental Material, p. 3 (doi:10.1289/ehp.1002986)]. Rats were exposed 4 hr/day, 5 days/week, for 1 month to air or DE at concentrations of 0, 0.5, or 2 mg/m 3 , which are higher than typically encountered in ambient air but may be achieved during heavy traffic or occupational situations. This is an established model commonly used to explore the effects of air pollution (Gottipolu et al. 2009;Stevens et al. 2008).
Intratracheal (IT) DEP adminis tration. Male Sprague-Dawley rats received either phosphate-buffered saline, pH 7.4 (control) or DEP (SRM 2975; 20 mg/kg) suspended in saline, as previously described (Arimoto et al. 2005) [for details, see Supplemental Material, pp. 3-4 (doi:10.1289]. Although DEP are adminis tered in a single bolus at a concentration (20 mg/kg) that is higher than typical environ mental exposures, this well-defined, established model (Gottipolu et al. 2009) provides data on the possible effects of particulate exposures via the lungs, as opposed to exposures through nasal entry to the brain. DEP preparation for in vitro studies. Nanometer-sized DEP were used as a model of ultra fine particulate matter (PM) and were prepared as described previously ) [for details, see Supplemental Material, p. 4 (doi:10.1289]. The precise amount of PM reaching the brain is currently unknown. However, studies have demonstrated that 0.01-0.001% of inhaled nanometer-sized iridium and carbon particulate remain in the brain 24 hr after exposure (Kreyling et al. 2009). Based on the in vivo models used in the present study (DEP; 0.5 mg/m 3 , 2 mg/m 3 , and 20 mg/kg), the in vitro concentrations of nanometer-sized particles (5-50 μg/mL) fall within the current estimates of what may reach the brain.
Cell lines. The rat microglia HAPI (highly aggressively proliferating immortalized) cells were a generous gift from J.R. Connor (Cheepsunthorn et al. 2001) and were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified incubator with 5% CO 2 /95% air.
Immunostaining in vitro. Microglia were stained with the polyclonal anti body raised against IBA-1 protein, and DA neurons were detected with the polyclonal anti body against TH, as reported previously [see Supplemental Material, p. 5 (doi:10.1289/ehp.1002986)].
Immunostaining in vivo. Brains from rats treated with saline or DEP via IT were fixed in 4% para formaldehyde and processed for immuno staining as described previously [for details, see Supplemental Material, p. 6 (doi:10.1289/ehp.1002986)].
ELISAS (enzyme-linked immuno sorbent assays). We measured levels of TNFα, interleukin-6 (IL-6), macro phage inflammatory protein-1α (MIP-1α), IL-1β, fractalkine, Figure 1. Inhalation exposure to DE elevates markers of neuro inflammation. WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m 3 DEP (n = 3/treatment group). TNFα (A) and MIP-1α (B) mRNA levels (mean ± SE) were determined in olfactory bulbs using quantitative real-time RT-PCR; values represent the fold increase from control 2 -ΔΔCT normalized with α-tubulin and expressed as a percentage of control. Representative images show changes in IL-6 (C) and IBA-1 (D) in whole-brain homogenate assayed by Western blotting; GAPDH was used as a loading control. (E) Whole-brain homogenate was also assayed for nitrosylated protein by ELISA; values shown are mean ± SE. *p < 0.05, compared with controls. and RAGE (receptor for advanced glycation end products) in cell culture supernatant and brain homogenate using commercially available ELISA kits (R&D Systems, Minneapolis, MN). Levels of protein nitration in brain homogenate, a common marker of oxidative stress, were also measured by ELISA, per manufacturer instructions (Millipore, Temecula, CA). For all ELISAs, brain regions were homogenized in Cytobuster lysis buffer (EMD Chemicals), and 100 μg total protein was assayed per well.
We developed an indirect ELISA to quantitate relative amounts of IBA-1 expression in brain homogenate [for details, see Supplemental Material, p. 6 (doi:10.1289/ehp.1002986)]. We meas ured the amount of DA present in midbrain tissue using snap-frozen, dissected midbrain tissue homogenized in 0.1 M HCl and 0.1 mM EDTA and a commercially available kit from Genway Biotech Inc. (San Diego, CA), following manufacturer instructions.
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).
We meas ured levels of TNFα and MIP-1α mRNA by RT-PCR. Total RNA was extracted from the mouse olfactory bulbs using the RNA Easy kit (Qiagen, Valencia, CA) as described previously Statistical analysis. Data are expressed as raw values, percentage of control, fold increase from control, or the difference from control, where control values were set to 100%, 1, or 0 accordingly. Data for the treatment groups are expressed as mean ± SE. We assessed statistical significance with a one-or two-way analysis of variance followed by Bonferroni's post hoc analysis using SPSS software (SPSS Statistics 19; IBM, Armonk, New York). A value of p < 0.05 was considered statistically significant.
Inhalation exposure. DE and neuroinflammation.
To discern whether DE caused neuro inflammation at all, we mea sured pro inflammatory mRNA expression in the olfactory bulb in rats exposed for 1 month by inhalation. DE caused a significant increase in both TNFα and MIP-1α mRNA expression in the olfactory bulbs (p < 0.05) ( Figure 1A,B).
To explore whether neuro inflammation was generalized throughout the brain, we tested whole-brain homogenate from rats exposed for 1 month by inhalation. Data show that DE exposure increased expression of IL-6 ( Figure 1C) and IBA-1 (microglial marker; Figure 1D), as meas ured by Western blot. DE also caused a significant elevation of protein nitration in whole-brain homogenate ( Figure 1E). At the time of sacrifice, no cytokines were elevated in the serum in DE-exposed rats compared with controls (data not shown) (Gottipolu et al. 2009). These data suggest that DE exposure caused generalized neuro inflammation and oxidative stress that extended throughout the entire brain.
DE and brain-region-specific neuroinflammation. We next addressed the effects of DE on neuro inflammation in brain regions known to be affected by neuro degenerative disease: the cortex (AD), midbrain (PD), and olfactory bulb (AD and PD) (Hawkes et al. 1997;Kovacs et al. 1998). Several cyto kines were evaluated in tissue homogenates by ELISA. All three brain regions showed significant increases in TNFα protein expression in response to DE, with the greatest increase occurring in the midbrain (Figure 2A). The pro inflammatory cytokine IL-1β was not increased in the olfactory bulbs but was significantly increased in the midbrain and cortex in response to both levels of DE exposure, with the most pronounced response in the midbrain ( Figure 2B). All three brain regions showed increased expression of IL-6 protein in response to DE ( Figure 2C) and increased levels of MIP-1α chemo kine, with the highest MIP-1α levels expressed in the midbrain ( Figure 2D).
In addition, we examined the effect of DE on levels of RAGE, which is elevated in the substantia nigra and frontal cortex in cases of early stages of parkinsonian neuro pathology (Dalfo et al. 2005) and is key to how microglia identify many neuro toxic stimuli (Fang et al. 2010). Interestingly, DE elevated RAGE expression, but only in the midbrain ( Figure 2E).
Notably, in controls, the midbrain showed signifi cantly higher levels of the microglial marker IBA-1 compared with either the cortex or the olfactory bulb ( Figure 2F), suggesting that numbers of microglia are higher in the midbrain under normal conditions. IBA-1 protein was significantly increased in the cortex after high-DE exposure (2.0 mg/m 3 ) but was increased in the midbrain in response Figure 2. DE elevates microglial markers, cytokines, and chemo kines in brain regions. WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m 3 DEP (n = 4/treatment group). Protein levels of TNFα (A), IL-1β (B), IL-6 (C), MIP-1α (D), RAGE (E), and IBA-1 (microglial marker; F) from the olfactory bulb (OB), cortex, and midbrain were measured by ELISA. In controls, IBA-1 was significantly higher in the midbrain compared with other brain regions, indicating higher levels of microglia in the absence of exposure. Notably, the midbrain region also showed the highest levels of cytokines, chemokines, and microglial markers in response to DE. *p < 0.05, compared with the corresponding region in controls. **p < 0.05 for IBA-1 in midbrain, compared with olfactory bulbs and cortex levels in controls. to both DE exposure levels ( Figure 2F). Combined with evidence of higher levels of microglia and a more pronounced proinflammatory response in the midbrain, these findings support a greater vulnerability of the midbrain to air-pollution-induced neuroinflammation compared with other brain regions.
DEP and neuro inflammation and systemic inflammation in vivo.
Air pollution comprises numerous compounds, including gases and PM. Because the DE inhalation exposure contained particulate and gas-phase components (primarily carbon monoxide and nitric oxide; see Gottipolu et. al. 2009), it is unclear which components of DE are responsible for CNS effects. To test the ability of PM to induce neuro inflammation, we administered the particles (DEP; SRM 2975) to rats by IT installation and measured TNFα levels in serum and whole-brain homogenate by ELISA. DEP caused significant TNFα elevation in both the serum and the brain (Figure 3A,B). Immunostaining of IBA-1 showed that DE did not cause acute changes in microglial morphology at 20 hr post treatment but did up-regulate IBA-1 expression (indicated by darker staining in Figure 3C), consistent with mild microglial activation. These results indicate that DEP can cause systemic TNFα elevation, increase brain TNFα, and activate microglia in vivo.
DEP and microglia. It is likely that nano sized DEP particulates and leachable components trans locate to the systemic circulation through the pulmonary capillary bed (Valavanidis et al. 2008). Recent studies have shown that PM (Wang et al. 2008) from air pollution (Calderón-Garcidueñas et al. 2008b) actually reaches the brain, which may be one mechanism by which CNS effects occur.
Using an in vitro model, we also tested the ability of DEP to modulate on going neuroinflammation. Microglia cultures pre treated with DEP had significantly enhanced levels of TNFα ( Figure 4A) and nitrite ( Figure 4B) in response to LPS. We also observed this DEP priming response in primary neuronglia cultures, where a low, non neuro toxic concentration of DEP (5 μg/mL) amplified LPS-induced TNFα ( Figure 4C) and nitrite ( Figure 4D). Together, these data indicate that DEP prime microglia, increasing their sensitivity to additional pro inflammatory stimuli. Additional pro inflammatory stimuli could include ongoing neuro degeneration or perhaps the peripheral cytokine response as it transfers to the brain.
DE and DA neuro toxicity in vivo and in vitro. We have previously shown in vitro that DEP are selectively toxic to DA neurons through microglial NADPH oxidase activation and the consequent production of extra cellular super oxide ). Interestingly, we did not observe evidence of DA neuro toxicity 24 hr after IT DEP exposure based on immuno histochemistry and staining of TH neurons (data not shown). Brain tissue sections were not available for the DE inhalation study, but we did not observe significant differences in the DA content in the midbrain region based on DA ELISA and α-synuclein content (Western blot) after DE exposure (data not shown).
We also investigated whether the low nonneuro toxic concentration of DEP (5 μg/mL) that enhanced the microglial pro inflammatory response to LPS ( Figure 4C,D)affected DA neuro toxicity in response to LPS. Pretreatment with DEP for 30 min significantly enhanced LPS-induced loss of DA neuron function, as measured by DA uptake ( Figure 5A). This suggests that exposure to low levels of air pollution that fail to initiate neuro toxicity alone may instead enhance additional proinflammatory triggers or on going neurodegenerative processes.
In an effort to understand why DA neuron damage was absent in the DE in vivo models, we began to explore the effect of DE on compensatory mechanisms in the brain that could counter act the neuro toxic effects of neuroinflammation. Fractalkine is a chemo kine expressed by neurons, and the receptors are exclusively on microglia (Cardona et al. 2006). Solublized fractalkine is a key regulator of the microglial pro inflammatory response, where it is has been shown to protect against microgliamediated DA neuro toxicity in vitro and in PD models in vivo (Re and Przedborski 2006). Here, we show that fractalkine was elevated only in the midbrain region after 1 month of DE exposure (2.0 mg/m 3 ) via inhalation ( Figure 5B). In vitro studies revealed that soluble fractalkine attenuated DE-induced microglia H 2 O 2 production ( Figure 5D) and DEP-induced loss of DA neuron function in vitro ( Figure 5C). Although elevated fractalkine expression did not abolish DE-induced neuro inflammation in vivo, fractalkine may attenuate the pro inflammatory response to non neuro toxic levels.
Discussion
There is increasing evidence that environmental inhalation exposures may result in neuro inflammation and DA neuro pathology (Antonini et al. 2009;Choi et al. 2010;Sriram et al. 2010;Verina et al. 2011), but the mechanisms are poorly understood. The present study employed in vivo and in vitro DE models to explore the mechanisms through which air pollution causes neuro inflammation and microglial activation, as well as the relevance of DE exposure for DA neuron survival. Here, we show that DE caused oxidative stress (i.e., protein nitration) and activated microglia in vivo (Figures 1-3). After 1 month of inhalation exposure, the IBA-1 microglial marker was up-regulated, particularly in the midbrain region, which contains the substantia nigra (Figure 2). At 24 hr after IT DEP adminis tration, immuno histochemical analysis showed that DEP up-regulated IBA-1 on microglial cells, without obvious differences in morphology or cell number (Figure 3), a response similar to microglial activation previously observed in response to systemic LPS administration (Qin et al. 2007). Because immuno histochemical analysis of the monthlong DE exposure was unavailable, we could not determine whether IBA-1 levels increased because of increased microglial numbers or because of up-regulated IBA-1 protein in the microglial membranes. However, DEP triggered H 2 O 2 production from microglia in vitro ( Figure 5). Further studies are needed to determine whether air pollution causes increased mono cyte trafficking to the brain, qualitative changes in recruited cell populations (circulating monocytes vs. bone marrow), or proliferation of parenchymal microglia in vulnerable brain regions.
Our present work provides evidence of generalized pro inflammatory cytokine elevation throughout the brain (Figure 1), with the greatest pro inflammatory response to DE observed in the midbrain (Figure 2). This distinction is important because, consistent with previous reports (Kim et al. 2000), the midbrain also expressed the highest levels of microglial markers at rest and the greatest elevation of these markers in response to DE, which suggests that microglia may mediate a regional vulnerability to the neuro inflammatory effects of air pollution ( Figure 2). These data also suggest that DE-induced neuro inflammation may be due in large part to a systemic response that affects the entire brain, rather than a local effect mediated solely by direct exposure through the olfactory bulb, a favored pathway of PM entry into the brain (Oberdörster et al. 2004). In fact, although the olfactory bulb showed elevation of some pro inflammatory factors with DE exposure, it also failed to show upregulation of IBA-1, RAGE, fractalkine, or IL-1β in response to DE, indicating a less pronounced pro inflammatory response in this region ( Figure 2). Notably, IT adminis tration of DEP also resulted in increased TNFα production in whole-brain homogenates and activated microglia morphology in the substantia nigra (Figure 3), further supporting the hypothesis that nasal entry through the olfactory bulb may not be necessary for DE to cause neuro inflammation.
There is increasing evidence that systemic inflammation may contribute to neurodegenerative diseases (Perry et al. 2010). We (Qin et al. 2007) and others (Ling et al. 2002(Ling et al. , 2006Wang et al. 2009) have previously shown that systemic LPS adminis tra tion causes neuro inflammation that persists long after the peripheral pro inflammatory response has resolved, resulting in delayed and progressive (Qin et al. 2007) DA neuro toxicity. Consistent with our prior studies using LPS in adult animals that showed pronounced neuroinflammation that persisted in absence of the initiating peripheral pro inflammatory trigger (Qin et al. 2007), we failed to see peripheral cytokines after 1 month of DE inhalation exposure, despite evidence of elevated neuro inflammation. In contrast, we observed increases in both serum and brain TNFα (6 hr and 24 hr) after IT administration of a single large dose of DEP ( Figure 3). However, differences in cytokine responses between the two models may be due to kinetics, differences in the concentration of DEP versus DE, and/ or chemical differences between the two exposures (i.e., gaseous components such as carbon monoxide and nitrogen oxides). Microglia and astro cytes did not produce cytokines or chemokines in response to DEP in vitro, suggesting that although PM reaching the brain may cause microglia-derived oxidative stress, , cortex, and midbrain of rats; WKY rats were exposed to 0 (air control), 0.5, or 2.0 mg/m 3 DEP (n = 4/treatment group) for 1 month, and fractalkine levels were measured by ELISA. (C and D) Primary mesencephalic neuron-glia cultures were pretreated for 30 min with fractalkine (100 pg/mL) followed by DEP (50 μg/mL) exposure (n = 3/treatment group). (C) DEP-induced loss of DA neuron function was reduced by fractalkine 7 days after treatment as measured by DA uptake assay. (D) DEP-induced H 2 O 2 in microglia was reduced by fractalkine 3 hr after treatment. *p < 0.05, compared with controls. **p < 0.05, compared with LPS alone. # p < 0.05, compared with DEP alone. systemic effects may be necessary to produce a comprehensive neuro inflammatory response that includes pro inflammatory factor production. Further, DEP enhanced microglial responses to pro inflammatory effects of LPS (Figure 4), indicating that inter action between DEP and systemic cyto kines may amplify neuro inflammation. Thus, DE exposure caused increased levels of systemic cytokines that may contribute to microglial activation and the pro inflammatory milieu of the brain.
We have previously shown in vitro that DEP activate microglia and are selectively toxic to DA neurons through microglia-derived reactive oxygen species ). In the present study, DE activated microglia, elevated neuro toxic cytokines in the midbrain, and induced oxidative stress in vivo (Figures 1 and 2), but we found no evidence of DA neuro toxicity in vivo. However, the longest exposure was only 1 month, and our prior research indicated that both aging and chronic microglial activation are needed to culminate in DA neuron death in vivo, suggesting that longer exposures and/or aging may be necessary for DE-induced neuro inflammation to initiate neuro degeneration in vivo.
To discern why DE failed to cause DA neuro toxicity in vivo, we shifted our focus to homeo static mechanisms designed to regulate microglia activation. Fractalkine is a chemokine produced by neurons that is cleaved to become a soluble anti inflammatory signal for microglia (Cardona et al. 2006). In fact, microglia are reported to be the only CNS cell type that expresses fractalkine receptors, and fractalkine-knockout mice have enhanced neuro inflammation and elevated DA neuro toxicity in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo (Re and Przedborski 2006). In the present study, DE elevated fractalkine expression only in the midbrain in vivo. In vitro, fractalkine inhibited DE-induced H 2 O 2 production from microglia and protected against DE-induced DA neuro toxicity in midbrain neuron-glia cultures. Recent reports indicated that fractalkine (Duan et al. 2008) and fractalkine receptors (Wynne et al. 2010) decrease in the aged brain, which further supports the premise that aging may be critical for air-pollution-induced neuro inflammation to cause neuro toxicity in vivo. In addition, lower and seemingly benign concentrations of DEP in vitro shifted microglia to a primed phenotype, resulting in a more pronounced pro inflammatory response and amplified neuro toxicity with additional stimuli ( Figure 5). Thus, even when not immediately toxic, air pollution may amplify ongoing neuro inflammation and associated neuron damage. Further studies are needed to discern the role of aging, fractalkine, and priming in the deleterious effects of air pollution.
Conclusion
Here we show that DE caused microglial activation and up-regulation of oxidative stress, pattern recognition receptors, neuro toxic cytokines, and chemokines in the rat brain. The midbrain expressed the highest levels of the IBA-1 microglial marker in control animals and produced the greatest response to DE, suggesting regional vulnerability. DEP activated microglia in vitro without increasing cytokine or chemokine production, but IT administration of DEP elevated both serum and brain TNFα levels in vivo, suggesting a key role for systemic inflammation. Indeed, our findings suggest that DEP may inter act with ongoing inflammation to amplify the pro inflammatory response (i.e., priming), which may be neuro toxic. This may be particularly relevant for individuals with active systemic inflammation or neurodegenerative disease. Although we did not detect significant loss of DA neurons in vivo in the models tested here, results suggest that fractalkine, which was elevated after DE exposure in vivo, may be responsible for adaptation by inhibiting DE-induced DA neurotoxicity, at least temporarily. Together, these findings reveal complex, inter acting mechanisms responsible for how air pollution may cause neuro inflammation and DA neurotoxicity [see Supplemental Material, Figure 1 (doi:10.1289/ehp.1002986)] and may be particularly relevant to the etiology of PD. | v3-fos-license |
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} | pes2o/s2orc | Allosteric Inhibition of Human Porphobilinogen Synthase*
Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.
the biosynthesis of the tetrapyrroles such as heme, low activity mutations in the human population are associated with the disease ALAD porphyria (2); all eight porphyria-associated PBGS mutations increase the propensity of the human protein to exist in the low activity hexameric assembly, thus establishing the physiologic relevance of the quaternary structure equilibrium to human health (3). In addition to ALAD porphyria, PBGS inhibition by divalent lead is a primary consequence of lead poisoning. Factors that stabilize the hexamer will further inhibit PBGS activity and, thus, potentiate the physiologic effects of lead poisoning.
The arrangement of the subunits in the PBGS hexamer creates a surface cavity that is not present in the octamer or the dimers (see Fig. 1b) (4). Ligand binding to this cavity is posited to stabilize the hexamer, draw the quaternary structure equilibrium toward the hexamer, and thus, inhibit function. The residues that comprise this surface cavity are phylogenetically variable. We have shown that small molecules that bind selectively to a plant PBGS hexamer, presumably by binding in this surface cavity, can act as species-specific PBGS inhibitors whose mechanism of action is stabilization of a low activity oligomer (4). Herein we apply these principles to the inhibition of human PBGS with the understanding that hexamer stabilizing molecules can potentiate diseases related to low PBGS activity. The approach used is computational docking of commercially available molecules to a hexameric crystal structure of the low activity human PBGS variant F12L (PDB code 1PV8 (5)). From the docking results we select a family of dissimilar structures for in vitro validation and discover three small molecules that inhibit human PBGS by stabilizing the low activity oligomer. Herein we use the term "morphlock" to describe a small molecule that stabilizes a specific functionally distinct alternate quaternary structure assembly, in this case the low activity hexamer.
Physiologically significant morphlocks can derive from environmental contaminants, natural products, or drugs in clinical use. A drug that functions as a morphlock toward an off-target protein could provide an unprecedented structural explanation for the side effects of the drug. One discovered morphlock, which we have called ML-3H2, is chemically similar to two amebicidal drugs currently in clinical use, clioquinol and iodoquinol. We address the ability of these drugs to stabilize the human PBGS hexamer and draw a putative correlation between these results and the neuropathic side effects of both drugs (6).
Most porphyric diseases are episodic, and physiologic mechanisms contributing to porphyric attacks are not fully understood. Morphlocks discovered to stabilize the wild type human PBGS hexamer are predicted to have increased potency against the naturally occurring ALAD porphyria-associated variants; we tested and confirmed this hypothesis with two such variants, E89K and A274T. The long range utility of this study is the identification of chemical structures that stabilize the human PBGS hexamer and that may potentiate diseases related to diminished PBGS activity.
EXPERIMENTAL PROCEDURES
Materials-The programs MACROMODEL, GLIDE (Version 3.5), LIGPREP, and QIKPROP and the graphical user interface MAESTRO were from Schrödinger, L.L.C. (New York, NY). The candidate inhibitors were from Life Chemicals, Inc. (Burlington, ON, Canada). All other chemicals were from Fisher or Sigma and were of the highest purity available. Electrophoresis equipment and reagents and chromatography equipment and resins were from GE Healthcare.
In Silico Library of Compounds-Two-dimensional representations of the Life Chemicals kinase-targeted and G-protein-coupled receptor-targeted libraries of compounds (69,593 compounds) were provided in SD format by the vendor. The structures were converted to MAESTRO format, and entries that contained metal ions or atom types other than carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur and halogens were discarded. Hydrogens were added as appropriate for the structures, generating a single uncharged stereoisomer per compound. The structures were energy-minimized using MACROMODEL with the MMFF force field (7) and then expanded to include all forms likely to be present in the pH range 5-9 using the LIGPREP utility, resulting in a total of ϳ111,000 structures. The structures were again energy-minimized using MACROMODEL, which yielded output files that were suitable for docking with GLIDE.
In Silico Docking-Docking was performed using the Fox Chase Cancer Center computer cluster running GLIDE (32 licenses) (8). The docking target was a hexamer-specific surface cavity of the human PBGS crystal structure of the F12L variant (PDB code 1PV8 (5)) shown in Fig. 1b. The F12L hexamer comprises three identical asymmetric dimers, and the hexamerspecific surface cavity is located at the interface of three subunits. Thus, the docking boxes contained components from three subunits (see Fig. 1c). A cube (25 Å dimension) was used to define a region where the entire molecule must fit to be scored by GLIDE. Within this box a smaller cube (14 Å dimension) was defined to restrict the location of the center of the docked compounds. The asymmetry of the crystallographic dimer produces binding sites in the hexamer that are identical in sequence but structurally distinct. In the crystal structure of the F12L variant the subunits are labeled alphabetically as A-F. One of the inhibitor binding sites is formed by subunits A, B, FIGURE 1. The structure and behavior of the alternate quaternary structure assemblies of PBGS. a, the dynamic equilibria between the quaternary structure assemblies of human PBGS are shown. The two predominant forms in solution are the high activity octamer (PDB code 1E51) and the low activity hexamer (PDB code 1PV8); interconversion between these forms requires dissociation to a dimer, which can take on alternate conformations. The interchange between dimer conformations involves alterations in a small number of / angles. b, shown is a space-filling model of the human PBGS hexamer (PDB code 1PV8) highlighting the hexamer-specific cavity formed by chains A, B, and C, illustrated in dark blue, light blue, and gray, respectively. The remaining three subunits are shown in yellow. Small molecule binding to this cavity is proposed to stabilize the hexamer and shift the equilibrium toward this low activity form, inhibiting human PBGS activity. c, the small molecule docking boxes are illustrated at the interface of chains A, B, and C (colored as in b). The entire docked molecule must be contained in the 25-Å cube (purple); the center of the molecule must be contained in the 14 Å cube (green). d, shown is the pH activity profile of the wild type human PBGS (taken from Ref. 1). The basic portion of the curve reflects changes in the quaternary structure equilibrium, favoring hexamer with increasing pH (1). The K m value of 0.2 mM shown at pH 7 represents the K m of the octamer (at pH 7), which predominates at neutral pH. The K m value of 0.02 mM shown at pH 9 represents the K m of the octamer (at pH 9), whereas the K m value of 4.5 mM shown at pH 9 represents the K m of the hexamer. and C (ABC), and the other is formed by subunits B, A, and F (BAF). Three of the six binding sites in the hexamer are equivalent to the ABC site, and three are equivalent to the BAF site. Each compound library was docked both to the ABC site and the BAF site. The positions of the docking boxes at the ABC interface are shown (see Fig. 1c). All compounds were docked in Standard Precision mode, and the docked pose of each compound was given a score based on a proprietary modification of the CHEMSCORE program. Standard Precision docking in GLIDE mainly evaluates goodness of fit based on geometries. The highest ranking compounds (the best 10%) in the Standard Precision mode were reevaluated in Extra Precision mode, where polarity and hydrophobicity are taken into account. Again, compounds with the highest ranking 10% of scores in this round were considered for purchase based on several additional criteria as follows. We asked if the docked molecule made van der Waals contacts or hydrogen bonds with all three subunits and had a predicted solubility (log S) estimate of at least Ϫ6 (calculated using QIKPROP (9)). To limit cost and yield a manageable number of compounds for in vitro analysis, the purchased set was further limited to molecules of dissimilar structure that docked at various locations within the relatively large docking box.
Compound Solutions-Compounds purchased from Life Chemicals were dissolved in dimethyl sulfoxide (DMSO) to yield a concentration of 10 mM. Solutions were stored in darkness at room temperature.
Native Gel Electrophoresis-Electrophoresis was performed using a PhastSystem with PhastGel native buffer strips. Either 6-lane (4 l per lane) or 8-lane (1 l per lane) sample applicators were used to load the samples. Separations were performed using 12.5% polyacrylamide gels. After separation, gels were developed on the PhastSystem using Coomassie Blue or activity stain as previously described (4,11). For the gel shift screen of putative morphlocks with human PBGS, samples were prepared by mixing 8 l of protein (0.3 mg/ml, 8.3 M subunits) in 0.1 M Bis-Tris propane-HCl, pH 8.0, 10 mM -mercaptoethanol, and 10 M ZnCl 2 with 2 l of 10 mM compound in DMSO. The resultant samples, which contained 20% DMSO and 2 mM compound, were incubated at 37°C in for 30 min, 6 h, and 24 h before loading and running the gels. Compounds that substantially stabilize the human PBG hexamer in 6 h were then incubated under the screening conditions for 0, 2, 4, and 6 h. These compounds were also evaluated for dose-response relationships, where human PBGS (0.3 mg/ml) was incubated under the screening conditions for 6 h, but with varied compound concentrations as indicated. In all cases the final concentration of DMSO was maintained at 20%. Hexamer stabilization was also evaluated as a function of pH, where the samples were incubated as described for the screening conditions for 6 h but at varied pH as indicated. For the native PAGE experiments containing the PBGS substrate 5-aminolevulinic acid (ALA), the gel samples were prepared as for the dose-response evaluations, and the ALA and compounds were both present for the dura-tion of the incubation. Where indicated, gels were stained for PBGS activity as previously described (4).
Quantification of PAGE results by densitometry was carried out using the program ImageJ (12). Three separate determinations were made to quantify the density of each gel band. The quantified data for each gel lane is presented as % hexamer, which was defined as the amount of protein present in the hexamer band relative to the total protein in the hexamer and octamer bands. For the dose response by gel shift evaluations, the data were fitted either to a simple hyperbolic binding equation ( where % Hexamer max is derived from the fit as the highest fraction of hexamer, [I] is the concentration of the inhibitor, K 0.5 is the concentration of inhibitor at the midpoint of the curve, % Hexamer o is the fraction of hexamer in the starting sample, and n is the Hill coefficient. Activity Assays-The activity of human PBGS was assayed by monitoring the production of the product, porphobilinogen. The concentration of porphobilinogen was determined based on the absorbance at 555 nm (⑀ 555 ϭ 60,200 M Ϫ1 cm Ϫ1 ) of its complex with -dimethylamino-benzaldehyde that is formed upon treatment with modified Ehrlich's reagent. The assay volume was 1 ml, and all assays were performed at 37°C. The assay buffer was 0.1 M Bis-Tris propane-HCl, pH 8.0, 10 mM -mercaptoethanol, and 10 M ZnCl 2 . Reactions were initiated by the addition of 100 l of 0.1 M ALA-HCl to 900 l of assay buffer containing PBGS and allowed to proceed for 5 min before the addition of 500 l of STOP reagent (20% trichloroacetic acid, 0.1 M HgCl 2 ). The stopped reactions were vortexed and centrifuged, the supernatants were treated with modified Ehrlich's reagent, and the absorbance at 555 nm was measured as described previously (13).
Inhibition-Inhibition was assessed using the activity assay after incubation of 90 l of enzyme in the appropriate assay buffer with 10 l of compound solution (stock 10 mM in DMSO) or DMSO alone at 37°C for specific time periods. After this preincubation, 800 l of the appropriate assay buffer was added, and the mixture was allowed to equilibrate at 37°C for 15 min before the addition of 100 l of 0.1 M ALA-HCl. The compound concentrations reported in the text are those in the final 1-ml assay volume. Inhibition data were plotted as fractional activity relative to the absence of compound, and inhibition curves were fitted to a hyperbolic function, Equation 4. DECEMBER 18, 2009 • VOLUME 284 • NUMBER 51
Hexamer Stabilizing Allosteric Inhibition of Human PBGS
where FA is the fractional activity, FA max is the fractional activity in the absence of compound (set as 1), [I] is the concentration of compound, and IC 50 is the [I] at 50% inhibition. All kinetic data were fit using the program SigmaPlot (Systat Software, Inc., San Jose, CA).
Chemical Characterization of Hexamer-stabilizing Compounds-
The purity and identity of ML-3A9, ML-3F2, and ML-3H2, as purchased from Life Chemicals, were assessed using a Waters liquid chromatography-mass spectrometry system and 1 H NMR as detailed in supplemental Table 1.
Identification of the ML-3H2 Analogs in Clinical Use-The chemical structures of both ML-3A9 (1,6- Fig. 2b) were used as entries in the search engine CAS Scifinder. Two drugs currently in clinical use, clioquinol (5-chloro-8-hydroxy-7-iodoquinoline) and iodoquinol (5,7-diiodo-8-hydroxyquinoline), were found to be chemically similar to ML-3H2. These compounds were purchased for analysis and evaluated using methods identical to the compounds from Life Chemicals.
RESULTS
Docking and Compound Selection-Computational docking was used as a prescreening method on large libraries of "druglike" compounds available from Life Chemicals. The prescreening allows selection of compounds for in vitro analysis that have an increased probability of binding to the target relative to a random selection of compounds. However, one cannot know whether the optimally docked stereoisomer is actually contained in the commercially available compound, as stereochemistry is often not specified.
The structure of the hexameric F12L variant of human PBGS (PDB code 1PV8 (5)) was used as the target for in silico docking (see Fig. 1, b and c). Docking of the ϳ111,000 structures derived from the Life Chemicals "G-protein-coupled receptor-targeted" and "kinase-targeted" libraries using GLIDE in SP mode resulted in a distribution of scores such that 10% of the compounds had good scores, 80% scored similarly to each other but worse than the top 10%, and 10% scored badly. Docking of the top 10% of compounds from the SP screen using GLIDE in XP mode gave a similar distribution of scores. The compounds with the top 10% of GLIDE XP mode scores (ϳ1100 structures) were further processed to select diverse docking positions and diverse structures. This resulted in the selection of ϳ100 compounds for purchase and in vitro evaluation as stabilizers of the human wild type PBGS hexamer. Only a portion of those selected, 77 compounds, were readily available from the vendor. Supplemental Table 2 includes the structures of the compounds that were purchased and evaluated.
Native Gel Mobility Shift Screening of Purchased Compounds-The docking process described above was designed to provide a selection of compounds likely to perturb the quaternary structure equilibrium of human PBGS toward the hexameric assembly. Such a perturbation can be evaluated using a native PAGE mobility shift assay, as this method separates the octameric, hexameric, and dimeric assemblies (4,13). Coomassie staining has proven the most reliable means of visualizing gel shift results; however, the sensitivity of this staining technique requires protein concentrations (0.3-1 mg/ml) that are higher than what would be found physiologically in erythrocytes (1-5 g/ml) or that are used for in vitro kinetic assays (5-10 g/ml). If the desired mechanism of inhibition is operative, high concentrations of protein will disfavor hexamer formation and increase the apparent K 0.5 of hexamer-stabilizing compounds apparent from the gel shift assay relative to an IC 50 based on activity assays. Thus, compound concentrations used in the gel shift assay are higher than those that might be used for a screen based upon inhibition of PBGS activity.
For human PBGS, the gel shift assay involved incubation of 2 mM compound with 0.3 mg/ml enzyme (8.3 M subunit) at 37°C for a fixed period of time and subsequent separation of the assemblies by 12.5% native PAGE. No significant change in PBGS oligomer distribution was observed for any of the compounds when using a 2-h incubation at pH 7. Although the high activity octameric assembly dominates the wild type human PBGS quaternary structure equilibrium at neutral pH, at higher pH values the hexamer is more highly favored (1,10). Thus, the gel shift screen was repeated at pH 8 where the intrinsic population of the wild type hexamer is measurable, and the probability of trapping this species is, thus, increased. Screening at pH 8 was carried out using incubation times of 30 min, 6 h, and 24 h. Minimal effects were observed at 30 min, and significant conversion to hexamer in the presence of solvent DMSO alone was observed at 24 h. Consequently, the 6 h gel shift assay was used as a first criterion for selection of hexamer-stabilizing compounds. Under these conditions we identified 12 compounds that promoted some degree of hexamer formation, most of which were at a level of 5 -30% conversion to hexamer (see Supplemental Fig. 1). However, three compounds, herein called ML-3A9, ML-3F2, and ML-3H2, significantly shifted the wild type human PBGS quaternary structure equilibrium 90 -100% toward the hexamer and were selected for further characterization. 1 H NMR and liquid chromatography-mass spectrometry analyses of the three compounds confirmed the chemical identity and purity of ML-3A9 and ML-3H2 (as detailed in supplemental Table 1); however, liquid chromatography-mass spectrometry of ML-3F2 revealed multiple peaks suggesting an impure sample, and 1 H NMR data were not fully consistent with the reported identity of this compound. Further analyses of the hexamer-stabilizing compounds, therefore, focused on ML-3A9 and ML-3H2. The structures of ML-3A9 and ML-3H2 and their docked poses in the putative hexamerspecific small-molecule binding site are shown in Fig. 2.
Native PAGE Analyses of the Effects of ML-3A9 and ML-3H2 on the Quaternary Structure Equilibrium of PBGS-To determine the time required for these small molecules to perturb the quaternary structure equilibrium of wild type human PBGS toward the hexamer in the gel shift assay, samples were incubated with 2 mM compound for 0, 2, 4, and 6 h at pH 8.0 as illustrated and quantified in Fig. 3a. We observe that 2 h is insufficient to reach complete conversion to hexamer; however, 4 -6 h is sufficient. Thus, we used a 6-h incubation time for further evaluation of the compound-PBGS interactions.
A dose-response analysis was carried out at pH 8.0 and a PBGS concentration of 0.3 mg/ml for ML-3A9 and ML-3H2 using the native PAGE gel shift assay after a 6-h incubation time with compound concentrations of 30 M, 100 M, 300 M, 1 mM, and 2 mM (Fig. 3b). No bands indicative of species smaller than hexamer (e.g. dimer) were present. The dose-response gel shift results for ML-3A9 fit to a hyperbolic equation from which we estimate the inhibitor concentration at which the protein is 50% hexamer (K 0.5 ) to be 140 Ϯ 10 M. Interestingly, the conversion of octamers to hexamers induced by ML-3H2 fits better to the Hill equation, from which we extract a K 0.5 of 120 Ϯ 10 M and a Hill coefficient of 1.7 Ϯ 0.1. These K 0.5 values are significantly higher than we expect to see in an enzyme inhibition assay, which is carried out at significantly lower protein concentration, as observed previously (4).
We have previously established that the hexameric assembly of wild type human PBGS is rare at pH 7 but becomes a substantial part of the equilibrium ensemble at higher pH and provides a structural basis for reduced activity at basic pH (see Fig. 1d) (1). This predicts that the apparent K 0.5 as analyzed by the gel shift dose-response analysis will also be pH-dependent. The dose response by gel shift evaluation was, therefore, repeated at pH 7.35 and 9.05 for both compounds (Fig. 3c). At pH 7.35, the dose response of ML-3A9 was hyperbolic, yielding a K 0.5 value of 260 Ϯ 50 M, whereas the dose response of ML-3H2 again fit better to the Hill equation with a K 0.5 of 200 Ϯ 30 M and a Hill coefficient of 2.5 Ϯ 0.7. At pH 9.05, the analysis became more complicated, as the protein is ϳ50% hexamer in the absence of compound. As with the lower pH data, the dose response of ML-3A9 was hyperbolic, yielding a K 0.5 value of 770 Ϯ 450 M, whereas the dose response of ML-3H2 fit to the Hill equation with a K 0.5 of 100 Ϯ 10 M and a Hill coefficient of 3.7 Ϯ 1.7. Note that the highest concentration of ML-3H2 shown on the gel was not included in the fitting analysis, as this condition induced formation of higher order assemblies of unknown structure. Although all of the data are generally well described by the fit equations, the error relative to the absolute value of the derived parameters is large. As such, the reported K 0.5 values should be considered estimates. A more clear indication of the pH-dependent phenomenon derives from the apparent end point of each of the fits included in Figs. 3, b and c. At the pH values 7.35, 8.0, and 9.05, these endpoints with ML-3A9 are, respectively, 71 Ϯ 4, 83 Ϯ 2, and 100 Ϯ 10% hexamer for ML-3A9. For ML-3H2, these respective values are 61 Ϯ 4, 95 Ϯ 2, and 97 Ϯ 2% hexamer. In general these data indicate that trapping the hexameric assembly is more facile at the high pH values, as was predicted by the higher equilibrium concentration of hexamer at basic pH in the absence of compound. We have also varied the pH of the incubation while holding each compound at its apparent K 0.5 at pH 8.0 and observed the expected increase in the amount of hexamer present as a function of pH (not shown).
We note that prior studies consistently demonstrate that incubation of PBGS (from many sources) with the substrate ALA serves to stabilize the octameric assembly (4,13). Substrate addition promotes a closed conformation of the active site lid, which in turn stabilizes a subunit interaction that is found in the octamer and not the hexamer. Thus, the opposing effects of the substrate on hexamer stabilization by ML-3A9 and ML-3H2 were examined by native PAGE. The dose-response experiment at pH 8.0 (shown in Fig. 3b) was repeated with the inclusion of 1 mM ALA in the samples. As shown in Fig. 3d, the octamer-stabilizing effect of ALA impairs the hexamerstabilizing ability of both compounds, although the magnitude of this effect differs. In the presence of ALA, the titration curves for both compounds fit poorly to a hyperbolic function, and the data for both were fit to the Hill equation (see Fig. 3d). For ML-3A9, the fit yielded a K 0.5 of 190 Ϯ 10 M and a Hill coefficient of 2.7 Ϯ 0.2. The data for ML-3H2 (for which only three of the gel lanes presented detectable hexamer) yielded a K 0.5 of 1150 Ϯ 20 M and a Hill coefficient of 2.2 Ϯ 0.1.
Hexamer Stabilization of Human PBGS Porphyria-associated Variants by ML-3A9 and ML-3H2-We propose that chemical entities that stabilize the hexameric assembly of human PBGS can potentiate the symptoms of ALAD porphyria. To further test this hypothesis, we evaluated ML-3A9 and ML-3H2 with two porphyria-associated human PBGS variants, E89K and A274T. Although the wild type protein purifies as Ͼ95% octamer, the porphyria-associated variants, E89K and A274T, are associated with an increased percentage of hexamer (3). This suggests that, relative to wild type, these variants accumulate more easily in the inactive hexameric form and would be more sensitive to hexamer stabilizing inhibition. The percentages of octamer in the E89K and A274T starting samples used in this study were 54 and 78%, respectively. Dose-response evaluations for ML-3A9 and ML-3H2 (Fig. 4, a and b) were set up under conditions identical to the wild type pH 8.0 gels in Fig. 3b. Consistent with our hypothesis, the K 0.5 values for both compounds are lower for the variants relative to the wild type. The dose-response curve for ML-3A9 fit to a hyperbolic equation for both variants, yielding K 0.5 values of 90 Ϯ 20 and 80 Ϯ 20 M for E89K and A274T, respectively. As we had observed for the wild type protein, the dose response for ML-3H2 fit to the Hill equation for both variants, yielding for E89K a K 0.5 of 70 Ϯ 10 M and Hill coefficient of 3.1 Ϯ 0.7 and for A274T a K 0.5 of 80 Ϯ 10 M and Hill coefficient of 3.1 Ϯ 0.1.
Kinetic Evaluation of ML-3A9 and ML-3H2 as Inhibitors of
Human PBGS-Stabilization of the low activity hexamer as demonstrated by the gel shift analyses is predicted to correlate with inhibition of human PBGS. Accordingly, ML-3A9 and ML-3H2 were evaluated as inhibitors of wild type human PBGS activity at a final protein concentration of 10 g/ml and a preincubation time of 6 h. The dose-response curves in Fig. 5 are fitted to a simple hyperbolic decay and correspond to IC 50 values of 58 Ϯ 6 and 10 Ϯ 1 M for ML-3A9 and ML-3H2, respectively. Based on time course and pH studies of the gel shift phenomenon as shown in Fig. 3, the apparent IC 50 values are predicted to be lower than observed if a longer protein-inhibitor preincubation time or a higher pH for the preincubation and assay had been used. Correspondingly, a shorter preincubation time and/or low incubation or assay pH would be expected to result in a larger apparent IC 50 value. Thus, these IC 50 values are a relative measure of how well each of the active compounds draws the human PBGS equilibrium toward the hexamer under assay conditions. As it is established that substrate serves to stabilize the octameric assembly ( Fig. 3d and Refs. 4 and 13), the quaternary structure equilibrium within the assay mixture is a result of competing forces to stabilize the inactive hexamer by the small molecule inhibitors and to stabilize the octamer by substrate.
Our ability to inhibit PBGS activity by trapping the inactive hexameric assembly reinforces our prior deduction that the hexameric assembly of human PBGS is a native assembly state in dynamic equilibrium with the octameric assembly (1). The behavior of the hexamertrapping inhibitors, as shown by the pH dependence in native PAGE gel mobility analyses, further validates our hypothesis that the pH dependence of the oligomeric equilibrium is responsible for diminished activity of human PBGS at basic pH (Fig. 1d).
Evidence That the Discovered Morphlocks Bind Specifically to PBGS Hexamers-To establish that the inhibitors promote hexamer formation by binding preferentially to and stabilizing the hexamer, samples treated with both inhibitors were analyzed by native PAGE gels stained for PBGS activity as detailed in supplemental Fig. 2. We have previously demonstrated that the hexameric component of PBGS (in the absence of hexamer-stabilizing inhibitors) will stain for PBGS activity because the substrate-mediated conversion to the active octamer occurs within the gel matrix (4,11). The absence of PBGS activity in the hexamer bands in the samples treated with ML-3A9 and ML-3H2 demonstrates that these compounds bind specifically to the hexamer, remain bound during the electrophoresis, and inhibit the in-gel substrate-mediated hexamer to octamer conversion.
Native PAGE Analyses of the Effects of Clioquinol and Iodoquinol on the Quaternary Structure Equilibrium of PBGS-
The amebicidal clioquinol (Fig. 6a) and iodoquinol (Fig. 6b) are structural analogs of ML-3H2 and were evaluated to determine whether human PBGS hexamer stabilization is an off-target effect of these drugs. Dose-response analyses for both of these compounds versus wild type human PBGS and the porphyriaassociated variants E89K and A274T were carried out at pH 8.0 (Fig. 6c) These data support the hypothesis that inhibition of human PBGS is an off-target side effect of these drugs and may contribute to clinically observed side effects.
Comparative Analyses of Compounds and Docking Sites in Human Versus Pea PBGS-The same set of ϳ111,000 structures that were docked against the human PBGS crystal structure were previously docked against the analogous region of a pea PBGS homology model. From this we discovered a hexamerstabilizing compound that shifted the quaternary structure equi- DECEMBER 18, 2009 • VOLUME 284 • NUMBER 51 librium of pea PBGS, which we called morphlock-1 and whose chemical identity is 2-oxo-1,2-dihydro-benzo(cd)indole-6sulfonic acid[2-hydroxy-2-(4-nitro-phenyl)-ethyl]-amide (4). It is important to note that only 3% of the top 100 compounds ranked by the GLIDE scoring function are common between docking of these ϳ111,000 structures to the pea versus the human PBGS (4). Furthermore, neither ML-3A9 nor ML-3H2 was ranked by the GLIDE scoring function in the top 1000 compounds docked to the pea PBGS, and morphlock-1 was not ranked in the top 1000 compounds docked to human PBGS. Herein we provide a comparison among the docked poses of the structures of ML-3A9, ML-3H2, and morphlock-1, which are all structurally dissimilar. There is some spatial overlap between the docked poses of ML-3H2 and morphlock-1, whereas neither of these compounds overlaps the docked pose of ML-3A9 (Fig. 7, a and b). A sequence alignment of residues within the docking box used for pea and human PBGS shows that there is significant sequence variation (Fig. 7c), particularly with regard to residues that make contact with each of the compounds as docked, thus providing a rationale for why each compound is effective only for PBGS from the targeted species. For a related application, we have considered sequence variations between human PBGS and that of human pathogens within the residues that compose the morphlock docking box. This variation predicts species-selective inhibition of heme biosynthesis by hexamer trapping inhibitors as an approach to development of antimicrobial agents (4).
Hexamer Stabilizing Allosteric Inhibition of Human PBGS
Evaluation of in Silico Docking as a Library Refinement Tool-In silico methods are becoming increasingly utilized in the identification of protein ligands such as drugs. The percentage of lead compounds identified from a library that is prescreened by in silico docking is estimated to increase some 10-fold compared with a high throughput only screen (14). In the case of this study, in silico docking was used as part of the process used to select a reasonable number of compounds for purchase and in vitro analysis from a library of 110,000 structures derived from nearly 70,000 commercially available compounds. The goal of the docking was to enrich the compounds selected for purchase with molecules likely to inhibit PBGS by the hypothesized hexamer-stabilizing mechanism. In vitro gel shift evaluation of 77 purchased compounds identified 12 as hits. Thus, in silico docking appears to provide an efficient means to target oligomer-specific sites in alternate quaternary structure assemblies of proteins.
A question that arises is this: Are we more likely to find hits among compounds selected from the docking than we would from a set of compounds purchased randomly? Although we do not have a random set of compounds, we have a set of 76 compounds that had previously been purchased as potential hexamer-stabilizing inhibitors of pea PBGS in a screen of the same commercial library to the analogous hexamer-specific surface cavity. The phylogenetic variation in the amino acids that make up this surface cavity is substantial when comparing human PBGS to pea PBGS (see Fig. 7c and Ref. 4). We evaluated the ability of the compounds selected to stabilize the pea PBGS hexamer for their ability to stabilize the human hexamer using the 6-h preincubation protocol described above. With one exception, compounds selected for pea PBGS hexamer stabilization did not perturb the human PBGS quaternary structure equilibrium toward the hexamer greater than a few percent (not shown). The single exception revealed that one of the compounds purchased for in vitro testing with pea PBGS was chemically identical to a compound also purchased for in vitro testing with human PBGS. This compound (coincidentally, the impure ML-3F2) was cataloged by the vendor under two different catalogue numbers with a difference of 1 dalton in molecular mass. The results of this important control suggest that we are, indeed, more likely to find compounds that stabilize the human PBGS hexamer among the compounds selected by docking to the human PBGS hexamer than to the pea PBGS hexamer.
Human PBGS Quaternary Structure Equilibrium and
Human Health-Human PBGS exists in an equilibrium of alternate quaternary structure assemblies, as illustrated in Fig. 1a (1, 3, 5, 10, 13). The physiologic relevance of this equilibrium is established by a correlation between human PBGS FIGURE 7. Comparison of the predicted binding sites for ML-3A9, ML-3H2, and morphlock-1. a, a ribbon diagram of the A, B, and C subunits of hexameric human PBGS (PDB code 1PV8) is shown in gray, and the subunits are labeled A, B, and C, respectively. A purple box indicates the search region that was used for docking. The molecule positions and orientations are illustrated as predicted by docking and are indicated by stick representations of ML-3A9 (orange), ML-3H2 (green), and morphlock-1 (magenta). Morphlock-1 was positioned by overlapping the hexameric assemblies of pea PBGS on human PBGS and transferring the docked location and orientation of morphlock-1. b, a close-up view of a in the same orientation is provided; in this case the ribbons are colored according to subunit (A in blue, B in light blue, and C in gray). c, a sequence alignment of human PBGS and pea PBGS includes only those residues within the targeted docking boxes. The criterion for a "contact" between an atom of the morphlock and an atom of the protein is a ratio of the distance between the two atoms divided by the sum of the van der Waals radii of the two atoms where that ratio does not exceed 1.30. The sequence alignment is colored (as shown in the key) to indicate contacts between the morphlock and its targeted PBGS and sequence conservation between human and pea PBGS for residues that are involved in side-chain contacts.
variants and the disease ALAD porphyria (3). All ALAD porphyria-associated variants shift the distribution of PBGS quaternary structural forms toward the lower activity hexamer, providing a structural basis for why PBGS activity is reduced in patients carrying these alleles. Herein we provide evidence that small molecules can stabilize the low activity hexameric assembly of human PBGS using both wild type protein and ALAD porphyria-associated variants. We posit that such small molecules will inhibit PBGS activity and potentiate disease states associated with diminished PBGS activity. Although ALAD porphyria is rare, diminished PBGS activity also results from the much more common condition of lead poisoning. In both disease states symptoms are related to the toxic effects of increased concentrations of the PBGS substrate ALA, which accumulates as a result of the lowered PBGS activity.
The structural similarity between ALA and the neurotransmitter 4-aminobutyric acid is related to the neurologic sequelae of porphyrias and lead poisoning. The fact that patients experience debilitating symptoms only sporadically throughout life is one poorly understood aspect of porphyria. It is possible that exposure to compounds like the morphlocks discovered herein could contribute to the sporadic nature of the disease. It is also possible that established drugs that act as morphlocks toward human PBGS could raise metabolic ALA levels and cause neurologic side effects. Thus, it is intriguing that the discovered morphlock ML-3H2 has a history in drug development (15). ML-3H2 was synthesized in the 1950s in an effort to improve the effectiveness and solubility of the halogenated quinolinols, which are a principal class of amebicidal agents. An iodide at position 7 of an existing amebicidal agent was replaced with a diethylaminomethyl group to produce ML-3H2. This modification did not produce a more potent amebicidal agent, and the original iodinated compound and a diiodinated derivative remain in use under the names clioquinol and iodoquinol (Fig. 6).
Most intriguing is that side effects associated with clioquinol and iodoquinol include deleterious effects on the nervous system (6). Our demonstration that both drugs do indeed stabilize the low activity hexameric assembly of human PBGS, as depicted in Fig. 6c, supports a relationship between the side effects of these drugs and inhibition of human PBGS. Both compounds induced dose-dependent hexamer formation and were more potent against the porphyria-associated variants than wild type human PBGS. The concentration of clioquinol in the plasma of patients using topical preparations of the drug has been measured at 1-2 M (16), which is substantially lower than the experimentally determined K 0.5 values derived from our native PAGE assay (40 -100 M). However, the concentration of PBGS used in the gel shift assays (0.3 mg/ml) is also much higher than the 1-5 g/ml that can be estimated for erythrocytes (17,18), where PBGS expression is enhanced over the normal housekeeping levels (19). We have demonstrated that increased protein concentration causes an inflated apparent K 0.5 for inhibitors that function by stabilizing PBGS hexamers (4). If it is determined that clioquinol is able to induce PBGS hexamer formation (and concomitant inhibition of PBGS activity) in vivo, this would constitute an unprecedented explanation for a drug side effect. It is possible that other known drugs, toxins, or environmental contaminants could have a similar hexamer-stabilizing inhibitory effect on human PBGS.
Significant Differences between Plant and Human PBGS-
The current study follows the discovery of hexamer trapping agents for plant PBGS, but the required time for in vitro hexamer stabilization was found to be significantly longer for human PBGS. This difference stems from a phylogenetic variation in the use of an allosteric magnesium ion, used in plant PBGS, which binds at a subunit interface that is present in the octamer but not present in the hexamer (5). Magnesium binding to this site stabilizes the octamer and increases activity by increasing the mole fraction of octamer in the quaternary structure equilibrium. In contrast, human PBGS does not contain the amino acids that compose the allosteric magnesium binding site. Instead, there is a guanidinium group of an arginine residue that is spatially equivalent to the magnesium of plant PBGS and that we have shown acts to stabilize the human PBGS octamer (3,10). Presumably because the allosteric magnesium can dissociate (K d ϳ 2 mM) and the guanidinium group cannot, the interchange of quaternary structure assemblies of human PBGS is much less facile than for plant PBGS. We have shown that human PBGS assemblies are metastable and interconvert only under specific circumstances, with relatively long halftimes on the order of 1-3 h (10, 13). The slow interchange of quaternary structure assemblies for human PBGS dictated considerably longer preincubation times for the in vitro analysis of putative morphlocks relative to pea PBGS.
The Morpheein Model of Allosteric Regulation-In the current understanding of protein dynamics, proteins are viewed as existing in an ensemble of conformations (20 -26). Within this context, quaternary structure dynamics as a fundamental basis for allosteric control of function is an extension of what we already understand about how protein motion is related to function. Although the behavior of PBGS was initially unexpected, it is now well established that the octamer can come apart, the dissociated form can change conformation, and the altered conformation can reassociate to a functionally distinct hexamer (or vise versa) (1,4,5,10,13). In the case of PBGS this equilibrium forms the basis for allosteric regulation, and thus, we introduced the morpheein model of allostery (27,28) whose kinetic characteristics are distinct from the classic models of allostery (29 -31). Regardless of the model put forward, the common theme in allostery is the binding of a ligand at one location of a protein (e.g. not the active site) affecting the behavior of the protein at another location (e.g. the active site). For plant and many microbial PBGS, the magnesium ligand that binds at an octamer-stabilizing site distinct from the active site, defines these proteins as allosteric. For human PBGS, the current study, which shows for the first time that small molecules can bind at a hexamer-stabilizing site distinct from the active site, defines human PBGS as allosteric.
Although PBGS is the first protein that has been unequivocally established to exist as a dynamic, fully reversible equilibrium of oligomers that can come apart, change shape, and reassemble differently, there are other proteins whose published characteristics are consistent with using the morpheein mechanism of allostery. We have suggested morpheein as a general term to describe homo-oligomeric proteins that can dissociate, change conformation, and reassociate into structurally and functionally distinct assemblies (4,27,28). The implications of identifying a protein as a morpheein are significant. Small molecule stabilization of alternate oligomers of proteins that exist in an equilibrium of quaternary structure assemblies (morpheein forms) provides a largely untapped resource for discovery of new drugs (4) and for understanding off-target side effects. A morpheein-trapping mechanism is distinct from a mechanism that simply interferes with the process of assembly. In the case of PBGS and perhaps also for other morpheeins, the targeted oligomer-stabilizing surface cavity has a large volume and structurally complex protein components, allowing ligands to bind in a variety of locations. Thus, unlike for an enzyme active site, one can consider diverse structures as ligands. We have previously suggested the proteins human immunodeficiency virus integrase, tumor necrosis factor ␣, and mammalian ribonucleotide reductase as morpheein drug targets (4); for each of these proteins there is evidence of functionally distinct alternate quaternary structure assemblies (32)(33)(34). The results presented herein suggest that morpheeins be considered as a possible explanation for off-target side effects of drugs in clinical use. | v3-fos-license |
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} | pes2o/s2orc | Alkali Promotion in the Formation of CH4 from CO2 and Renewably Produced H2 over Supported Ni Catalysts
In Power‐to‐Methane (PtM) plants, the renewable electricity supply can be stabilized by using green electrons to produce H2 via H2O electrolysis, which is subsequently used to hydrogenate CO2 into CH4. In this work PtM is studied in a cascade fashion, from simulated solar light to methane production in an all‐in‐one setup, which was newly developed for this work. This setup was used to assess the effects of H2 stream purity on the activity of Ni/SiO2 catalysts in CO2 methanation. An activity effect in downstream methanation is shown to be onset by aerosols that evolve from the electrochemical splitting of water. Small amounts of K are shown to affect CH4 production positively, but only if they are deposited in situ, via KOH aerosols. K‐doped Ni/SiO2 catalysts prepared in an ex situ manner, by impregnation with a KOH solution, showed a decrease in activity, while the same amount of KOH was deposited. Operando FT‐IR spectroscopy reveals that increased back‐donation to CO‐containing intermediates and carbonates formation likely causes catalyst deactivation in ex situ samples as often reported in literature for Ni/SiO2 catalysts. The mechanism for in situ promotion is either an increased rate in the hydrogenation of CHx (X=0–3) fragments, or a more facile water formation or desorption as CO‐containing reaction intermediates are unaffected by in situ promotion. These results are relevant to PtM from a fundamental standpoint explaining the effect of potassium on nickel methanation, but also from a practical standpoint as the presented effect of in situ promotion is difficult to achieve via standard synthesis methods.
Introduction
The cascade synthesis of CH 4 from solar light (i. e., Power-to-Methane, PtM) is an attractive concept that will allow us to decrease CO 2 emissions, simultaneously demodulating the mismatch in renewable electricity demand and supply and stabilizing the electricity grid. [1,2] In the PtM concept first renewably produced electrons are used to split H 2 O into H 2 and O 2 . While this renewable H 2 can, in theory, be used as an energy buffer, storing H 2 is approximately an order of magnitude more costly than storing CH 4 , and thus particularly for long-term (seasonal) storage of electricity, is not necessarily costefficient. [3] Hence the conversion of this H 2 into CH 4 (also called e-gas), which can be stored safely in large quantities through infrastructure that already exists, becomes interesting from an economic standpoint. [3][4][5][6] Furthermore, methanation is already a crucial element of any engineering solution involving on-site H 2 production through low-temperature catalytic reforming of organic substrates (e. g. alcohols, formic acid). [7] To the best of our knowledge, at the time of writing, the Audi e-gas plant in Wertle, Germany is the largest commercial methanation plant worldwide and produces methane starting from CO 2 captured from close by produced biogas via amine scrubbing, and from H 2 generated by three 2 MW KOH-based alkaline electrolyzers powered by renewable energy. In this plant, the hydrogen produced from the electrolysis is filtered to eliminate KOH aerosols, and subsequently dehydrated and compressed to 10 bar to be stored in a buffer tank worth 1 h of methanator operation, decoupling the operation between the electrolyzer and the methanator units. The produced methane is dried and fed into the natural gas grid. Water is cycled back in the electrolyzers, while oxygen produced at the anode of the electrolyzer is vented out. The methanation reactor is cooled by molten salts and the heat is used to regenerate the amine scrubber, bringing the process efficiency from 54 % (without heat recovery) to 72 %. [3] Despite the high efficiency, the process is not yet profitable, mainly due to electricity costs and fees, showcasing how cheap electricity, but also policies can play a big role for the commercialization of PtM technologies. As described for the operating PtM plant above, several important steps occur for the application of this PtM process, which are illustrated separately in Figure 1A. Solar light is first converted into electrons via photovoltaics (step 1). Then, these green electrons are converted into H 2 via electrolysis of H 2 O (step 2, both the Hydrogen Evolution Reaction (HER) and Oxygen Evolution Reaction (OER) have to be considered here). [8,9] CO 2 streams are captured and purified (step 3), and can then be converted into CH 4 via renewably made H 2 (step 4 in Figure 1). This CH 4 is then stored and distributed (within a closed-cycle process, step 5). CO 2 reduction to methane may be regarded as an established process as French chemist Paul Sabatier (1845-1941) discovered the reaction over 100 years ago. [10,11] Nevertheless, there are novel questions that arise from the application of this process within the concept of electrification of the chemical industry described above. An obvious question is that of the effect of sequential process steps on one another. To study such questions in detail, a setup was built bringing together all elements of this process in a single, newly constructed experimental setup, combining simulated solar light with an electrolyzer to split water, and a methanation unit. Photographs of this setup can be seen in Figure S1.
The overall process efficiency of the PtM process is highlighted in Figure 1B,C. Figure 1B highlights the theoretical values of power storage (56 %) and subsequent power generation (36 %), as calculated for an optimized production plant. In Figure 1C we highlight the all-in-one setup, developed in our laboratory, that will be discussed in this work and is shown in Figure S1. The yields of each step in our (non-optimized) labscale setup are shown in Figure 1C. Obviously, the setup that we will discuss in this work is far from what an optimized industrial plant would be, mainly due to electrode size constraints and inefficient heating of the methanation reactor, but also because the kinetic conditions applied (i. e. low CO 2 conversion) were purposely chosen for the final catalytic methanation step to study activity trends. The applied setup has an overall efficiency of 0.018 %. As a comparison, the theoretical maximum efficiency for photosynthesis is 11 %. However, most plants yield an efficiency of approximately 0.1-3 % as plants do not absorb all incoming radiation. [12] Although the comparison between our setup and natural photosynthesis is not entirely fair, as plants convert CO 2 generally into much more useful or valuable, complex carbohydrates, it illustrates the efficiency of nature in a process that has been optimized over hundreds of millions of years.
In this work we examine the effect of hydrogen production (step 2 in Figure 1A) on methane production (step 4 in Figure 1A). More specifically, it is interesting to consider the possible presence of aerosols in the H 2 feed, which may evolve from the HER in alkaline medium. This should be done, as NASA literature from 1974 already reports the possible presence of aerosols in the feed of electrolysis systems, [13] and industrially often aerosol filters are applied as mentioned above.
The water splitting reaction is performed in alkaline media, as the kinetically limiting step in the electrolysis of water -the OER -benefits from a high pH. [14] Hence the aerosols that may be present in the gas feed from the split water may contain alkaline material. Literature shows both promoting, and deactivating effects for the addition of alkaline dopants to thermocatalytic methanation reactions, [15][16][17][18][19][20][21][22][23] and relevant reaction steps thereof, such as the Reverse Water-Gas Shift (RWGS) reaction. Table S1 lists some of this relevant literature, and their normalized promotional or deactivating effect on the turnover frequency of CO and CO 2 methanation. In general, alkali promotors (most commonly potassium) are added to methanation catalysts either to poison support acidity, or to catalyze coke removal via hydrogen or steam reactions. [15] Xu et al. show a "volcano-shape-curve" between the Mg/Al molar ratio and catalytic activity, explained by an optimal surface basicity induced by the Mg. [19] The activity and selectivity of K promoted nickel catalysts seem to mainly depend on the support. [20] On Ni/Al 2 O 3 a promotional effect is found, whereas on Ni/SiO 2 , an exponential decrease in total activity with increased promotor concentration is seen which is claimed not to be due to a decrease in the dispersion. [20] Campbell et al. also note an increase in activity for Al 2 O 3 -supported Ni, but a decrease in activity for SiO 2 supported Ni. [23] While Huang et al. find a promotional effect for Ni/SiO 2 . [15] Thus, fundamentally understanding the nature of this potential promotional effect is of direct practical and academic relevance.
In this work we will study the effect of K promotion on Ni/ SiO 2 catalysts of different mean nickel particle sizes, as well as different forms of addition of K to the Ni/SiO 2 catalysts via operando FT-IR spectroscopy, and the use of XRD, STEM-EDX and ICP-AES. To the best of our knowledge, no systematic study has been performed to distinguish the possible particle-size dependent effect of alkali promotion, while it seems a relevant variable in Table S1, and no study has considered the effect of in situ deposition of potassium onto a Ni-based methanation catalyst as a potential promotor. Table 1 lists an overview of the fresh, or "parent" Ni/SiO 2 catalyst samples that are studied in this work.
Catalyst materials
The Supporting information (e. g. Table S2), and previous literature [24] gives more information on the characterization of these materials. The effect of in situ doping with KOH-containing aerosols which evolve from hydrogen production on the catalyst samples listed in Table 1 was compared to an ex situ doping procedure where a 0.6 wt % K loading was achieved by impregnation, via suspension of the catalyst materials in a KOH solution and subsequent evaporation.
Hydrogen production via electrolysis and cascade methanation reaction
Pt mesh electrodes (MaTecK, 99.9 %, 25 × 50 mm, 3600 mesh/cm 2 , 0.04 mm wire diameter) interwoven with a Pt wire (MaTecK, 99.9 %, 1 mm diameter, 100 mm length) were suspended in a 1 M KOH electrolyte (ACS reagent, > 85 % pure,~15 % water) loaded into a homemade H-cell. This cell consists of GL14 glass tubes with an internal volume of 20 mL for the electrolyte on each side, resulting in 40 mL electrolyte in total. The HER and OER reactions are spatially separated by a Nafion 117 perfluorinated membrane (Aldrich, 0.007 in. thick). Prior to use the membrane is activated by immersion in nitric acid for several hours, while the Pt electrodes were cleaned by using a butane flame. The electrolyte was purged with 2 mL/min Ar, 0.1 mL/min Kr on the HER side and 2 mL/min N 2 , 0.1 mL/min Kr on the OER side for at least 2 h prior to the experiments.
The Ni/SiO 2 catalysts (3 mg, sieve fraction 75-150 μm) were loaded in a plug-flow reactor (borosilicate capillary, 1 mm diameter) and reduced at 550°C for 60 min, with a heating rate of 5°C/min under a flow of 1 mL/min of pure H 2 . Afterwards, the catalysts were cooled down to 400°C (5°C/min) under a flow of pure N 2 . This flow was maintained for 3 h to remove residual H 2 from the system. Electrochemistry (Ivium CompactStat) was then started potentiometrically at a current of 4 mA, requiring ca. 1.69 V potential (the Nafion membrane yields a high resistance in the system), 1.69 V also being the output of a small solar cell module of three cells in series. See also Figure S3 for chronopotentiometry of the water splitting with two Pt electrodes, and the GC response to the application of current. The flow of Ar/Kr and N 2 /Kr was constantly maintained as a carrier gas for the produced H 2 and O 2 . The feed from the HER was led over the plug flow reactor containing the Ni/ SiO 2 catalyst, optionally filtered by an aerosol filter.
After 15 min of flow from the H-cell an 800 ppm CO 2 feed was opened over the Plug Flow Reactor (PFR) at a rate of 1 mL/min and was mixed with the HER feed prior to injection on the PFR. The reactor output gas was again filtered by an aerosol filter before being injected on an InterScience CompactGC every 4 min and analyzed by FID. The flow was split over two column systems optimized for measure hydrocarbons on one end and CO/CO 2 /CH 4 on the other. The latter was equipped with a methanizer to visualize each component. On a separate channel the flow from the oxygen halve of the system was injected and measured by a TCD every 4 min. This state was maintained for several hours, up to 2 or 3 days depending on the experiment.
Aerosol measurement and formation
To measure the aerosol formation from the electrochemical cell, a Water-based Condensation Particle Counter (WCPC, TSI model 3785) was attached to the output of the cell, in place of the methanation reactor. This is shown in Figure S2. In a typical experiment, a 2 mL/min flow of N 2 was bubbled through the electrochemical cell filled with 1 M KOH, and the outlet stream was diluted with 1 L/min filtered air before being fed to the particle counter. The effect of flow, applied potential and KOH concentration was also studied, see Figure S4. Control experiments carried out bypassing the cell or using water instead of 1 M KOH didn't show any aerosol formation.
Operando FT-IR spectroscopy with on-line gas chromatography
Operando FT-IR spectroscopy was performed using a Bruker Tensor 37 FT-IR spectrometer and OPUS software with DTGS detector. [24] Spectra were recorded every 30 s. Gases were introduced with Brooks mass flow controllers. Self-supported catalyst wafers for operando transmission FT-IR spectroscopy were prepared using roughly 4 tons of pressure. The catalyst wafers (15 mg in weight) were loaded into a Specac high-temperature transmission FT-IR reaction cell, and subsequent in situ reduction was performed with 1 : 1 H 2 :N 2 flow of 20 mL/min each for 1 h at 550°C. Temperature increased at a rate of 5°C/min and atmospheric pressure was held throughout. The cell was cooled to 100°C before CO 2 hydrogenation commenced, after which temperature was increased at 5°C/min to 400°C where it was held for an hour. Gas rates used in CO 2 hydrogenation without water were 6.25 mL/min N 2 , 5.00 mL/ min H 2 , and 1.25 mL/min CO 2 . For experiments with water in the gas feed, water was fed through a stainless steel saturator, by bubbling inert gas. Milli-Q water was fed at 21°C, which at 6.00 mL/ min N 2 ensured 2.8 % (or 0.36 mL/min) was added to a feed of, 5.00 mL/min H 2 , and 1.25 mL/min CO 2 . Operando FT-IR experiments were performed at atmospheric pressure, with online gas chromatography. Global Analyst Solutions CompactGC 4.0 and Thermo Scientific Dionex Chromeleon 7 software were used for online gas chromatography, with FID and TCD detectors. Product and transients continued to be captured for 15 min after CO 2 and 5 min water saturator was closed until H 2 was closed, all while at 400°C.
KOH aerosol semi-quantitative characterization
The Ni/SiO 2 catalysts tested for up to 3 days in the methanation reaction using the H 2 produced by the HER showed a content of 0.6 � 0.2 wt.% K by ICP-AES analysis. Assuming that all K from the gas feed gets adsorbed on the catalyst, this would give a value of 4-11 μg/L KOH (4 mg of sample, 2 mL/min gas carrier flow).
Comparable to the KOH content reported by NASA (3-5 μg/L). [13] Considering pure KOH density (2.12 g/mL) we can calculate the volume (in mL) of KOH per mL:~2-5 × 10 À 9 . Which, divided by the observed number of particles (about 30 per mL in the flow analyzed in the counter, which is diluted 500 times with N 2 , corresponding to 15000 per mL in the starting 2 mL flow), gives a volume per particle of~0.3-0.6 × 10 À 12 mL, i. e.~0.3-0.6 μm 3 . Assuming a spherical shape, this corresponds to 0.4-0.5 μm radius,~1 μm diameter. [25] Setup efficiency calculation Table 2, or Figure 1A for an overview.
Results and Discussion
We have studied a set of SiO 2 supported nickel catalysts, labelled from 1-6 in accordance with increasing particle size (see also Table 1). Previous literature [24] lists full details on the catalyst samples, which were characterized by several techniques such as H 2 chemisorption, N 2 physisorption, HAADF-STEM, temperature programmed reduction, X-ray absorption spectroscopy. A benchmark 1 M KOH alkaline solution is used for the production of renewable H 2 via H 2 O electrolysis, [14,26] and we focus on its effect on the catalytic hydrogenation of CO 2 into CH 4 over the mentioned catalyst samples. H 2 O splitting to H 2 and O 2 was performed with two Pt electrodes and done in a stable manner at 1.69 V at a current of 4 mA. O 2 production was used to control the water splitting efficiency, and was stable at a Faradaic efficiency of 99.6 % ( Figure S3). By use of a H 2 O-based condensation particle counter, which is shown in Figure S2, the presence of aerosols could be detected in the HER-produced H 2 feed. The concentration of aerosols formed is shown in Figure 2A. Figure 2B shows that the hydrogen produced by electrolysis has an effect on the activity of the sample shown (catalyst sample 5 in Table 1). More specifically, the H 2 produced via electrolysis, seemed to have a promoting effect on the activity of the Ni/ SiO 2 catalyst in CO 2 methanation at 400°C and ambient pressure, as shown in Figure 2B, when compared to clean hydrogen from the bottle under the same reaction conditions. In Figure 3, the effect of the H 2 from HER on different Ni/ SiO 2 catalyst samples with varying mean nickel particle sizes is shown, as tested in the all-in-one setup. The investigated Ni/ SiO 2 catalysts show stable methanation activity at 400°C, over a prolonged period of time when pure H 2 from a cylinder is used as a feedstock, but this changes when H 2 from the HER is used. The observed turnover frequency (TOF) values using "clean" hydrogen in this setup are comparable to previously shown results, which were measured in a different setup, an operando Table 2. Summary of energy input and output of each of the three steps with the energy efficiency per step as well as the total efficiency in the last column.
Step FT-IR spectroscopy setup. [24] Additional activity data is given in Figures S5-S8. Interestingly, when the reaction proceeds with H 2 produced via the HER, i. e. with alkaline aerosols, some trends are observed for catalysts with different mean nickel metal nanoparticle sizes. Over catalyst sample 5, which has 4.4 nm mean nickel particle size diameter supported on SiO 2 , a strong transient increase in methanation activity is observed, followed by a decay in activity over time, which nonetheless remains considerably higher than the values observed using pure H 2 (Figure 3 middle). On the other hand, over larger nickel metal nanoparticles (i. e., catalyst 6, 6.0 nm mean nickel particle size), no promotion is observed and the catalyst steadily deactivates over time (Figure 3, bottom). The smallest mean nickel metal nanoparticle size under study, catalyst 1, 1.2 nm Ni/SiO 2 , shows increased activity as well (Figure 3, top) but less than catalyst 5 sample. Figure 2A also shows that when a filter is placed before the methanation reactor, the aerosol can be effectively removed from the H 2 stream. In order to rule out a possible direct effect of the aerosol on the catalytic activity, e. g. by reaction with CO 2 in the gas-phase leading to carbonate formation (which is known to occur in mere seconds in the case of NaOH [27] ), an aerosol filter was installed on a by-pass between the electrochemical cell and the methanation unit (before the CO 2 feed), and the H 2 feed was switched from unfiltered to filtered during the reaction. Notably, the activity promotion on catalyst 5 (4.4 nm Ni/SiO 2 ) was maintained, which strongly suggests that the aerosol itself does not play an active role in the activity enhancement, which therefore will be solely due to K deposition on the catalyst. Strikingly, this also means that one can dose just the right amount of KOH to promote methanation activity and then remove the aerosol at will, to avoid further K accumulation, which will eventually be detrimental to the activity. In these experiments, TOF values are proportional to activity as the particle size distribution does not change significantly for the spent catalysts using filtered ("clean") H 2 , and unfiltered H 2 (containing aerosols), as shown by TEM analysis in Figures S8-S10. Quite obviously, impurities in the H 2 feed from HER have an effect on the downstream catalytic methanation reaction. As a result, 0.6 � 0.2 wt.% K was observed by ICP-AES analysis of the Ni/SiO 2 catalysts after 2 days of reaction when using HERproduced H 2 , while on the undoped sample, and the catalyst tested with pure H 2 K amount was below the detection limit. These values correspond to expected KOH concentrations with respect to aerosol particle sizes in accordance with literature (4-11 μg/L KOH, 1 μm diameter particles, see the experimental section). [25,27] Furthermore, it should be noted that the particle count is proportional to the gas flow through the electrocatalytic cell, while it did not detectably vary with the potential that is applied to carry out the HER ( Figure S3). Based on ICP-AES and BET results, one can hereby calculate an average K + concentration on the surface of the catalysts of 0.2 K + /nm 2 . Considering a K + weight loading of 0.6 % and the measured BET surface area of the Ni/SiO 2 samples (482.7 m 2 /g), one can calculate the average distribution of K + on the catalyst surface. About 6 mg K + ions are deposited per g of catalyst, which corresponds to roughly 12.4 μg/m 2 , 0.3 μmol/m 2 , 0.2 K + atoms/nm 2 f or catalysts 1, 5 and 6. Assuming hemispherical nickel metal nanoparticles of 1.2, 4.4 and 6.0 nm in diameter, this gives a nickel surface area of about 2.5, 30 and 57 nm 2 per particle, i. e., 0.5 (implying about 1 K atom every two particles, on average), and 6 and 12 K + atoms/particle, respectively. Considering 10 nickel atoms/nm 2 , this gives a K : Ni surface atom ratio of 1/50. Accordingly, an effect on nickel activity at such low K/Ni ratios is consistent with results in literature on Ni(100) surfaces, for which CO and CO 2 methanation were affected starting from well-below Monolayer (ML) coverage (0.05 ML). [28,29] To benchmark the promotional effects that were reported in the literature, and those we have observed via in situ doping, K-doped Ni/SiO 2 catalyst samples 1-6 were prepared by impregnation using a KOH solution, to achieve a loading of 0.6 wt % K (i. e. comparable to the amount of K + deposited from the aerosol stream after 2 days). These samples will henceforth be called "ex situ doped" differentiated from the samples that are doped in situ via aerosol deposition. To prepare and simultaneously study in situ doped samples, a pure H 2 stream was bubbled through a saturator filled either with a 1 M KOH aqueous solution or with DI water.
To gain further insight into the nature of the (particle size dependent) effect of K on CO 2 methanation over nickel via both of these preparation methods, operando FT-IR spectroscopy was carried out at 400°C for the sets of undoped (catalyst 1-6), ex situ doped samples (ex situ doped catalysts 1-6), and in situ doped (in situ catalysts 1-6). Catalyst sample 6 has very high nickel weight loading (60 %) which makes the self-supported catalyst wafers of this sample needed to study FT-IR prone to breakage. The ex-situ catalyst preparation procedure apparently increased the fragility of this sample to an extent where it could not be studied in this setup. Figure 4A shows the TOF trends as measured for the different samples. Ex situ doping has a clear deactivating effect, while in situ doping has a positive effect on the activity of Ni/ SiO 2 catalysts in the methanation of CO 2 at 400°C. Furthermore, it can be seen in Figure 4A and 4B that the initial doping effect of the aerosol is higher than when it reaches steady state. This was also observed in the all-in-one setup as described above, and shown in e. g. Figure 2. It is important to note that no clear particle size trend to the in situ promotion effects can be observed. While this appeared to be the case for the three particle sizes shown in Figure 3, the full set of catalyst samples tested here shows less of a distinctive trend. The general differences in promotion/deactivation trends, however, that are shown in Figure 4 are reflected in the FT-IR spectra, of which examples are shown in Figure 5. The supporting information shows the full set of FT-IR spectra in Figures S11 and S12. Figure 5 shows the region from 1400-2200 cm À 1 of the FT-IR spectra recorded during CO 2 hydrogenation at 400°C for undoped, in situ, and ex situ doped catalyst samples 1, 4 and 5. In this region the CO stretching vibrations and also formate type species can be found, another important spectral feature which may be linked to the deactivating effect of ex situ doped catalysts. [24,30] Notably, ex situ doped samples show a lower relative intensity at around 2020 cm À 1 , which ascribes to linearly adsorbed CO atop a single nickel atom [31][32][33][34] and was shown to be an important descriptor for catalyst activity in Ni/SiO 2 in recent work. [30] Deactivation is accompanied by the disappearance of the CO (ads,top) signal with maximum at 2020 cm À 1 . CO is an important intermediate in the formation of methane via the RWGS reaction. It is then subsequently converted to methane either via H-assisted CO dissociation or intermediate carbide hydrogenation. The binding strength, and coverage of CO is thus crucial in the formation of methane. [24,30] A trend in intensity of CO ads-top can be observed in Figures 5AÀ C going from in-situ promoted > undoped > ex-situ doped catalysts. For the in situ promoted samples, this shift is much more subtle (if noticeable at all), while the change in activity is much more significant. Figures S13-S15 further show that no real trends can be ascribed to the effect of in situ doping on peak positions of CO ads species. Notably, a higher relative intensity of a broad feature around 1750 cm À 1 , ascribed to carbonate species, is observed for the ex situ samples. The detectable presence of these species only in the ex-situ doped samples, and the fact that they are not hydrogenated to formate species, suggests that their formation could be due to K 2 CO 3 formation over K deposits when exposed to air. In any case we can say that electronic effect of K on ex-situ doped Ni/ SiO 2 seems to be more significant, which results in more backdonation to CO, thus effectively shifting the signal towards lower wavenumbers, which is in line with recent literature. [21,22] The deactivating effect of K on Ni/SiO 2 is also shown in work by Campbell et al., in which K was only shown to deactivate Ni/ SiO 2 , even in very small amounts (0.05 wt.%). [20,23] Yet, also here, the catalysts were preimpregnated with a K-containing solution, or prepared via co-impregnation. In fact, each of the catalyst samples where a deactivating effect was observed (Table S1), was treated with hydrogen at high temperature in the activation procedure of the catalyst. Also in our case, the ex situ doped samples were prepared in such a way that the K and nickel underwent a reduction procedure together. This reduction procedure in the presence of K has an obvious effect already on the particle size of some catalysts as could be seen Figure 5. A) The TOF of Ni/SiO 2 catalysts of different mean nickel particle size in different states of KOH promotion (unpromoted, in situ, and ex situ doping) against the position of the peak maximum in operando FT-IR. The dotted lines are drawn as eye-guides, showing that with in-situ promotion the TOF increases but there is no significant change in the CO top position, and with ex-situ promotion there is no significant increase in the TOF, but there is a significant change in the CO top peak position. BÀ D) Operando FT-IR of the activity measurements shown in Figure 4, showing the catalysts of different mean nickel particle sizes B) catalyst 1, C) catalyst 4, D) catalyst 5) in undoped state, and doped in situ and ex situ with KOH. These spectra were baseline-subtracted and normalized to the peak at 1853 cm À 1 .
in the TEM analysis presented in Figure S9. This could be explained by an electronic interaction of K with Ni, or by the decomposition of KOH with temperature to yield K 2 O and water at around 400°C, [35] which is known to affect Ni sintering. [36] Another explanation could be the formation of bicarbonates after exposure to atmospheric CO 2 . Their decomposition to K 2 CO 3 , CO 2 and water proceeds at low temperature (100-200°C). [35] Poisoning via these strongly adsorbed species could inhibit the formation of product. During in-situ promotion, the reaction temperature is higher than the melting and decomposition temperature of KOH, which could explain why carbonates are not formed on the in situ doped catalysts (besides the low K concentration on the surface, as previously stated).
The effect of K on the activity could also be dependent on the K + distribution, as shown by recent observations in the coking resistance in catalytic methane steam reforming (the inverse reaction of CO 2 methanation) over Ni/Al 2 O 3 catalysts. [37] Yet no observable agglomeration could be found when the catalysts were examined by SEM-EDX ( Figure S16). When examined by higher resolution STEM-EDX ( Figure 6), there was also no observable difference in the comparison between the distribution of K throughout ex situ doped samples and in situ doped samples, which can be expected for such low amounts of K.
As no signs of agglomeration were detected by STEM-EDX and the operando FT-IR suggests a difference in electronic structure between in situ and ex situ doped samples, two plausible possibilities of the promotional effect of aerosols over ex situ promotion remain; first as already previously mentioned, alkali promotors are added to industrial methanation catalysts to suppress the formation of carbon deposits. The suppression of coke formation itself is not very relevant to our work as no aromatic carbon vibrations (as aromatics are often considered as precursor molecules for coke deposit formation) could be observed in FT-IR for promoted or unpromoted reactions, and the promotional effect is larger initially than at steady state. The second possibility explaining the promotional effect of the in situ deposition of KOH via aerosols is a mechanism where the KOH promotes the removal of oxidizing species on the nickel surface via the formation of water. Both of these mechanisms require further investigation.
Conclusions
By combining operando FT-IR spectroscopy and a well-defined set of Ni/SiO 2 catalysts, we have shown both alkali promotion and poisoning of carbon dioxide hydrogenation over Ni/SiO 2 catalysts by KOH. In situ deposition of KOH-containing aerosols, realized by constructing an all-in-one setup capable of combining the H 2 O electrolysis and CO 2 methanation steps, shows downstream promotion effects in the CO 2 methanation reaction. In contrast, the ex situ promotion of the Ni/SiO 2 catalysts with the same amount of KOH shows a deactivating effect for all catalyst samples under study. This deactivation can be rationalized in terms of the electronic effect of K in combination with the Sabatier principle, as ex situ promoted samples likely bind the reaction intermediate CO more strongly than is required. The mechanism for in situ promotion is either an increased rate in the hydrogenation of CH x (X = 0-3) fragments, or more facile water formation or desorption as the COcontaining reaction intermediates in FT-IR spectroscopy are unaffected by in situ promotion.
Practically this information translates to the application of a method of doping which is similar to dosing a certain amount of K via a H 2 feed coming directly from H 2 O electrolysis in KOH electrolytes. In this way, the catalytic activity of nickel metal nanoparticles supported on SiO 2 in the Sabatier reaction can be efficiently enhanced. Although the mechanism as to what this can be attributed to requires further investigation, the work shows how a small amount of alkali promoters can dramatically change catalyst performance in CO 2 methanation . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Keywords: Power-to-methane · aerosol · alkali promotion · CO 2 hydrogenation · nickel | v3-fos-license |
2018-08-14T19:12:27.141Z | 2018-08-06T00:00:00.000 | 51930023 | {
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} | pes2o/s2orc | Hydrogels of Polycationic Acetohydrazone-Modified Phosphorus Dendrimers for Biomedical Applications: Gelation Studies and Nucleic Acid Loading
In this work, we report the assemblage of hydrogels from phosphorus dendrimers in the presence of biocompatible additives and the study of their interactions with nucleic acids. As precursors for hydrogels, phosphorus dendrimers of generations 1–3 based on the cyclotriphosphazene core and bearing ammonium or pyridinium acetohydrazones (Girard reagents) on the periphery have been synthesized. The gelation was done by the incubation of dendrimer solutions in water or phosphate-buffered saline in the presence of biocompatible additives (glucose, glycine or polyethylene glycol) to form physical gels. Physical properties of gels have been shown to depend on the gelation conditions. Transmission electron microscopy revealed structural units and well-developed network structures of the hydrogels. The hydrogels were shown to bind nucleic acids efficiently. In summary, hydrogels of phosphorus dendrimers represent a useful tool for biomedical applications.
Introduction
Local drug delivery is a powerful approach to treat a wide number of diseases, including cancer, inflammation and so forth. Indeed, the targeted application of therapeutic permits to achieve locally high concentration of a drug, whereas avoiding potential systemic toxicity, drug-drug interactions, side effects and related complications [1]. Hydrogels are considered prospective drug carriers for local delivery. Indeed, hydrogels can be prepared by simple procedures, they possess high drug loading capacity; by optimizing the chemical structure of precursor molecules, hydrogels can be made non-toxic and biodegradable. Being abundantly hydrated, hydrogels can be easily applied to tissues and organs of choice [2][3][4].
Dendrimers-symmetric hyperbranched polymers-are considered prospective tools for nanomedicine [5]. In particular, they have been recently used as building blocks for hydrogels. Choosing the architecture of dendrimers, one can program the properties and structure of hydrogels, such as reversibility (physical or chemical gels [6]), self-assembly patterns [7] and so forth. Recently, one dendrimer-containing gel drug, Vivagel ® , has been approved by the US FDA for the use in condom lubricants to prevent the HIV infection [8,9]. It is subjected for clinical trials as a therapeutic to treat bacterial vaginosis as well [10,11]. This recent success encourages the development of new dendrimer-containing constructions and materials for innovative medicine.
Among the wide variety of dendrimer architectures, phosphorus dendrimers represent a unique object for nanomedicine [12]. They possess biological activity per se (such as anti-cancer [13,14], immunostimulatory [15] and anti-prion [16] as well as act as efficient carriers for plasmids [17] or therapeutic nucleic acids [18][19][20][21]. The ability of phosphorus dendrimers to be both structural block and functional element of a therapeutic construction can be fully realized in the design of dendrimer-based hydrogels. Such hydrogels can be a promising material for tissue engineering and drug delivery. However, till now, known examples of phosphorus dendrimers-based hydrogels [22] were mainly prepared using additives that could be cytotoxic when taken in high concentrations. Obviously, this approach needs optimization to produce novel biomaterials for medicine. Herein, we report the assemblage of hydrogels of phosphorus dendrimers formed in the presence of biocompatible precursors, study of their structural features and loading with a macromolecular cargo (model oligonucleotide).
Oligonucleotides were synthesized using the solid-phase phosphoramidite method in an ASM-800 automated synthesizer (Biosset, Novosibirsk, Russia) from commercially available phosphoramidites (Glen Research, Sterling, VA, USA) according to the protocols optimized for the given equipment and purified by 15% PAGE assay.
Synthesis of Girard T, P-Modified Phosphorus Dendrimers
All manipulations were carried out using standard dry argon-high vacuum technique. Organic solvents were dried and distilled prior to use. Aldehyde-terminated phosphorus dendrimers of generations 1-3 were obtained by growing from a cyclotriphosphazene core as described in [23]. Girard T or P-modified dendrimers were synthesized according to the procedure reported for the modification of dendrimers having thiophosphate as a core [22]. 1 H, 13 C{ 1 H} and 31 P{ 1 H} NMR spectra were recorded using AV400PAS, AV400LIQ spectrometers (Bruker, Karlsruhe, Germany). The attribution of the NMR signals of the dendrimer branches was made by analogy with Refs. [23,24], the attribution of the NMR signals of the amines on the periphery was made by analogy with Ref. [22]. To assign the 13 C NMR signals, Jmod, HMBC, HBQC NMR experiments were additionally done, if necessary. The atom numbering used for the signals attribution is given in Figure 1. Mass spectrometry was not used to prove the purity of these dendrimers because of spontaneous rearrangements in the phosphorhydrazone structure during the analysis [25], thus, the purity of the dendrimer samples was assessed only by NMR [26].
Grafting of Girard Reagents onto the Periphery of Dendrimers: General Procedure
To a solution of aldehyde-terminated dendrimer Gn (300 mg, 0.1 mmol (G1), 0.044 mmol (G2), 0.02 mmol (G3)) in 20 mL chloroform, a solution of Girard reagent T or P (1.05 eq. per aldehyde function, 1.26 mmol (G1), 1.11 mmol (G2), 1.01 mmol (G3)) in 10 mL methanol was added dropwise upon stirring. Several drops of acetic acid were added to reach the pH~5. Reaction mixture was stirred overnight at room temperature. The completeness of conversion was proven by the disappearance of free aldehyde signals in 1 H NMR (DMSO-d6) at 9.98 ppm. The reaction mixture was evaporated to dryness, the solid residue was washed with chloroform (25 mL), diethyl ether (2 × 25 mL) and dried. Girard T or P-modified dendrimers were obtained as pale-yellow powders in quantitative yield. Dendrimer PG2. Synthesis of this dendrimer has been described previously in [22]. 1
Preparation of Hydrogels
10 mg of a dendrimer TGn or PGn was dissolved in 500 µL of a dispersant upon vortexing and incubated in a 1.5 mL Eppendorf type plastic test tube at 65 • C. As a dispersant, the following were used: deionized water; PBS; 10% glucose (Glc) in deionized water or PBS; 10% glycine (Gly) in deionized water or PBS; 10% polyethylene glycol-4000 (PEG) in deionized water or PBS. The gelation was considered finished when the appearance of gels in test tubes ceased to change. Depending on the physical properties of a final gel, the gelation was considered complete (rigid, coherent gel), incomplete (gel and turbid supernatant) or partial (incoherent gel).
Transmission Electron Microscopy
Hydrogels were visualized in transmission electron microscopy (TEM) using two approaches: negative staining of native gels and ultrathin sections. Copper TEM grids covered with formvar film preliminary stabilized with carbon evaporation were used for the sample adsorption.
To prepare negatively stained samples, semifluid hydrogels were adsorbed on a TEM grid for 1 min. In the case of more rigid hydrogels, a piece of about 2 mm 3 was mixed with 2 µL of distilled water using a needle. Then, a grid was placed onto gel for 1 min, after that visible pieces of gel were gently removed by a needle. The grids with adsorbed samples of hydrogels were contrasted for 10 s on a drop of 2% phosphotungstic acid (pH 0.5). At least 5-6 grid-fields were examined for each sample in TEM.
To prepare ultrathin sections, hydrogels fixed in 4% paraformaldehyde were postfixed in 1% OsO 4 , routinely dehydrated in ethanol and acetone and embedded in epon-araldit mixture. Ultrathin sections were prepared on a Leica EM UC7 ultratome (Leica Microsystems, Wein, Austria) and routinely contrasted with uranyl acetate and led citrate. All grids were examined in JEM 1400 TEM (JEOL, Tokyo, Japan), digital images were collected with a Veleta camera (EMSIS, Muenster, Germany). At least 10 individual ultrathin sections of each sample were examined in TEM.
Fluorescence Intensity Measurements
Fluorescence intensity measurements of the samples containing 3 -fluorescein-labelled oligonucleotide 5 -ACCCTGAAGTTCCGGCAAGCTG-FAM-3 were carried out using CLARIOSTAR microplate reader (BMG Labtech, Ortenberg, Germany) in black 96-well half-area microplates (Costar, Thermo Fischer Scientific, Waltham, MA, USA). Steady-state fluorescence spectra of fluorescein were recorded upon excitation at 495 ± 4 nm in the range 500-600 nm. The fluorescence intensity values at the maximum (518 nm) were taken for the calculations.
Oligonucleotide Binding by Hydrogels
A piece of a hydrogel (1.0 mg) containing TGn or PGn prepared as described in Section 2.3 was rinsed with deionized water and then 40 µL of PBS was added. Fluorescein-labelled oligonucleotide 5 -ACCCTGAAGTTCCGGCAAGCTG-FAM-3 was added in portions (350 pmol in 5 µL PBS each), followed by the gentle stirring for 10 min at 25 • C. After each addition, fluorescence intensity of the supernatant was measured, as described above. The amount of free oligonucleotide in solution was calculated from the fluorescence intensity values at 518 nm using a calibration curve ( Figure S2, Supplementary Materials) obtained for the same quantities of the oligonucleotide as being added to a hydrogel (n(total)). The amount of bound oligonucleotide n(bound) was calculated as n(total)-n(non-bound) and plotted versus n(total). Binding profiles were fitted using a model derived from the Langmuir isotherm [27], fitting was considered satisfactory if R 2 > 0.95; saturation values were extracted from the fitting.
Oligonucleotide Release from Hydrogels
A piece of a hydrogel (1.0 mg) containing TGn or PGn prepared as described in Section 2.3 was rinsed with deionized water and then 40 µL of PBS was added. Fluorescein-labelled oligonucleotide 5 -ACCCTGAAGTTCCGGCAAGCTG-FAM-3 was added (2100 pmol in 30 µL PBS) followed by the gentle stirring for 10 min at 25 • C. Then fluorescence intensity of the supernatant was measured and the amount of bound oligonucleotide was quantified using a calibration curve as described in Section 2.6. The corresponding fluorescence intensity value was taken as a reference for the calculation of the amount of released oligonucleotide.
Oligonucleotide-containing hydrogel was rinsed with deionized water (3 × 50 µL). 50 µL of 10 mM phosphate buffer adjusted to the pH 4.5; 5.0; 5.5; 6.0; 6.5; 7.0 was added and the sample was incubated at 25 • C upon gentle stirring. Aliquots of the supernatant were taken at given time points within 48 h of incubation; their pH was adjusted to 7.0. The fluorescence intensity of samples was measured and the amount of released oligonucleotide was calculated.
Dendrimer Synthesis
Cyclotriphosphazene core-based phosphorus dendrimers bearing trimethylammonium acetohydrazone (Girard T reagent) or pyridinium acetohydrazone (Girard P reagent) moieties on the periphery were synthesized by grafting of corresponding hydrazides onto the surface of aldehyde-terminated dendrimers upon mild acid catalysis [22]. The presence of an aromatic fragment on in the vicinity to the hydrazone stabilizes the Schiff base formed. The functionalized dendrimers were obtained in quantitative yield as mixtures of inseparable isomers around the hydrazone fragment -CH=N-NH-C(O)-CH 2 -, as revealed by 1 H and 13 C NMR spectroscopy. The structures of these dendrimers are given in Figure 2.
Gelation Studies
Girard-modified dendrimers possess numerous =N-NH-C(O)-fragments on the periphery and thanks to that, they are able to self-assemble into hydrogels. The association of dendrimers into hydrogel occurs by the hydrogen bonding between hydrazone groups on the surface of different dendrimers' branches. To achieve that, dendrimer solutions were incubated at 65 • C that induces dendrimer hydration and intermolecular interactions. The hydrogels formed are thus classified as physical gels, suggesting that the gel network is potentially reversible.
The gelation is greatly facilitated by the use of hydrophilic additives that contribute to the hydrogen bonding network linking dendrimers together more efficiently. In particular, metal salts, Tris base, EDTA, ascorbic acid and so forth were reported to dramatically decrease the gelation time [22]. Herein, we used biocompatible additives, glucose, glycine and polyethylene glycol-4000, to assist the gelation. Gelation time and properties of the gels were found to depend on the dendrimer generation, structure of the acetohydrazone on the surface of dendrimer and on the nature of the additive. Table 1 summarizes the data on the properties of dendrimer hydrogels obtained. The formation of a rigid, coherent gel was considered to be complete gelation, otherwise incomplete (gel and turbid supernatant) or partial gelation (incoherent gel). In general, dendrimers PGn bearing pyridinium groups on the periphery appear to be better gelators than dendrimers TGn, that is in agreement with our previous findings [28]; glucose drives the faster formation of coherent, rigid gels. In the absence of additives, hydrogels were incoherent and in some cases, no gelation was observed within two weeks of incubation. It is worth noting that continuous incubation at relatively high temperature (65 • C) leads to the maturation of hydrogels resulting in their condensation and water loss [29]. Such gels are more rigid, the dendrimer content is higher than in others. Examples of dendrimer hydrogels are shown in Figure S1 (Supplementary Materials).
TEM Studies of Hydrogels
We tried to understand the three-dimensional structure of hydrogels by analysing two groups of transmission electron microscopy (TEM) data obtained by independent TEM methods (negative staining and ultrathin sectioning). We supposed that the TEM would allow to reveal the structure of hydrogels, to establish their structural units as well as to get the idea of the hydrogel alignment (porosity of gel network, alignment of gel fibres etc.). To the best of our knowledge, this is the first complete TEM characterization of dendrimer hydrogels: both structural elements and bulk morphology are shown.
The study of gel samples adsorbed onto the TEM grid followed by standard negative staining with phosphotungstic acid (see Section 2.4) permitted to observe common structural elements of dendrimer hydrogels. Two types of structural units have been found: spherical particles and fibres (Figure 3).
Both types consist of dendrimer molecules co-aggregated with additives. Spherical particles have high electron density after staining; their mean diameter is 8-10 nm. In the hydrogel network, they are associated in several layers being in close contact with each other. The fibres have low to moderate electron density, less associated; they act as cross-links between spherical particles. Spherical particles, their associates and fibres are co-assembled into a three-dimensional porous network. To understand the alignment of dendrimers into hydrogel networks better, hydrogel samples were fixed by the paraformaldehyde cross-linking. Such a treatment permits to preserve the structure of complex objects; thanks to that, it became a standard technique for the preparation of biological samples for the TEM study. The following treatment does not distort the structure of the samples (see Section 2.4); ultrathin sectioning permits to observe even small objects, including supramolecular associates. The analysis of the consecutive sections provides the idea of the three-dimensional structure of samples.
Examination of hydrogel ultrathin sections (~70 nm thickness) has shown that all the gels under study have homological network structure differing by the pore size ( Figure 4). The bigger were the electron-transparent regions in hydrogels (pores), the higher was the electron density of the network elements. The distribution of electron-transparent regions in samples correlates with the dendrimer content in hydrogels. Dendrimer generation contributes less in the density and morphology of gels.
Thus, the combination of two independent TEM methods (negative staining and ultrathin sections) permits to observe the structural elements of dendrimer hydrogels, visualize their alignment into a three-dimensional structure as well as qualitatively estimate differences in the morphology and structure of different hydrogels. The data obtained suggest that the gelation starts from the formation of dendrimer associates in solution further aggregating into developed networks composed of spherical particles and fibres. The TEM data are in good agreement with previously obtained SEM and cryo-TEM data [22]. Nevertheless, further physico-chemical and structural characterization of hydrogels would be needed to get more detailed information on the porosity of gel network, pore size, thickness and length of gel fibres, regularities of their alignment into a hydrogel and so on.
Oligonucleotide Binding and Release
Since Girard reagents have quaternized nitrogen atoms, dendrimer hydrogels have strong positive surface charge. This feature was used to load hydrogels with a model cargo. It would be interesting to test the strength of electrostatic forces for the drug loading, that is why an oligonucleotide, namely oligodeoxyribonucleotide 5 -ACCCTGAAGTTCCGGCAAGCTG-3 , has been used to study the hydrogel loading. 3 -Fluorescein-labeled oligonucleotide was added portionwise to hydrogels until the saturation is reached. At each step of the oligonucleotide addition, the amount of bound oligonucleotide was estimated from the residual fluorescence intensity.
In general, hydrogels bound oligonucleotides quite efficiently, with the efficiency correlating with the density of the hydrogel network. Figure 5 shows representative binding curves obtained for dendrimers TG3 and PG3. Binding profiles were fitted using Langmuir binding models, the saturation values (i.e., the highest amount of oligonucleotide that can be bound) were estimated from the fitting curves. The cargo loading capacity was 1.5-3 µmol/g depending on the type of dendrimer and additive. An important aspect of the design of drug-containing biomaterials is the rate of cargo release. Indeed, the rate and completeness of the release define potential applications of materials. Here, oligonucleotide-saturated hydrogels were incubated in 10 mM phosphate buffer at pH 4.5-7.0 to examine the release of the cargo at the pH occurring in different tissues. Oligonucleotide-containing aliquots were taken at given time points within 48 h of incubation and the percentage of released oligonucleotide was calculated from the fluorescence measurements. The representative release kinetics plots are given in Figure 6.
The rate of release does not vary much with the pH change, the amount of free oligonucleotide in solution increases during the first 16 h of incubation and then stabilizes. Nevertheless, the oligonucleotide release appeared to be pH-dependent, the release efficiency increases at pH < 6. Unfortunately, even at acidic pH, the overall amount of the released oligonucleotide does not exceed 10%. This is likely explained by multiple electrostatic interactions between oligonucleotide molecules and hydrogel surface. It should be noted that in real hydrogel-tissue interfaces, the rate and efficiency of cargo release can be different due to the presence of other biopolymers displacing oligonucleotide from the gel.
Conclusions
In this work, physical hydrogels of phosphorus dendrimers containing biocompatible additives have been obtained for the first time. The hydrogels have well-developed network structure, with elementary units of these networks being distinctly observed by TEM. Hydrogels efficiently bound oligonucleotides, however, their release is slow. This may occur due to numerous cationic groups exposed to the surface of hydrogels. The use of gels of less charged dendrimers may allow to control the cargo release rate better.
It is interesting to note that the gelation time and hydrogel properties depend on the nature of an additive, however, oligonucleotide binding and release are defined presumably by the dendrimer percentage in a final gel. It would be interesting to study the biological effects of hydrogels containing different additives on different cell lines. Mechanistically speaking, due to the high surface charge, dendrimer hydrogels could increase the cell adhesion [30] thus serving as matrices for directed cell growth and development.
In summary, hydrogels based on cationic acetohydrazone-functionalized phosphorus dendrimers hold considerable interest and prospects for nanomedicine as potential biomaterials for tissue engineering and drug delivery. Our preliminary data suggest that physico-chemical and mechanical properties of the hydrogels as well as their biocompatibility and interactions with cell cultures and tissues deserve further investigation. | v3-fos-license |
2016-05-21T08:49:11.215Z | 2016-05-19T00:00:00.000 | 16803965 | {
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} | pes2o/s2orc | Binding Analysis of Human Immunoglobulin G as a Zinc-Binding Protein
Human immunoglobulin G (IgG) binding with zinc ions was examined using zinc ions immobilized on chelating Sepharose beads (Zn-beads). Human IgG bound to Zn-beads but not to Sepharose beads (control beads). Mouse, rat, bovine and equine IgGs also bound to Zn-beads, similar to human IgG. The human IgG F(c) fragment showed zinc ion–binding activity whereas the Fab fragment did not. Ethylenediaminetetraacetic acid (EDTA)-treated Zn-beads no longer bound human IgG; however, washing the beads, followed by the addition of zinc ions, restored the binding activity towards human IgG. Zn-beads saturated with human fibrinogen could bind human IgG, and Zn-beads saturated with human IgG could bind fibrinogen. These results suggest that animal IgGs, including human, specifically bind zinc ions, probably through a zinc-binding site in the F(c) fragment and not in the Fab fragment. In addition, IgG and fibrinogen interact with each other and/or bind zinc ions through different mechanisms.
Introduction
It was recognized in 1869 that zinc is necessary for the growth of Aspergillus niger [1][2][3] and later its importance for the growth of plants and animals was documented [1], but it was not until 1961 that it was accepted that zinc deficiency could occur in humans [1][2][3]. Nutritional deficiency of zinc in humans occurs worldwide, particularly in areas where people eat cereal proteins containing a high concentration of organic phosphate compounds such as phytate, which hinder the absorption of zinc [1]. Zinc deficiency manifests as growth retardation, testicular and ovarian dysfunction, neurosensory disorders, immune dysfunction and cognitive impairment [1,2]. Zinc administration improves these syndromes and zinc acts as an antioxidant and anti-inflammatory agent [1][2][3][4].
Immune functions are very sensitive to zinc restriction [2]. Zinc is essential for T cell differentiation, suggesting that it affects the up-regulation of mRNAs of factors such as IFN-γ, IL-12 receptor β 2 and T-bet [5]. High concentrations of zinc inhibit the production of pro-inflammatory cytokines in monocytes/macrophages, resulting in the down-regulation of TNF-α, IL-1 and IL-6 [6]. Zinc relieves oxidative stress by acting as an inhibitor of NADPH oxidase and the co-factor of super oxide dismutase, and by inducing metallothionein production [1,2]. Furthermore, zinc supplementation augments the antitumor effect of tumor chemotherapy by enhancing p53 function [7]. Homeostasis of the intracellular zinc level is strictly regulated by the zinc transporter [8]. There are many zinc-binding proteins in human blood such as albumin, α 2 -macroglobuin, haptoglobulin, ceruloplasmin, immunoglobulins (IgG, IgM and IgA), complement C 4 , prealbumin, C-reactive protein, and fibrinogen [9][10][11][12][13]. Zinc-binding proteins may act as zinc storage compounds for maintaining immunoregulatory and oxidative balance [10]. IgG is believed to preferentially change conformation to allow for zinc transport through its zinc-binding ability and to distribute zinc ions in the cell [11].
A number of zinc ion binding proteins have been identified, and the cellular uptake of zinc ions, the effect of zinc ion uptake on cellular function, and the essential need of immune cells and enterocytes for zinc have been revealed. However, the binding mechanism of zinc ions by circulating zinc ion binding proteins remains unclear. This study presents a binding analysis of zinc ions with human IgG and speculates on the zinc-binding form of the protein in circulation.
Binding of Mammalian IgGs to Zn-Beads
Human IgG was incubated with zinc ion immobilized on chelating Sepharose beads (Zn-beads) or Sepharose beads (control beads: CB), and then the suspension was centrifuged. Human IgG was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in the supernatant of CB but not Zn-beads (Figure 1a): the CB supernatant showed two bands corresponding to the H (55 kDa) and L (23 kDa) subunits of human IgG comigrated. In the Zn-beads supernatant, the IgG H and L subunit bands were detected in the pelleted beads, indicating the binding of human IgG to zinc ions. On the other hand, natural antibodies such as anti-carbohydrate antibodies are found in normal human serum [14], and, as described below, when CB was used, some of the IgG proteins could be detected by the interaction with the carbohydrate chain in the CB rather than its precipitation by centrifugation due to insufficient washing. Mouse, rat, bovine and equine IgGs also showed zinc ion-binding activity ( Figure 2b). Animal IgGs, including human, were slightly detected in the pelleted CBs, probably due to insufficient washing of the beads and non-specific binding and/or carbohydrate binding of IgG to CB. The intensity of the Coomassie staining of IgG is species-dependent ( Figure 1b). For example, equine IgG H and L subunits were less stained as compared with the IgG from other mammals, but a part of the IgG molecule appears to recognize the carbohydrate chain immobilized on the beads. The presence of a band with a higher molecular weight than the H subunit band in IgG from each mammal seems to be an artifact. These results indicate that mammalian IgGs have similar zinc ion binding activities. Zn-beads and CB were treated with or without 50 mM EDTA (pH 7.4). After washing, PBS (980 µL) containing human IgG (25 µg) was added to aliquots of the treated and untreated Zn-beads and CB (net volume of beads per sample: 20 µL). Different aliquots of EDTA-treated CB and Zn-beads were washed and incubated with 1 mL 50 mM ZnSO4, rotated overnight, and then the ZnSO4-treated beads were washed and suspended at 50% (v/v) in PBS. Aliquots (1 mL) of IgG (25 µg) and ZnSO4-treated beads (Zn-beads or CB) in PBS were prepared (net volume of beads per sample: 20 µL each), and incubated at 4 °C overnight. As described in the "Experimental Section", the obtained bead sample was analyzed by SDS-PAGE; human IgG (IgG) was also applied to the gel (2 µg/lane), and the separated H and L subunits were denoted H and L, respectively. M represents marker proteins.
Binding Mechanism of Human IgG to Zn-Beads
The zinc-binding activity of human IgG to EDTA-treated Zn-beads was decreased compared to its binding to untreated beads ( Figure 2). The removal of EDTA and the addition of zinc ions to the beads restored the IgG-binding activity to the Zn-beads. EDTA also removed IgG bound to the Znbeads (data not shown). This study demonstrates that the binding of IgG to Zn-beads is zinc iondependent.
The binding of mammalian IgGs, including those of humans, to zinc ions is a common characteristic of IgGs, and therefore the binding of the Fab and F(c) fragments of human IgG to zinc ions was examined (Figure 3a,b). Human IgG Fab and F(c) fragments were detected by SDS-PAGE at Zn-beads and CB were treated with or without 50 mM EDTA (pH 7.4). After washing, PBS (980 µL) containing human IgG (25 µg) was added to aliquots of the treated and untreated Zn-beads and CB (net volume of beads per sample: 20 µL). Different aliquots of EDTA-treated CB and Zn-beads were washed and incubated with 1 mL 50 mM ZnSO 4 , rotated overnight, and then the ZnSO 4 -treated beads were washed and suspended at 50% (v/v) in PBS. Aliquots (1 mL) of IgG (25 µg) and ZnSO 4 -treated beads (Zn-beads or CB) in PBS were prepared (net volume of beads per sample: 20 µL each), and incubated at 4˝C overnight. As described in the "Experimental Section", the obtained bead sample was analyzed by SDS-PAGE; human IgG (IgG) was also applied to the gel (2 µg/lane), and the separated H and L subunits were denoted H and L, respectively. M represents marker proteins.
Binding Mechanism of Human IgG to Zn-Beads
The zinc-binding activity of human IgG to EDTA-treated Zn-beads was decreased compared to its binding to untreated beads ( Figure 2). The removal of EDTA and the addition of zinc ions to the beads restored the IgG-binding activity to the Zn-beads. EDTA also removed IgG bound to the Zn-beads (data not shown). This study demonstrates that the binding of IgG to Zn-beads is zinc ion-dependent.
The binding of mammalian IgGs, including those of humans, to zinc ions is a common characteristic of IgGs, and therefore the binding of the Fab and F(c) fragments of human IgG to zinc ions was examined (Figure 3a,b). Human IgG Fab and F(c) fragments were detected by SDS-PAGE at very low levels in pelleted CB; in contrast, the F(c) fragments were strongly detected in a sample of pelleted Zn-beads but the Fab fragment was not, suggesting that the F(c) fragment is likely involved in the binding between IgG and zinc ions. Human anti-ferritin autoantibodies (IgG, IgM, IgA) bind with zinc ions [12], indicating that all classes of immunoglobulins bind zinc ions. Further studies are required to clarify the binding between zinc ions and the F(c) fragments of other mammalian IgGs, and the binding of zinc ions with other classes of human immunoglobulins.
Binding of Human IgG and Fibrinogen to Zinc Ions
Zn-beads were saturated with human fibrinogen; after centrifugation, unbound fibrinogen was detected in the supernatant (data not shown). In addition, human IgG was added to a suspension containing Zn-beads saturated with fibrinogen and containing unbound fibrinogen. Human fibrinogen was separated by SDS-PAGE into three bands corresponding to the Aα, Bβ and γ subunits, with molecular masses of 67 kDa, 56 kDa and 50 kDa, respectively, as shown in purified sample (F). The IgG H band appeared to comigrate with the γ band of fibrinogen present in the supernatant (S) of CB and present in the pelleted Zn-beads (B), as shown in Figure 4a. Fibrinogen bands were detected in the supernatant of Zn-beads whereas the IgG L band was not. This study demonstrates that the addition of human IgG to a suspension containing Zn-beads saturated with fibrinogen and containing unbound fibrinogen results in human IgG binding to the Zn-beads, even though they are saturated with fibrinogen. Zn-beads saturated with IgG were used to examine whether fibrinogen binds to IgG-bound beads. A strongly stained IgG H subunit band made it impossible to identify the
Binding of Human IgG and Fibrinogen to Zinc Ions
Zn-beads were saturated with human fibrinogen; after centrifugation, unbound fibrinogen was detected in the supernatant (data not shown). In addition, human IgG was added to a suspension containing Zn-beads saturated with fibrinogen and containing unbound fibrinogen. Human fibrinogen was separated by SDS-PAGE into three bands corresponding to the Aα, Bβ and γ subunits, with molecular masses of 67 kDa, 56 kDa and 50 kDa, respectively, as shown in purified sample (F). The IgG H band appeared to comigrate with the γ band of fibrinogen present in the supernatant (S) of CB and present in the pelleted Zn-beads (B), as shown in Figure 4a. Fibrinogen bands were detected in the supernatant of Zn-beads whereas the IgG L band was not. This study demonstrates that the addition of human IgG to a suspension containing Zn-beads saturated with fibrinogen and containing unbound fibrinogen results in human IgG binding to the Zn-beads, even though they are saturated with fibrinogen. Zn-beads saturated with IgG were used to examine whether fibrinogen binds to IgG-bound beads. A strongly stained IgG H subunit band made it impossible to identify the Bβ and γ bands of human fibrinogen (Figure 4b) but the Aα band of fibrinogen was detected specifically in the supernatant (S) of CB and pelleted Zn-beads (B). IgG bands were also detected in the supernatant of Zn-beads but fibrinogen bands were not. The binding of human fibrinogen to zinc ions was previously studied using fibrinogen-immobilized Sepharose beads [13] and it was also shown that human fibrinogen binds to zinc ion-immobilized beads and is thus a zinc-binding protein. Interestingly, IgG and fibrinogen were detected bound to Zn-beads even after the addition to Zn-beads saturated with fibrinogen and IgG, respectively. In the present study, the addition of IgG and fibrinogen to beads was performed using a solution containing unbound proteins and the Zn-beads were pre-saturated with fibrinogen and IgG, respectively, because these bound proteins were removed from the beads by washing (data not shown). These results suggest that IgG molecules bind fibrinogen [15], and that these proteins bind zinc ions through two different mechanisms. Further study is needed to examine whether the binding activities of these proteins with zinc ions may change depending on the pH, solvent, chelator and spacer [16]. study is needed to examine whether the binding activities of these proteins with zinc ions may change depending on the pH, solvent, chelator and spacer [16]. Zinc ions affect thrombin absorption with fibrin and shorten blood clotting time [17]. Fibrinogen interacts with IgG and enhances IgG-mediated phagocytosis [15]. This study also demonstrated the interaction between IgG and fibrinogen. However, it remains unclear whether IgG and fibrinogen compete in the circulation to bind to zinc ions. Fibrinogen is an antioxidant and is susceptible to oxidative stress [18][19][20]. Oxidative modifications of fibrinogen cause structural changes, suggesting that the zinc-binding activity of fibrinogen changes according to oxidative conditions in the circulation [20]. Fibrinogen binds iron and copper ions which cause oxidative stress [13,[18][19][20]. Further study is required to clarify whether zinc ions compete with iron or copper ions for the zincbinding site on the surface of fibrinogen [1,2]. Binding of human IgG and fibrinogen to Zn-beads which were saturated with fibrinogen and IgG, respectively. After binding of human fibrinogen (600 µg) or IgG (500 µg) to Zn-beads or CB (net volume of beads per sample: 20 µL each), aliquots (20 µL) of human IgG (100 µg) or fibrinogen (100 µg) in PBS were added to protein-saturated Zn-beads and incubated with: fibrinogen in (a) and IgG in (b). As described in the "Experimental Section", the supernatant (S) and bead samples (B) obtained after centrifugation were subjected to SDS-PAGE. Human fibrinogen (F) and IgG samples were also applied (2 µg/lane). Their separated subunit bands derive from fibrinogen (Aα, Bβ and γ) and IgG (H and L). M represents marker proteins.
Zinc ions affect thrombin absorption with fibrin and shorten blood clotting time [17]. Fibrinogen interacts with IgG and enhances IgG-mediated phagocytosis [15]. This study also demonstrated the interaction between IgG and fibrinogen. However, it remains unclear whether IgG and fibrinogen compete in the circulation to bind to zinc ions. Fibrinogen is an antioxidant and is susceptible to oxidative stress [18][19][20]. Oxidative modifications of fibrinogen cause structural changes, suggesting that the zinc-binding activity of fibrinogen changes according to oxidative conditions in the circulation [20]. Fibrinogen binds iron and copper ions which cause oxidative stress [13,[18][19][20]. Further study is required to clarify whether zinc ions compete with iron or copper ions for the zinc-binding site on the surface of fibrinogen [1,2].
Zinc supplementation is effective in relieving oxidative stress and in decreasing the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-10 [1][2][3][4]6]. The clinical effects of zinc ions are very impressive and have a major impact on human health [1][2][3]21]. The present study suggests that IgG and fibrinogen interact with each other and/or bind zinc ions with different mechanisms. Zinc ions are taken up intracellularly by transporters but may also be indirectly taken up by receptors for zinc-binding proteins [22]. Further research is required to elucidate how important variables such as body condition, aging, species specificity and various zinc-binding proteins affect zinc availability.
Chemicals
Human and rat IgGs were purchased from Invitrogen Corp.
SDS-PAGE
SDS-PAGE was performed essentially according to the method of Laemmli [23] using slab gels consisting of a 4.5% polyacrylamide stacking gel and a 12% polyacrylamide running gel.
Binding of Mammalian IgGs to Zinc Ion-Immobilized Beads
Zinc ion was immobilized to Chelating Sepharose Fast Flow beads using 0.2 M ZnSO 4 according to the manufacturer's instructions and the beads were suspended to 50% (v/v) in phosphate-buffered saline (PBS, 150 mM NaCl, 20 mM sodium phosphate, pH 7.2). One mL PBS containing IgG, human IgG Fab or IgG F(c) fragments (25 µg each) and a suspension of 40 µL 50% (v/v) zinc ion-immobilized (Zn-beads) or Sepharose 4B (CB: control beads) beads was prepared (net volume of beads per sample: 20 µL) and the mixture was rotated at 4˝C overnight. The mixture was centrifuged at 14,000ˆg for 7 min at 4˝C, providing supernatant and pelleted beads for SDS-PAGE. One mL of washing solution (0.5 M NaCl, 20 mM sodium phosphate buffer, pH 7.2) was added to the pelleted beads and the suspension was centrifuged as described above. One mL of washing solution was then added, the beads were suspended, and centrifugation was repeated to further wash the beads. This washing step was repeated three times in total and the final pelleted beads were suspended in sample buffer for SDS-PAGE and used for SDS-PAGE (net volume of beads per lane: 8 µL).
The effect of EDTA on the binding between human IgG and Zn-beads was investigated by preincubating Zn-beads and CB in the presence and absence of 1 mL of 50 mM EDTA (pH 7.4), then IgG was added and the beads were incubated at 4˝C overnight. The treated and untreated beads were exhaustively washed as described above and the beads were suspended to provide a 50% (v/v) suspension in PBS. Human IgG was also incubated with treated and untreated Zn-beads and CB as previously described. In another experiment, the EDTA-treated Zn-beads or CB were exhaustively washed, then 1 mL PBS containing 50 mM ZnSO 4 was added to the pelleted beads. The mixture was rotated at 4˝C overnight, then the beads were exhaustively washed, suspended to 50% (v/v) with PBS, and incubated with IgG (25 µg) as described above. In all experiments, beads were washed and centrifuged, the resulting supernatant and the pelleted beads were treated as described above, and these samples were subjected to SDS-PAGE.
Comparison of Binding Affinity of Human Fibrinogen and IgG to Zn-Beads
One mL PBS containing human fibrinogen (600 µg) or IgG (500 µg) and a suspension of 40 µL 50% (v/v) Zn-beads or CB beads was prepared (net volume of beads per sample: 20 µL) and the mixture was rotated at 4˝C overnight. The presence of human fibrinogen or IgG was detected in the supernatant, suggesting that the Zn-beads were saturated with fibrinogen or IgG (data not shown). A 20 microliter aliquot of human IgG (100 µg) and fibrinogen (100 µg) in PBS were added to a suspension of beads saturated with fibrinogen and IgG, respectively, and further incubated as described above. The mixture was centrifuged at 14,000ˆg for 7 min at 4˝C to provide supernatant and pelleted beads for SDS-PAGE. Pelleted beads were applied to gel as much as possible due to natural sedimentation. CB were treated and analyzed in an identical manner.
Conclusions
Animal IgGs, including humans, bind with zinc ions immobilized on Sepharose beads (Zn-beads). The human IgG F(c) fragment bound with Zn-beads whereas the Fab fragment did not, suggesting that IgG binds Zn ions through the F(c) fragment. EDTA-treated Zn-beads could not bind human IgG, but regeneration with zinc ions recovered the zinc-binding activity with human IgG. Human IgG retains zinc-binding activity even after addition to Zn-beads saturated with fibrinogen and fibrinogen binds to Zn-beads saturated with IgG. These results demonstrate that human IgG specifically binds zinc ions, suggesting a zinc-binding site in the Fc fragment, and IgG binds fibrinogen and/or zinc ions through a different mechanism. | v3-fos-license |
2019-08-27T14:07:47.304Z | 2019-08-26T00:00:00.000 | 201757557 | {
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} | pes2o/s2orc | Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients
Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s40883-019-00114-5) contains supplementary material, which is available to authorized users.
Introduction
Our ability to understand the complex biology and physiology of cells and tissues is being advanced through innovative approaches to reproduce their multicellular three-dimensional (3D) interactions, structure, and function in vitro. With the relatively simple approach of culturing different cell types, together with specific signaling factors and natural or synthetic scaffolds, cells can be encouraged to self-assemble and form organized 3D cellular structures in vitro that mimic native tissues. Organoids and organ-on-chip technologies are now enabling investigation of specific developmental, physiological, and disease processes and to dissect the effects and mechanisms of biochemical factors or drug molecules in a controlled manner [1][2][3][4][5][6][7][8].
The level of precision and accuracy of control of cell interactions and delivery of bio-instructive signals that can be achieved is varied among these technologies, and the ability to work interactively and precisely at the scale of individual cells and their microenvironment is a particular challenge [9]. Meeting this challenge, we have established the use of holographic optical tweezers to enable simultaneous imaging and micromanipulation of multiple cells, as well polymer microparticles within 3D culture environments [10]. These can be precisely positioned and assembled, within a matter of minutes, into predetermined 3D microtissue structures, with accuracy of control from micron-to millimeterlength scales (10).
In this study, we use HOTs to create biochemical gradients within 3D microenvironments by precise positioning of microparticles loaded with a biochemical factor and induction of highly localized responses in mouse embryoid bodies (EBs). The formation of EBs from embryonic stem (ES) cells has been used extensively as a model to investigate in vitro differentiation of particular cell types in response to specific Electronic supplementary material The online version of this article (https://doi.org/10.1007/s40883-019-00114-5) contains supplementary material, which is available to authorized users. biochemical factors [11][12][13][14][15]. Various approaches have been used to stimulate differentiation, including simply adding biochemical factors to the culture medium [11][12][13] through to aggregating ES cells together with microparticles doped with biochemical factors [14,15]. While such studies demonstrate directed and localized differentiation of ES cells and EBs in response to bio-instructive signals, the results are often highly variable, with minimal predictable and repeatable targeted control of differentiation within defined cell populations or locations within the 3D cellular aggregates.
To demonstrate the potential of HOTs to achieve precision delivery of bio-instructive signals with concomitant biological responses at defined locations within EBs we have used a robust and highly sensitive retinoic acid (RA) inducible genetic system. Recent work has shown that mES cells are sensitive to low concentrations of RA resulting in marked increases of two RA responsive elements (RAREs), stimulated by RA gene 8 (Stra8) and deleted in azoospermia-like (Dazl) [16,17]. The expression of these two RAREs is tightly linked to RA concentration and up to 800-fold increases have been reported after just 24 h of exposure [16,17]. This presents a robust model to demonstrate highly localized release of RA and expression of Stra8.
In this study, we show the formulation of RA encapsulated microparticles with controlled release profiles, together with an approach to enable selection of microparticles with high RA loading efficiency. Subsequently, we show the precise positioning of small numbers of RA encapsulated microparticles to defined X, Y, Z locations and distances around mEBs and the resulting highly localized induced expression of Stra8, correlating directly with the positioning of RA release source.
Lay Summary (98/100) Advances in growing cells in a culture dish and forming organized 3D cellular structures that mimic tissues in our body are greatly improving the study of biological processes in health and disease. Using an instrument called holographic optical tweezers, we have shown how a nondamaging light source can literally work as a pair of microscopic tweezers. We demonstrate the ability to interactively and precisely build microscopic tissues and to deliver biochemical or drug molecules and induce highly localized responses in the cells. This technology has great potential for building accurate tissue models to test and develop new drugs.
Future Work (50/50)
To build more complex 3D models of biochemical gradients, including localized delivery of single or multiple factors and dynamic monitoring of localized release kinetics and cell responses. Recent studies on spontaneous in vitro assembly of embryo-like structures present a robust model to build localized biochemical gradients and interrogate developmental processes.
Holographic Optical Tweezer (HOT) Systems
Experiments were performed with the HOTs instrument as described previously [10] and a commercially available portable system (CUBE; Meadowlark Optics, USA). Both systems are based on an infra-red laser for bio-applications. All software-based control functions were programmed using LabVIEW software (National Instruments) as described previously [10]. The CUBE system was housed in a standard cell culture incubator (37°C, 5% CO 2 ) facilitating HOTs controlled patterning of cells and microparticles under standard culture conditions.
Formation of Embryoid Bodies (mEBs) by Hanging Drop
Prior to aggregation, mES cells were transiently serum started by culturing in mES cell culture medium without FCS for 24 h to minimize any RA-induced responses before the controlled release experiments. Uniformly sized cell aggregates, mES cells were created by the hanging drop method [13]. Cell suspensions were diluted to 2 × 10 4 cells/mL in the same medium as above without LIF and then, using a multi-flow pipette (8 tips), 25-μL volumes were deposited onto the underside of a 60 mm Petri dish lid to form eight rows. The resulting 64 droplets containing mES cells were then inverted and the lid is placed on the Petri dish containing PBS. The droplets containing roughly 500 cells were cultured for 24 h and the resulting cell aggregates were collected and suspended in a pre-gel GelMA solution ready for patterning on the HOTS system.
Immunohistochemical Staining of Stra8 in mES Cells and mEBs
Dose-response effects of RA on expression of Stra8 were initially assessed by adding RA directly to culture medium and culturing ES cells or EBs in concentrations of RA at 0, 10, 100, and 1000 nM for 24 h. Subsequently, the experiments were repeated with solutions of RA released from microparticles allowing comparison of freshly prepared RA. ES cells and EBs were fixed in 3.5% paraformaldehyde for 20 min. Fixed cell samples were permeabilized in 0.1% (w/v) Triton X-100 (diluted in PBS) for 40 min at room temperature for cell monolayers and 90 min for cell aggregates. Following permeabilization, samples were covered in blocking solution for 30 min at room temperature. Aggregates were immunostained by incubation overnight at 4°C with rabbit anti-Stra8 primary (Abcam) diluted 1:100. After washing 3 × 10 min with PBS, the aggregates were incubated for 2 h at room temperature with anti-rabbit Alexa Fluor 546 fluorescent secondary antibody (Invitrogen) diluted 1:200. Immunostaining was visualized by conventional fluorescence and confocal microscopy. In all experiments, qualitative observations are presented and described and for each individual experiment images were collected at similar exposure settings.
Retinoic Acid (RA) Microparticle Encapsulation
Retinoic acid containing PLGA/TBIIF (70:30) polymer microparticles with an average size of 5 μm were produced using a single-emulsion, water-in-oil encapsulation method as previously described [19]. Briefly, 2 mg RA was solubilized in dichloromethane (DCM) along with 700 mg PLGA and 300 mg TBIIF, 10 mg FITC-BSA (or 10 mg unlabeled BSA), and 200 mL of 0.3% (w/v) poly vinyl alcohol and atRA, all trans retinoic acid homogenized at 4000 rpm for 2 min. The solution was stirred at 300 rpm for 4 h to allow the DCM to evaporate. The resulting microparticles were then collected by centrifugation, the particles were washed with distilled water three times and snap frozen in liquid nitrogen and freeze dried (Edwards Modulyo D, IMA Edwards, UK) for 2 days.
Encapsulation Efficiency of Microparticles
Using an adapted version of the method published by Sah (1997) [20], 15 mg of loaded PLGA microparticles was dissolved in 750 μL of DMSO and 2150 μL of 0.02% (w/v) SDS in 0.2 M NaOH for 1 h at room temperature and 150-μL aliquots of each solution were added to a well plate and a bicinchoninic acid (BCA) assay (Sigma-Aldrich) was performed. Appropriate standards of BSA were created, and after 2 h of incubation at 37°C, the plate was scanned at 562 nm on a plate reader. The total protein content was then calculated via a polynomial equation of the standard curve, and the encapsulation efficiency was calculated from the theoretical expected loading of the microparticles. The microparticle batches were lyophilized and the powder was vacuum packed and stored at 4°C until required. The size distribution of microparticle batches was determined by suspension in deionized water (20 mg/mL) and sized using a laser diffraction method (Coulter LS230; Beckman Coulter, UK).
For release kinetics, 25 mg of microparticles was suspended in 1.5 mL of PBS in glass tubes and then placed on a GyroTwister and gently rocked at 5 rpm at 37°C. At 24-h intervals, the tubes were centrifuged at 3000 rpm for 3 min. The supernatant was then carefully removed and stored at -20°C for analysis. The microparticles were then re-suspended in 1.5 mL PBS and returned to the incubator; this process was repeated over 12 days to ensure complete release from the microparticles.
Fluorescence-Associated Cell Sorting (FACs) or RA Microparticles
Microparticles containing FITC-BSA RA or BSA RA and 5-μm reference beads were suspended in PBS (25 mg/mL) and sonicated for 30 s to break up aggregates before being added to separate 5 mL FACS tubes under sterile conditions. Sorting and analysis were performed using a MoFlo Astrios Cell Sorter (Beckman Coulter, UK) equipped with a 488-nm laser at 100 mW of power. Forward scatter (FSC1) and side scatter (SSC) were collected through a filter and the FITC signal was collected in the FL1 channel through a 513/26 bandpass filter. A light scatter gate was drawn in the SSC versus FSC1 plot to include microparticles of a similar size to 5-μm reference beads. Cells within the gate were displayed within a SSC versus 488,513/26 intensity plot allowing a visualization of the fluorescence intensity distribution within the microparticle batches. A final selection gating was applied to sort based on fluorescence intensity. Microparticles were sorted over several sessions in separate batches to reduce the time spent suspended in PBS and were freeze dried for longterm storage.
HOTs Patterning Procedures with Embryoid Bodies and Microparticles
The movement and positioning of cells and microparticles was as described previously [10]. Briefly, the system uses a Nd:YAG, solid state, infrared (1064 nm), 3 W maximum output, continuous wave, class 4 3.2-mm-beam-diameter laser . This is then coupled with the optical tweezer system by imaging the SLM onto the back aperture of a high numerical aperture oil immersion microscope objective lens (40 × 1.3 NA Zeiss, Plan-NeoFluar). The resulting traps can then be focused anywhere within the field of view, with controlled holograms generated by the SLM giving full axial and lateral control over the trapping beam. All software-based control functions were programmed using LabVIEW software (National Instruments) as described previously [10]. A patterning, microfluidic-type gasket with multiple wells connected by channels was made (see supplementary material). This enabled EBs and microparticles, together with pre-GelMA solution, to be added to the wells and then moved and assembled by user-specified design with the HOTs. The GelMA was prepared as described previously [10] and then dissolved in 80°C photo-initiator (Irgacure 2959 0.5% (w/v)) to yield a 10% (w/v) GelMA solution, and stored in the dark at 4°C until use. This solution was warmed to 37°C and then added to the patterning gasket to a maximum volume of 100 μL. Cells and microparticles to be patterned were added directly to the patterning gasket as required under sterile conditions. Once patterning was completed, the GelMA solution was cross-linked with a 5-s burst of UV light from a distance of 5 cm resulting in an output of 30 W/ cm 2 using a UV lamp (Omnicure S2000; JentonUV, UK). The GelMA was left for 5 min to ensure complete crosslinking. If prolonged cell culture was required, cell culture medium was added on top of the cross-linked GelMA before incubation at 37°C with 5% CO 2 .
Retinoic Acid Dose Response-Induced Expression of Stra8 in mES Cells
The dose response experiment in Fig. 1 shows induced expression of mES cells to a range of RA concentrations, from 0 to 1000 nM. The cells were serum starved for 24 h prior to being exposed to RA for 24 h. The fluorescence staining intensity gives an indication of the level Stra8 protein expression and is seen to increase with increasing RA concentrations. Little or no staining was seen in absence of added RA. This contrasts with the low-level staining seen without transient serum starvation (data not shown).
Retinoic Acid Dose Response-Induced Expression of Stra8 in Mouse EBs
To show effective delivery and induction of Stra8 in 3D cell aggregates, mouse EBs were exposed to either 0 or 10 nM RA for 24 h. Figure 2 shows clear staining and expression of Stra8 in response to 10 nM RA, with negligible staining seen in the absence of RA. Prior to aggregation, the ES cells were serum starved for 24 h, and this data indicates that there were no deleterious effects on EB formation or Stra8 gene expression and background Stra8 expression remains negligible.
Retinoic Acid Encapsulation and Induction of Stra8 in mES Cells
The RA microparticles (BSA-RA co-loaded) had an average size of around 5 μm (Fig. 3a), and SEM imaging showed they had spherical, non-porous morphology (Fig. 5b). The in vitro release study (Fig. 5c) showed an initial burst release within the first 24 h, followed by a more gradual release over the next 10 days. The bioactivity assay (Fig. 3d) showed Stra8 staining in mES cells in response to freshly prepared RA at concentrations of 10 and 100 nM and a solution collected from a suspension of RAcontaining microparticles, estimated by microparticle mass and estimated encapsulation efficiency, to have a RA concentration of 100 nM. It can be seen that the solution collected from RA encapsulated microparticles showed a level 6 RA laden microparticles 8 RA laden microparticles Hoechst Anti-Stra8 AlexaFlour488 Hoechst Anti-Stra8 AlexaFlour488 of staining somewhere between that seen with fresh RA at concentrations 10 and 100 nM and confirmed the ability to deliver RA from microparticles. Figure 4 shows an example of the results of HOTs patterning experiments with a defined number of BSA-RA microparticles precisely positioned around mEBs. Immunostaining for Stra8 was only seen in approximately only 50% of experiments (n = 4) and was likely due to variability in loading efficiency in the microparticles. This data is included to show the evolution of the experiment and our work toward induction of a highly localized response in the EBs. The 6-and 8-microparticle patterns were arbitrarily chosen to produce an obvious, userdefined pattern and to achieve a localized or focused delivery source. There were no differences in observed effects between 6-and 8-microparticle patterns and the 6microparticle triangular configuration was used in all subsequent experiments.
Analysis and Sorting of the FITC Co-Loaded RA Microparticles
In order to be able to select small numbers of RA-laden microparticles and ensure high encapsulation efficiency of the selected microparticles, a method of co-loading FITC-BSA and RA was developed. Figure 5 shows analysis and selection of FITC co-loaded RA microparticles and sorting into different groups of microparticles with fluorescence intensity ranging from "High," "Medium," and "Low." The microparticles were initially gated based on their size versus 5-μm reference beads, assessed by forward and side scatter (Fig. 5a). As a means of comparison, the non-fluorescent RA-laden microparticles were also analyzed to provide a baseline fluorescence intensity. The use of these microparticles as a control ensured that any RA or polymer autofluorescence would be normalized from the FITC-labeled batch. When comparing these two batches in the "Batch fluorescent intensity distribution" (Fig. 5b), it can be seen that the FITC co-loaded microparticles had a much greater fluorescence intensity. Three separate groups were created to select for "High," "Medium," and "Low" fluorescence microparticles, as shown in the representative plots (Fig. 5c). Then by running a high-density suspension of the FITC co-loaded batch through the FACS process, the three separate suspensions ("High," "Medium," and "Low") and any sub-"Low" microparticles were separated.
Analysis of the FACS Selected "High" Loading Microparticles Figure 6 shows that FITC fluorescence correlates with encapsulation efficiency of BSA and by extension with RA. With increased FITC fluorescence, greater RA is estimated to be encapsulated within the batch. The "High" group was estimated to have an encapsulation efficiency of 89.5 ± 1.8 and collectively this was expected to achieve a robust and reliable selection and delivery process for the HOTs patterning experiments.
HOTs Patterning of FITC-BSA/RA Microparticles Around mEBs Figure 7a shows the release of RA from the FITC-BSA coloaded RA microparticles over 24 h. By the 24-h point, 39.5 ± 5.9% of the loaded RA was released. Figure 7b shows the positioning of six RA-releasing microparticles in a defined triangular pattern formed in close proximity (within 20 μm) to mEBs and stabilized by use of GelMA cross-linking. After 24 h, immunostaining for Stra8 can be clearly seen in localized regions within the EBs closely apposed with wherever the RA release source was placed around the EBs. Through the use of FACS sorted "High" loading RA microparticles, experimental success was raised from 50% (n = 4 separate experiments) to 80% (n = 5 separate experiments).
In this experiment, we focused entirely on positioning of the microparticles at defined locations around EBs and inducing highly localized responses while maintaining the same distance that the microparticles were positioned from the EBs at each set location. In a related study, we have investigated the ability to control specific cellular responses over defined distances using a model of chemotactic responses of mouse osteoblasts (see supplementary material).
Discussion
In this study, we have shown that we can build 3D cell microenvironments with defined, reproducible control and precision delivery, with micron resolution, of a biochemical signal gradient and to induce a highly localized biological response in multicellular aggregates directly in response to the biochemical signal.
The spatial and temporal induction of Stra8 in mEBs in response to RA [17,18] released from microparticles positioned at discrete locations around the 3D cellular structures has served primarily as exemplar of the HOTs technology to precisely deliver and induce a biological response. This builds on our previous work with HOTs on assembly of complex microenvironments [10] to show that the technology is also s t test analyses were performed, and the significant difference is indicated accordingly, ****p < 0.001, ***p < 0.0045, ***p < 0.01, and *p < 0.0253. EE, encapsulation efficiency very amenable to creating biochemical gradients. It also shows how the HOTs technology can complement other technologies on cellular assembly, as we have shown here by precision engineering of the microenvironment around EBs formed by the hanging drop method [13]. We envisage that this can be applied to organoids and organ-on-chip models and collectively can bridge gaps with the scale of cellular assembly, precision of cellular organization and accurate control of delivery of bio-instructive signals [1][2][3][4][5][6][7][8].
The sensitivity and relative simplicity of the RA-inducible Stra8 model [17,18] was extremely useful for refining the delivery of biochemical factors from microparticles and specifically adapting their use for selecting and positioning small numbers of microparticles. Formulation of microparticles and the controlled release of soluble biochemical factors and drug molecules is well established with wide-ranging, demonstrated applications [21][22][23][24][25][26][27][28]. In many cases, controlled release studies are performed with bulk volumes of microparticles, and while it is known that loading within individual microparticles can be highly variable, this is relatively unimportant unless there is a need to work with individual microparticles. As we found in our studies with HOTs positioning of RA microparticles, in some experiments Stra8 expression was induced in EBs, and in other experiments, no expression was seen, suggesting variable loading of RA in the HOTs selected microparticles.
We were able to overcome this limitation by co-loading microparticles with FITC-BSA and RA and through FACS we were able to specifically select particles with high loading efficiency and markedly increase the success rate in inducing localized expression of Stra8. The burst release kinetics of the microparticles, combined with the ability to reproducibly and accurately position them at defined locations around the EBs, stabilized with GelMA, facilitated highly localized RA-induced responses in regions of the EBs directly facing the RA
Bright Field
Hoechst microparticles. The choice of pattern of 6 microparticles, roughly in an equilateral triangle configuration with the "base of the triangle" facing the EBs, was an arbitrary choice intended to clearly show that it was precisely patterned and included a sufficient number of microparticles to achieve localized, focused release or RA toward the EBs sitting in close apposition.
As we have previously shown, the HOTs technology offers considerable scope and flexibility to create a wide range of defined patterns with microparticles, scaffolds, and cells [10].
Although we have not quantified the induced, localized expression of Stra8, the response is highly defined and striking and serves to demonstrate a key objective of this study. In related work, we have demonstrated and quantified chemotaxis of mouse primary calvarial osteoblasts to platelet-derived growth factor-BB (PDGF-BB), a known potent chemotactic factor for osteoblasts. Agarose beads doped with PDGF-BB (10 nM) and single mouse primary calvarial osteoblasts were positioned at defined distances apart (50, 100, and 150 μm) using HOTs and stabilized within gelMA (see supplementary material). As expected, there was very clear movement of the osteoblast toward the PDGF-BB-doped bead, with a trend of decreasing distance moved with increasing distance of separation. Over the 8-h time course of this experiment, it would have been reasonable to expect a more marked gradation of chemotaxis but, overall, there was no significant difference in net positive migration relative to distance from the release source. These observations serve to highlight the challenges of creating and quantifying biochemical gradients and the many variables involved, including the properties of the biochemical factor, the kinetics of release, and the properties of the environment through which the factor is distributed. What is absolute from this study is that the HOTs technology and the approach described here is highly amenable for precision assembly of tunable, complex microenvironments with spatial and temporal control of both cellular organization and delivery of bio-instructive signals.
In summary, we have demonstrated the creation of biochemical gradients within 3D cellular microenvironments and the precision delivery of bio-instructive signals by interactively positioning biochemical-laden microparticles around multicellular 3D aggregates at user-defined locations. While challenges remain, such as quantifying signal gradients within cellular microenvironments, this robust, highly interactive "cause-and-effect" type model is able to accurately target and provide insights into understanding and quantifying specific biological responses in 2D or 3D cell models.
Conclusion
The construction and interrogation of multicellular 3D models and microenvironments and the capability to recapitulate complex physiological and pathophysiological processes in vitro is being advanced significantly by various innovative techniques and technologies, yet our understanding remains far from complete.
Using HOTs, we can assemble complex microenvironments and create biochemical gradients with a level of control from micron-to millimeter-length scales. As we have also shown, this technology is very adaptable and can be used with other technologies such as organoids and microfluidics. Collectively, this shows that we can create more accurate in vitro representations of the native tissue structures and signaling interactions and to build multicellular 3D cell models that enable more detailed in vitro investigation and interrogation of physiological and pathophysiological responses. | v3-fos-license |
2021-05-21T16:56:19.276Z | 2021-04-30T00:00:00.000 | 234849739 | {
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} | pes2o/s2orc | Comparative study of the effects of Herbal and chemical Coagulants in the clarification of raw water
1 Laboratoire de Biochimie, Biotechnologie, Technologie Alimentaire et Nutrition (LABIOTAN), Université Joseph KI-ZERBO, 03 BP 848, Ouagadougou 03, Burkina Faso. 2 Laboratoire Sol-Eau-Plante, Institut de l’Environnement et de Recherches Agricoles (INERA), Ouagadougou, 01 BP 476 Ouagadougou 01, Burkina Faso. 3 Unité Mixte de Recherche Internationale Environnement, Santé et Sociétés (UMI 3189, ESS) 4 Office National de l'eau et de l'assainissement (ONEA), 01 BP 171 Ouagadougou 01, Burkina Faso.
INTRODUCTION
In Burkina Faso, as in many other developing African countries, access to water is an important issue for population. Unfortunately, groundwater reserves in the country are discontinuous because of the lateritic soil which is worsen over the years by demographic explosion in towns, the Burkinabe state has implemented the construction of many water reservoirs commonly called dams, to mobilize surface water and somehow alleviate the water scarcity that strikes the populations which does not retain water. To deal with water scarcity (Zoungrana and Combelem, 2016). However, once mobilized, this water is not drinkable, because it had been more or less polluted by various urban discharges. Henceforth, the need to undergo a series of treatments before consumption is necessary. The main steps in the usual treatment of surface water include crucial clarification. This is based on the phenomenon of coagulationflocculation and generally uses aluminum sulphate as a coagulant since this chemical appears to be the cheapest trivalent (Kowanga et al., 2016). However, the use of aluminum sulphate in water clarification raises significant health and environmental problems (Deshmukh and Hedaoo, 2019;Tietz et al., 2019). First, although it is relatively low-priced, it remains costly enough for developing countries like ours. Also, on environmental ground, its use inexorably generates metal residues, and its use exposes it to high risk of suffering Alzheimer, since aluminum residues in water are pointed by several scientific studies as responsible for this disease (Zhang et al., 2015). In view of this, the use of biological processes for the treatment of surface water could be a sustainable alternative because of the availability and non-toxicity of such treatment (Chong and Kiew, 2017). Thus, several studies on water clarification with natural coagulants like Moringa seeds (Adeniran et al., 2017;Camacho et al., 2017;Hoa and Hue, 2018) or other natural coagulants (Adewuyi and Adewumi, 2018;Benalia et al., 2019;Freitas et al., 2015;Hussain and Hydar, 2019;Jayalakshmi et al., 2017) have already been carried out. Other researches have shown the efficiency of addition of Opuntia ficus-indica extracts as coagulant aids or flocculant (Adjeroud-Abdellatif et al., 2020;Bouaouine et al., 2019;Karanja et al., 2017;Othmani et al., 2020). Bouaouine et al. (2019) revealed that quercetin and starch constitute the active agents found in O. ficus-indica. Currently, it is discovered that all plants being studied for flocculants production have mucilaginous constituents and it is predicted that some of the active ingredients in the mucilage (polysaccharides) are responsible for the flocculating property, but the most studied plant of the natural coagulant (Moringa oleifera) shows that the active agents are dimeric cationic proteins (Adjeroud-Abdellatif et al., 2020;Camacho et al., 2017). These biological molecules offer many advantages compared to conventional coagulants used for water treatment. The benefits are cost-effective, environmentally friendly, no pH alteration required, no need for the addition of alkalinity and the reduction of sludge volumes (Idris et al., 2016). Although studies conducted in Burkina Faso have proved the effectiveness of Moringa seeds in water treatment, they hardly consider its substitution with chemical coagulants in large-scale treatment. One of the factors hindering its use is the decanting time of the flocs which appears to be longer than with aluminum sulphate (Saleem and Bachmann, 2019). Hence, this prompted us to make a modest contribution by optimizing the treatment process, using successively Moringa seeds and cactus extract to reduce Konkobo et al. 533 treatment time. This study is conducted in such context and aims at evaluating the effectiveness of natural coagulants in the treatment and determining the optimal treatment conditions. More specifically, it was all about comparing the effectiveness of Moringa seeds to that of aluminum sulphate and optimizing the two coagulants by adding a chemical flocculant, then a natural one.
Sampling of surface waters
The raw water samples investigated on as part of this study are from Loumbila dam (12° 29 N, 01° 24 W). The samples were collected at Paspanga station of ONEA through pipes, draining the water from the dam to the station. The sampled water was screened and sand-freed, while being drained to the station. They are then able to undergo the clarification treatment in accordance with the conventional procedure of surface water treatment. The samples were collected in 20 L plastic cans and stored in a refrigerator at +4°C in the laboratory.
Biological material
The biological material used in this study consists of M. oleifera seeds and extracts of O. ficus indica. These two items stand respectively for the biological coagulant and the biological flocculant. The seeds of M. oleifera were obtained at the National Forest Seed Center (CNSF). They are mature and dry seeds from Garango and were harvested between February and March 2018. As for O. ficus indica, the whole plant was collected in a nursery located nearby dam n°3 at Tanghin. It was then transported to the laboratory (LABIOTAN) to be used for the extract preparation.
Preparation of the moringa and cactus extracts
The bio-coagulant used in this study is an aqueous solution prepared from the powder of M. oleifera seed. These seeds were shelled and grounded according to the method described by Folkard and Sutherland (2002). The extract was prepared at 4°C at a concentration of 100 g/L in distilled water, followed by filtration after a 2 h stirring. The stock solution containing the water-soluble substances from Moringa seeds is then stored at -18°C until use. This treatment allows a better preservation of the active ingredient.
As for the extract of cactus, the organic flocculant, a stock solution of 30 g/l in distilled water, was prepared. This solution is relatively stable and keeps its flocculation ability for several days without any preservation system.
Preparation of aluminum sulfate and synthofloc solutions
The stock solution of aluminum sulphate, the chemical coagulant, was prepared by dissolving its granules in distilled water (10 g/L). Synthofloc, a chemical flocculant, was prepared at a concentration of 1 g/L in distilled water. These are stock solutions used for the coagulation-flocculation tests.
Optimum dose of coagulants determination: Jar tests
Coagulation-flocculation tests were conducted using an electric flocculator (FC6S Jar-Test Velp Scientifica) with six beakers containing 1 L of raw water. Increasing doses of aluminum sulphate (0.025-0.05 g/L) or moringa (0.2-1.1 g/L) were added to these and the whole was then subjected to vigorous stirring at 150 rpm for 5 min and then slowly for another 5 min. It was left to settle for about deux hours, the clearer of each dosage was picked as optimum for that coagulant. When the coagulant used significantly reduces the pH, lime is used as a corrector of acidity in order to enhance it.
Determination of the minimum decanting time
The minimum decanting time was determined by setting the dose of moringa or aluminum sulfate and changing the decanting time. For this, beakers were filled with 1,000 ml of raw water after which fixed doses of moringa (0.9 g/L) or aluminum sulphate (0,05 g/L) were added to these, while the last beaker was kept as a control without any coagulant. The mixtures were stirred rapidly for 5 min; followed by 5 min of gentle mixing to aid sludge formation. Subsequently, the samples were transferred and left for decantation for 15; 30; 60; 90; 120; 720 min in sedimentation cones.
Flocculation optimization: Effect of cactus extract and synthofloc
Synthofloc and cactus extracts, with similar viscous aspect, were respectively used as chemical and biological adjuvants to optimize the flocculation of flocs formed by the isolated action of aluminum sulphate and Moringa. To do this, constant volumes of moringa solution (0.9 g/L) or aluminum sulphate (0.05 g/L) were added to the beakers containing the water to be treated, followed by a rapid stirring phase (150 rpm) for 5 min. Then increasing volumes of cactus extract or synthofloc were added to these samples, followed by further stirring at 45 rpm for 10 min, followed by decantation of 15 min.
Evaluation of the efficacy of the two coagulants and the two adjuvants
Supernatants trained after each treatment were collected and analyzed to evaluate and compare the effectiveness of different coagulants and adjuvants in clarifying surface water. Turbidity, pH, conductivity, Temperature, Alkalimetric Title (AT), Complet Alkalimetric Title (TAC), Total Hardness (TH), Calcium hardness (Tca 2+ ), Na + and K + as well as Total Coliforms, Streptococci and Escherichia coli were determined . Turbidity was measured with a WTW Turb 550 IR laboratory turbidimeter (nephelometer) as per French Norm NF ISO 7027 (2000). The pH was measured following the electrochemical method, using a pH meter/thermometer (330i WTW) equipped with a Sen Tix 41 combined electrode according to the NF 10523 (1994) method. Conductivity as well as Temperature was measured, using a conductivity meter coupled with a WTW mark thermometer. Alkalinity (TA) and Complet Alkalinity (TAC) were determined by titrimetric series according to French Norm NF T 9963: 1996. The concentrations of calcium and magnesium and the total hardness were also determined by means of titrimetric series consistent with French Norm, NF T 90-016: 1984 and NF T 90-003: 1984, respectively for calcium and magnesium and for total hardness. Na + and K + ions were determined, using a flame photometer. As for the microbiological parameters of water, they were all determined according to the method of membrane filtration and spreading on specifics culture media as per the French Norm NF ISO 9308-1 (2000). For the search for total coliforms and E. coli, Chromocult coliform Agar was used for an incubation temperature of 37°C, whereas for streptococci, Enterrococus agar was used at a temperature of 44°C.
Statistical analysis
The data obtained in the application of the various treatments have been subjected to statistical analysis. The XLSTAT software (2016 Version) was used for variance analysis (ANOVA) to compare the average values of the variables considered in each case of the study. The Turkey test was used to determine significant differences between the variables considered at the 5% threshold (P> 0.05).
Influence of the coagulant dose on turbidity
Water samples with initial turbidity of 352.80 NTU were used for coagulation-flocculation tests with increasing doses of aluminum sulphate and M. oleifera grains extracts. Changes in the residual turbidity were recorded in Figure 1A and B. The results showed that the reduction of turbidity was dose-dependent for aluminum sulfate, that is increasing aluminum sulphate dose decreased the water turbidity. As for moringa, turbidity was evolved in two phases. At low doses, the reductions were doses dependent. Beyond 0.9 g/L of moringa used, there was another phase which resulted in an increase in turbidity. Therefore, the optimal dose of moringa needed to treat the water sample was 0.9 g/L. At this dose, a residual turbidity of 63.26 NTU was obtained. The best results were obtained with addition of aluminum sulphate. For samples with equal turbidity of 352.80 NTU, more moringa coagulant (0.9 g/L) than aluminum sulphate (0.05 g/L) was required to obtain 63.26 and 25 NTU respectively. Although these values are above the norm NF ISO 7027-2000, Moringa could also be considered as good coagulant because it has eliminated 82.06% against 92.91% obtained with aluminum sulfate, the reference coagulant.
Influence of decanting time on turbidity
The results recorded in Figure 2 showed that the reduction in turbidity also depended on decanting duration. It was important during the first 15 min and low after 30 min of decanting. Standard turbidity was reached after 90 min with 0.05 g/L of aluminum sulphate against 12 h with 0.9 g/L of moringa. initial optimal concentration of 0.9 g/L of moringa seeds. With the aluminum sulphate, a value of 4.56 NTU was obtained for a concentration of 0.04 g/L of aluminum sulphate. Synthofloc has improved the clarification of water treated with either moringa or aluminum sulphate.
Optimization by addition of cactus extract
The results of clarification tests with equal doses of Moringa extracts followed by a flocculation stage with increasing doses of cactus extracts showed that turbidity reduction was dose-dependent ( Figure 4). As the dose of cactus extracts increased, the water turbidity decreased. Upon application of 0.6 ml of cactus extracts, clear water with 4.19 NTU turbidity was obtained. This complies with the norm (≤ 5NTU). With the 0.6 ml of cactus extract, a turbidity of 2.36 NTU was obtained with 0.05 g/l of aluminum sulphate ( Figure 5A and B). In view of these results, it could be said that the use of this new flocculant improves the action of the two coagulants in the like of synthofloc because it has resulted in a water turbidity complying with the norm (≤ 5 NTU).
Effects of treatments on the evolution of physicochemical parameters
With a view to assessing the effectiveness of the treatments, the evolution of some physicochemical parameters of the water samples were monitored and compared before and after treatment. The analysis of these parameters was carried out in triplicate and the averages obtained were recorded in Table 1. Analysis of the variances (at p> 0.05) showed that the applied treatments had a significant effect on almost all Physicochemical parameters of the water samples analyzed except for temperature and TA for which the variations were not significant. For the other parameters analyzed, the variations were significant regardless the coagulant and the flocculant used. For the pH value, there was no change during treatment with Moringa seeds, whereas with aluminum sulphate, the post treatment value decreased significantly, except for aluminum sulphate/synthofloc-based treatment, since lime was used as acidity corrector. Other parameters, such as calcium, sodium and total hardness, were found to be lower in waters treated with Moringa seeds than those treated with aluminum sulphate. The TAC and the potassium content were, however, higher in samples treated with Moringa seeds as compared to the ones treated with aluminum sulphate.
Effect of treatments on the evolution of microbiological parameters
Microbiological analyzes were also carried-out in triplicate and the averages are presented in Table 2. The microbial indicators (Escherichia coli, total coliforms) of the water samples varied significantly depending on the coagulant and/or flocculant used. This variation was significant for all treatments based on moringa seeds and cactus extract according to the ANOVA analysis at the 5% probability level (P> 0.05). For the other treatments based on coagulants and purely chemical flocculants (aluminum sulphate and / or synthofloc), the variation was not significant. Since streptococci were initially absent from the raw water, no variation was observed regardless of the treatment applied.
DISCUSSION
The effectiveness of moringa seeds in clarifying the water of the Loumbila dam was compared with that of aluminum sulphate, with a view to finding an alternative to the harmful consequences of the use of the latter on human health and the environment. Several studies have already been conducted on the use of Moringa seeds to clarify raw water (Adeniran et al., 2017;Camacho et al., 2017;Hoa and Hue, 2018;Zaid et al., 2019). Indeed, these ground seeds, added to water acts as primary coagulant and can clarify any water regardless of its degree of turbidity. However, no study has yet revealed the cumulative effect of moringa and synthofloc or moringa and cactus extracts in improving the clarification process. Optimization studies have indeed been conducted, but concerned the addition of Bombax costatum, a natural flocculant, and activated silica, a synthetic polymer in the like of synthofloc. Other combination studies have also been conducted but they combine alum with Moringa oleifera (Dehghani and Alizadeh, 2016) or Moringa oleifera with aluminum sulphate (Valverde et al., 2018).
At the end of the treatments carried out on the water samples from the Loumbila dam (turbidity 352.80 NTU) with increasing doses of the different coagulants, it was revealed that turbidity decreased with the increase of aluminum sulphate dose. These results are similar to those obtained by Valverde et al. (2018). On the other hand, with moringa seeds as a coagulant, the reduction in turbidity depended on the dose up to 0.9 g/l. Beyond this dose, turbidity tended to enhance. This can be explained by the re-stabilization of the colloidal particles, caused by the overdose of the coagulant. The excess makes it to play a reverse role, neutralizing all the particles. Indeed, these coagulants would contain a polypeptide, more specifically a set of active cationic polyelectrolytes (Camacho et al., 2017;Baptista et al., 2017). These positively charged poly-electrolytes thus neutralize colloids in murky waters, the majority of which are negatively charged (Valverde et al., 2018). At a very low dose of coagulant, the low level of turbidity reduction could be explained by the imbalance between the negative charges of colloidal particles and the positive charges of the coagulant. This results in strong adsorption of negative charges and prevents the appearance of flocs. It is then necessary to have balance between these charges to obtain optimum reduction of turbidity. Wandiga et al. (2017) have mentioned that once the optimal dosage is achieved, the excess polyelectrolyte proteins repel each other due to their charged nature, leading to the flocs floating or turbidity. Wandiga et al. (2017) have mentioned that once the optimal dosage is achieved, the excess polyelectrolyte proteins repel each other due to their charged nature, leading to the flocs floating or suspending in the water. Such floating flocs
Treatments
Turbidity ( could be filtered to achieve lower turbidity. In addition, Akpenpuun et al. (2016) have shown that the reduction is not only proportional to the dose of moringa but also depends on the quality of the seeds, that is, the concentration of active proteins contained in the seeds. A concentration of 0.9 g/L of moringa against 0.025-0.03 g/L of aluminum sulphate was necessary to obtain a reduction of approximately 80%. It appears from the statistical analysis that the difference in abatement for these two coagulants was not significant even though the dose difference between the two coagulants was significant. It is henceforth inferred that moringa seeds could be an as effective coagulant as aluminum sulphate in clarifying water. These results are explained by the fact that the active principle of moringa seeds is not the only constituent of these seeds. The raw protein extract not being separated from the rest of the non-protein fraction, then becomes less active and thus involves more powder than would be necessary if the active ingredient was isolated. Protein interactions may be the cause of this decline in moringa flocculating activity (Jayalakshmi et al., 2017). Aluminum sulphate, unlike moringa, has a higher proportion of aluminum (active ingredient) which, moreover, has a higher molecular weight.
Escherichia Coli
Monitoring turbidity evolution against the decanting time has shown that turbidity reduction depended on the decanting time. The minimum durations that allowed achieving turbidity at acceptable limits for drinking water were 90 min with 0.05 g/L of aluminum sulphate; against 12 h with 0.9 g/L of moringa seed extracts. Ngbolua et al. (2016) found a higher optimal decanting time (24 h) for the treatment of a turbid water of 9.06 NTU with 0.3 g/L of moringa. The process optimization by adding synthofloc to moringa and aluminum sulphate coagulants significantly reduced the decanting time to 15 min. When synthofloc which is a chemical polymer is combined with the optimal dose of coagulant, it agglomerates microflocs into larger flocs called macro-flocs after coagulation. These larger flocs are heavier and decant much faster and reduce the decanting time.
Comparing the water treatment with moringa and aluminum sulphate coagulants optimized with synthofloc, it shows a significant improvement in decanting time as compared to the same treatment without synthofloc. Thus, in the 15 min instead of 12 h of decanting, the turbidity obtained was complied with acceptable limits for drinking water. The use of a flocculant makes it possible to improve the performance of the flocculation and decanting process. These results are comparable to the ones of the study conducted by Dehghani and Alizadeh (2016), on the optimization of coagulation-flocculation using silica in addition to aluminum sulphate. Other studies by Mumbi et al. (2018) have also shown the positive impact of using a polymer in addition to aluminum sulphate on flocculation.
However, the variation in the concentration of aluminum sulphate in this study, which went from 0.05 to 0.04 g/L, could be explained by lime addition. The purpose of this was to raise the water pH, initially reduced by aluminum sulphate, which is known to modify pH, unlike moringa seeds. Since synthofloc is only effective for a pH between 6 and 7, this addition in the treatment with aluminum sulphate was necessary, hence the modification of the optimal pH of action of the flocculant, and therefore also that of the optimal Konkobo et al. 539 coagulant dose. Synthofloc substitution tests with cactus extracts as flocculants revealed satisfactory results. For a volume of 0.6 ml of cactus extracts the same effects expected with synthofloc was achieved. The difference with this new biofloculant is that it does not require any acidity corrector as is the case with synthofloc because it seems to work at any pH. Nharingo et al. (2015) highlighted the efficiency of cactus extracts as flocculant in the clarification of industrial effluents. Prodanović et al. (2020) also achieved satisfactory results, almost similar to the present ones with extracts of another plant, Bombax Costatum. These authors stated that the use of plants mucilage as flocculant significantly improves both turbidity and decanting duration in raw water clarification process. The effect of the treatments (based on moringa seeds and aluminum sulphate only, then optimized by synthofloc and cactus extracts) on some other parameters were allowed to deepen the comparison. As far as pH is concerned, there was no significant change across the various treatments with Moringa seeds as a coagulant (optimized or not), whereas with aluminum sulphate, the value of this parameter decreased significantly except for the case of treatment with synthofloc because of lime addition. It is inferred that the effectiveness of Moringa as a coagulant does not change the water pH, thus avoiding the use of acidity rectifiers (lime); while the use of aluminum sulphate as a coagulant still requires an acid rectification during the process if the water pH is to meet the norm. These results corroborate the ones of Shan et al. (2017) since their studies concluded that moringa-based treatment has little influence on the water pH and the variation of the latter is not statistically significant. Ngbolua et al. (2016) also reported the same observations with respect to pH variation for pond water from the Batéké Plateau (DRC) treated with moringa seed. Valverde et al. (2018) also found results similar to the ones of the present study as they concluded that there is no significant change in pH as a result of Moringa-based treatment, while aluminum sulphate-based treatment was maked it to vary from 7 to 6.
Microbiological analyzes revealed the absence of streptococci in the water after the different treatments. This is explained by their absence even in the initial raw water sample. On the contrary, total coliforms including Escherichia coli, initially present in the raw water sample have seen an increase in the Moringa-based treatment which settled for 12 h. This can be explained by the presence of organic substances in moringa. Theses organic substances served as food source for bacteria and fostered their proliferation during the long treatment time. Similar results were also obtained by Ngbolua et al. (2016). Valverde et al. (2018) have also shown in the same direction that M. oleifera seeds contain nearly 94% of organic substances and cause an increase of 100 to 400% of the rate of organic substances in the processed water. Other moringa-based treatment with 15 min decanting time, as well as aluminum sulphate-based treatment resulted in E. coli reduction by 100%. For aluminum sulphate-based treatment, this will be explained by the fact that it does not contain organic substances. As for Moringa seeds-based treatment, the short decanting time (15 min) does not allow the proliferation of microorganisms. For other coliforms, it is noted that the treatments with aluminum sulphate also resulted in a reduction of 100% of these microbial indicators. However, they were slightly present in moringa-based processed water and again when cactus extract was used as flocculant. The greater proliferation would be due to the fact that this biofloculant is also very rich in organic substances.
CONCLUSION AND PERSPECTIVE
This study proved that moringa seeds can be used as coagulant while cactus extracts can be used as flocculant. A comparison of the clarification capacity of this coagulant with aluminum sulphate shows that these coagulants are both effective. Also, the comparison of physicochemical parameters other than turbidity revealed that the pH of water decanted with moringa seeds remains nearly unchanged. This is an important advantage because its treatment does not require any acidity corrector, as opposed to aluminum sulphate. However, microbial indicators showed that treatment with aluminum sulphate was more beneficial because it eliminated all pathogenic microorganisms initially present in the water, whereas moringa seeds treatment such as a coagulant or even cactus extract as flocculant caused an increase in organic substances and induced a proliferation of bacteria over time. At the end of this study, it is inferred that moringa seeds are a good coagulant, but aluminum sulphate is better for large-scale water treatment. On the other hand, if some concerns were taken into account, the pair moringa seeds/ cactus extracts would be a viable bio alternative. It would be more effective than aluminum sulphate in the clarification of surface waters in our country. These concerns include identifying the active ingredients contained in both moringa seed and cactus extract; purifying these active ingredients in order to eliminate the organic substances and synthesizing them to obtain a more manageable and accessible product. | v3-fos-license |
2019-04-10T13:12:45.105Z | 2018-01-01T00:00:00.000 | 104354086 | {
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} | pes2o/s2orc | Fluorescent Sensors Based on Cu-Doped Carbon Quantum Dots for the Detection of Rutin
In this paper, Cu-doped carbon quantum dots (Cu-CQDs) were prepared by carrying out thermolysis of Na2[Cu(EDTA)] and hydroxylamine hydrochloride at 300 °C for 2 h. The asprepared Cu-CQDs were characterized by UV-Vis, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The Cu-CQDs exhibited good fluorescence property, high stability, excitationdependent emission behavior and a quantum yield of 9.8%. More significantly, the synthesized Cu-CQDs were developed for sensing rutin (RT) based on the fluorescence quenching of the prepared Cu-CQDs. A good linear relationship between the fluorescence quenching of Cu-CQDs and different concentrations of RT was obtained in the range of 0.1-15 μg mL with a correlation coefficient of 0.9959. The detection limit was as low as 0.05 μg mL (signal-to-noise ratio, S/N = 3). The possible mechanism of fluorescence quenching was explored, which was proved to be inner filter effect. Importantly, the proposed method was successfully applied in the detection of RT tablet samples and with satisfactory results, showing the practical applications.
Introduction
4][5][6][7][8] CQDs provided an exciting opportunity as alternative to heavy metal (e.g., Hg, Cd, Pb)based semiconductor quantum dots, 9,10 and were applied in biological imaging, 11,12 fluorescent sensors [13][14][15] and catalysis. 16The optical and fluorescence properties of CQDs arise from quantum confinement and edge effects, and also depend upon functional groups on the surface of CQDs. 179][20][21] Ethylenediaminetetraacetic acid (EDTA) is one of common starting materials for CQDs, which with a saturated Schiff-base-like structure could form EDTA chelate such as (Na 2 [Cu(EDTA)]), and the Na 2 [Cu(EDTA) pyrolyzed to form a flaky graphite conductive structure CQDs. 22utin (RT) (quercetin-3-ehamnosylglucoside, Figure 1) is a kind of flavonoid glycoside. 23Many studies have revealed that rutin is good for human health, such as antibacterial, antiulcer, anti-inflammatory, antiarrhythmia, antitumor, protective and expanding blood vessel. 24,25][35][36] In this work, Cu-doped carbon quantum dots (Cu-CQDs) were prepared by a facile one-step pyrolytic synthesis using Na 2 [Cu(EDTA)] as precursor.The saturated Schiff-base-like structure transforms into a Cu coordination complex chelated with graphene matrices during pyrolysis, which is confirmed by Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS).The formed Cu-CQDs were bluish green luminescence under 365 nm and showed emission maximum at 412 nm when excited at 354 nm.With the addition of rutin, the emission intensity at 412 nm of Cu-CQDs decreased.Based on the phenomenon, the method was established to determinate concentration of rutin.
Ultraviolet-visible (UV-Vis) absorption spectrum was performed on a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan).The fluorescence spectrum was recorded using molecular fluorescence spectrometer (Agilent Technologies, California, USA).The morphologies of the samples were characterized using a transmission electron microscope (Philips-FEI, Eindhoven, Holland).FTIR spectroscopy was performed on a TENSOR27 FTIR spectrometer (Bruker, Karlsruhe, Germany).XPS analyses were carried out on an X-ray photoelectron spectrometer (Kratos, Manchester, UK).XRD was recorded using a D8-advance X-ray diffractometer (Bruker, Karlsruhe, Germany), and a vortex mixer (Hanuo Instrument Co., Ltd., XH-B, Shanghai, China) was used in the experiment.
Synthesis of Cu-CQDs 3 g of Na 2 [Cu(EDTA)] and 2 g of hydroxylamine hydrochloride were put into the center of a quartz tube, and calcined at 300 ºC for 2 h at a heating rate of 5 ºC min -1 under N 2 atmosphere.Then the product was ground and dissolved in 100 mL water, and the suspension was treated with ultrasound (300 W, 40 kHz) for 15 min at room temperature.After centrifugation for 20 min at 10000 rpm, the upper brown solution was filtered with 0.22 μm membrane to remove the non-fluorescent deposited Na-salts.The solution was dialyzed with MD34 (3500 Da) dialysis tube for 48 h to remove the remaining salts and small fragments.The concentrated solution was dried at 60 ºC for 24 h, and Cu-CQDs powder was obtained.The amount of carbon dots obtained after purification was about 0.4-0.5 g.
Measure of fluorescence quantum yield
In general, quantum yield (QY) is measured by a standard fluorophore solution which shows a well-known quantum yield (Q).The QY of the Cu-CQDs was measured using quinine sulfate (QY of 54% at 360 nm, η = 1.33) as a reference, which was dissolved in a 0.1 mol L -1 H 2 SO 4 aqueous solution.The QY of the as-prepared Cu-CQDs was calculated according to equation 1: (1) where Q represents QY, A represents the absorbance at the excitation wavelength, I represents the integrated emission intensity, η represents the solvent refraction index.The subscript 'S' and 'R' refer to the samples and quinine sulfate, respectively.
Rutin tablet samples pretreatment 10 pieces of rutin tablets were ground to powder in a mortar, and 10 mg of the powder was dissolved in 25 mL of ethanol.Recovery experiments were performed by adding different concentrations of rutin to the treated samples.
Characterization of Cu-CQDs
The optical properties of the Cu-CQD were investigated by UV-Vis absorption and fluorescence emission spectra.The UV-Vis spectrum (Figure 2a) showed two shoulders at 269 and 321 nm.The absorption at 269 nm corresponded to π*-π* of C=C/C=N and the absorption at 321 nm was ascribed to n-π* transition of the C=O/C-NH 2 bond. 2 In addition, the color of the Cu-CQD solution was brown, and exhibited strong blue fluorescence (Figure 2a inset) when excited with a UV lamp (365 nm).As it can be seen in Figure 2b, the maximum emission wavelength of the Cu-CQDs solution was located at 412 nm with the excitation wavelength of 354 nm.Therefore, 354 nm was fixed as the optimal excitation wavelength for all the fluorescence measurements.Figure 2c exhibited the relationship between excitation and emission wavelength at room temperature.When excited Cu-CQDs by different wavelength at 314, 324, 334, 344, 354, 364, 374, 384 and 394 nm, the corresponding photoluminescence (PL) emission peak located at 398, 400, 404, 410, 412, 416, 426, 438 and 446 nm, respectively.Obviously, the emission wavelength was red-shifted with gradual lowering of PL intensity and with the increasing excitation wavelength.This excitation wavelength-dependent PL behavior could be contributed to the surface state of Cu-CQDs, which affected the band structure, and this phenomenon can be resulted by the different sizes, complex Cu doping or diverse surface emissive trap sites. 37Moreover, the fluorescent quantum yield (QY) of the Cu-CQDs in aqueous solution was measured to be ca.9.8% using quinine sulfate as reference.][40] As shown in Figure 3a, TEM image clearly revealed the uniform morphology and narrow size distribution of the synthesized Cu-CQDs.The prepared Cu-CQDs exhibited good dispersion and were uniform in shape with an average diameter of 4 nm.The high-resolution TEM image (Figure 3a, inset) revealed the crystallinity of Cu-CQDs with a lattice parameter of ca.0.21 nm, which is consistent with the basal spacing of graphite.Obviously, its dispersion and uniformity were in agreement with the fact that it actually dissolves.As shown in Figure 3b, XRD of the Cu-CQDs showed a strong peak at around 2θ = 27.7°,which corresponds to the graphitic structure. 41he thermal stability of synthesized Cu-CQDs was analyzed by thermogravimetry analysis (TGA), which was shown in Figure 3c.The TGA of Cu-CQDs showed a H 2 O evaporation weight loss below 100 °C and other sharp weight loss at around 350-600 °C, which is presumably due to the loss of CO and CO 2 from the oxygen-containing functional groups.Figure 3d showed the FTIR spectrum of the as-prepared Cu-CQDs.The broad band in the region of 3100-3500 cm -1 was assigned to stretching vibrations of C−H and N−H.The peak at 2939 cm -1 could be identified as the stretching vibrations of C−H.The peaks at 1662, 1404 and 1296 cm -1 were attributed to the stretching vibrations of C=O, C−N and C−O, respectively.The full XPS scan of Cu-CQDs was shown in Figure 3e, indicating that the synthesized Cu-CQDs contains C, O, N and Cu elements with the content ratios of 46.31, 27.14, 4.26 and 0.98%, respectively.In detail, Figure 3f shows the high resolution C1s spectrum of Cu-CQDs, the three peaks at 284.5, 285.7 and 287.6 eV could be assigned to C=C, C-N and C=O. Figure 3g shows the high resolution O1s spectrum of Cu-CQDs, the three main peaks at 530.5, 531.7 and 535.2 eV could be attributed to C=O, C-O-C and C-O-H.The detailed high resolution Cu 2p spectrum (Figure 3h) shows the existence of Cu in Cu-doped CQDs Cu-doped CQDs. 42,43The results from XPS analysis were in good agreement with FTIR.XPS and FTIR data demonstrated that the surface of Cu-CQDs were rich in water-soluble groups, such as hydroxyl groups, amino groups and carboxyl groups.
Mechanism of Cu-CQDs quenching
Inner filter effect (IFE) would occur when the absorption spectrum of quencher in the detection system overlapped with the excitation or emission spectra of CQDs.Generally, highly effective IFE requires that the absorption band of the absorbent should overlap sufficiently with the excitation of the fluorophore and/or the emission band.Therefore, it is important to choose an appropriate absorber and fluorophore pair in the IFE-based fluorescence nano-probe. 44Figure 4a showed the UV absorption spectrum of RT.It could be observed that there were two wide absorption peaks at 265 and 362 nm.The as-prepared Cu-CQDs were chosen as the fluorophore because of the large overlap between the excitation and emission spectra of the prepared Cu-CQDs and UV absorption of RT.The overlap would cause RT to shield excitation and emission light from Cu-CQDs.Therefore, the fluorescence of Cu-CQD could be successfully quenched by the absorbance enhancement of RT, which assures that the IFE occurs in a highly efficient way.Furthermore, fluorescence lifetime was considered to explore the possible mechanism of RT quenching the fluorescence of Cu-CQDs.As shown in Figure 4b, the average fluorescence lifetime of Cu-CQDs almost had no significant change after the addition of RT.The results indicated that the main possible mechanism is IFE. 45Besides, the essentially unchanged fluorescence lifetime of the system before and after the addition of RT also indicated that the fluorescence quenching is static quenching. 46The possible mechanism is shown in Scheme 1.
Effect of pH and ionic strength
To evaluate the stability of the Cu-CQDs, the effects of pH and ionic strength on the fluorescence intensity were further examined.As shown in Figure S1a (Supplementary Information (SI) section), the fluorescence intensity was obtained under different pH conditions.As the results suggested, the intensity of fluorescence of the prepared Cu-CQDs was increasing as the pH value rose from 2.0 to 5.0.Then the intensity was almost unaltered with the change of pH values in the range of 5.0-8.0.The pH owned a small impact on the fluorescence of prepared Cu-CQDs under weak acidic or alkaline condition, which may be due to the protonation or de-protonation of the functional groups on the surface of Cu-CQDs.Accordingly, it was demonstrated that the structure of Cu-CQDs was relatively stable and not simply ruined within the pH range of 5.0-8.0.We also have investigated the influence of ionic strength on the fluorescence of the Cu-CQDs.As shown in Figure S1b (SI section), different concentrations of NaCl solutions range from 0 to 2 mol L -1 were added into the Cu-CQDs.The fluorescence intensity maintained almost unchanged with the increasing NaCl concentrations, implying high stability of the Cu-CQDs even under high ionic strength environment.The stability of the effective fluorescence is conducive to the application of the Cu-CQDs.
Stability of Cu-CQDs for the detection of RT
Stability is an important point for Cu-CQDs to detect the RT.To confirm the fluorescence intensity of Cu-CQDs in the process of detecting RT, different incubation time was investigated.As shown in Figure 5a, the fluorescence intensity of Cu-CQDs decreased after the addition of RT, and maintained basically unchanged after 2 min.In addition, the fluorescence intensity could remain unchanged for 30 min.These results showed that the proposed method was fast and stable for the detection of RT.Therefore, the reaction time for the RT determination was set to 2 min.Moreover, Figure 5b showed the long stability of the as-prepared Cu-CQDs.It can be observed that the Cu-CQDs had good long stability.The Cu-CQDs still exhibited strong fluorescence intensity after 3 months storage at 4 ºC.Meanwhile, Cu-CQDs could be re-dispersed in water for using after being dried to powders in vacuum oven.The result showed that the fluorescence intensity had no obvious decrease.
Selectivity of RT detection
To evaluate the selectivity of Cu-CQDs as a fluorescent probe for the detection of RT, the fluorescence intensity of Cu-CQDs was examined in the presence of different possible interfering substances, including urea, Fe(NO 3 ) 3 , FeSO 4 , Hg(Ac) 2 , MgCl 2 , Mn(NO 3 ) 2 , Pb(NO 3 ) 2 , CdCl 2 , Zn(NO 3 ) 2 , baicalin, chlorogenic acid and quercetin.As shown in Figure 6, RT and quercetin could efficiently quench the fluorescence intensity of the Cu-CQDs, while the influence of the other possible interfering substances on the fluorescence intensity of the Cu-CQDs was negligible.However, the interference of quercetin against rutin would be avoided in the practical rutin detection in tablets samples.In following studies, the analytical capability of this fluorescence probe would be evaluated in rutin tablets without the potential coexistence of quercetin.As discussed in the characterization of prepared Cu-CQDs, the surface of the as-prepared Cu-CQDs was rich in hydroxyl groups, amino groups and carboxyl groups.The selectivity of Cu-CQDs towards RT is mainly due to the hydrogen bonding between the hydroxyl groups of RT and amine, amide and carboxyl groups of Cu-CQDs in addition to π-π interaction of the aromatic stack of Cu-CQDs with RT.
Sensitivity of RT detection
To estimate the sensitivity of Cu-CQDs toward RT, the fluorescence spectra were recorded with the addition of different concentrations of RT. Figure 7a showed that the fluorescence intensity centered at 412 nm of Cu-CQDs decreased gradually with the addition of various concentration of RT.It is discussed previously that this fluorescent quenching of Cu-CQDs after the addition of RT is owing to the inner filter effect (IFE).That is to say, the absorption of RT shields the excitation or emission wavelengths of Cu-CQDs, and lead to the fluorescence quenching.Additionally, this trend is in agreement with the fluorescence quenching photographs (insets in Figure 7b), in which the color of the solution gradually changes from bright blue to dark blue under the same conditions.A good linear relationship was obtained between the fluorescence quenching and different concentration of RT in the range of 0.1 to 15 μg mL -1 with a regression equation of F/F 0 = 0.059C + 0.0283 (correlation coefficient (R 2 ) = 0.9959) (insets in Figure 7a), where F 0 and F represent the fluorescent intensities of Cu-CQDs in the absence and presence of RT, and C is the different concentration of RT.The detection limit of RT is estimated to be 0.05 μg mL -1 at a signal-to-noise ratio of 3 (S/N = 3).The Cu-CQDs prepared in the work showed a relatively lower limit of detection when compared with other reported literatures which detected RT (Table 1).
Determination of RT in drug samples
To assess the practicality of the method, medicinal rutin tablets were chosen as real sample, which was ground by a mortar and dissolved in absolute ethanol to get the sample solutions.The sample solutions were diluted 50 times before using.Then the sample solutions were measured according to the as-proposed method with the data shown in Table 2.The recoveries were in the range of 97.5-102.5% and relative standard deviation (RSD) values were below 2.21%.The results indicated the reliable and efficient application of Cu-CQDs as fluorescent sensors for rutin determination.
Conclusions
In summary, highly fluorescent Cu-CQDs were synthesized by a green and low cost route from Na 2 [Cu(EDTA)] and hydroxylamine hydrochloride.The prepared Cu-CQDs showed good fluorescence properties and water solubility.Based on the phenomenon that the fluorescence intensity of Cu-CQDs could be selectively quenched by rutin, the Cu-CQDs were used as nano-sensors for the sensitive and selective detection of RT.This novel proposed method provided good linear RT detection in the range of 0.1-15 μg mL -1 with a detection limit of 0.05 μg mL -1 .The possible mechanism of fluorescence quenching was discussed, which was proved to be inner filter effect and static quenching.The successful application of the Cu-CQDs in the detection of RT in pharmaceutical products demonstrated the proposed method have great application prospect.
Figure 1 .
Figure 1.The molecular structure of rutin.
Figure 2 .
Figure 2. (a) UV-Vis absorption spectra (abs), inset: photographs of the solution of the Cu-CQDs taken under visible light (left) and 365 nm UV light (right); (b) fluorescence excitation (λ ex ) and emission (λ em ) spectra of the Cu-CQDs; (c) fluorescence emission spectra of Cu-CQDs at different excitation wavelengths from 314 to 394 nm in 10 nm increments.
Figure 5 .
Figure 5. (a) Fluorescence intensity of Cu-CQDs with RT at different reaction time and (b) fluorescence intensity of Cu-CQDs storage times.
Figure 6 .
Figure 6.Selectivity of the Cu-CQDs to different possible interfering substances.
Table 1 .
Comparison of different methods for the detection of RT
Table 2 .
Determination of RT in real samples (n = 3) RSD: relative standard deviation; HPLC: high-performance liquid chromatography. | v3-fos-license |
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} | pes2o/s2orc | Chemical , morphological , rheological and thermal properties of Solanum lycocarpum phosphorylated starches
Em vista da necessidade de amidos com características específicas, é fundamental o estudo de amidos não convencionais e de suas modificações, de acordo com as exigências do mercado consumidor . O objetivo deste trabalho foi estudar as características físico-químicas de amidos nativo e fosfatado da S. lycocarpum. O amido foi fosfatado com tripolifosfato de sódio (de 5 a 11%), sob agitação. Determinaram-se a composição química, a morfologia, a densidade, a capacidade de ligação à água fria, o poder de inchamento e o índice de solubilidade, a turbidez e a sinerese, as propriedades reológicas e calorimétricas. Não se detectou fósforo na amostra nativa, porém, a fosfatação gerou amidos modificados com teores de fósforo de 0,015, 0,092 e 0,397%, que se caracterizaram por absorver maior quantidade de água, a frio e a quente. Os resultados reológicos mostraram a forte influência do teor de fósforo na viscosidade dos fosfatados, apresentando temperatura de empastamento menor e viscosidade de pico maior que as do amido nativo. A
INTRODUCTION
Starch is an important ingredient for the industrial sector and is a major component of the human diet.It has diverse applications and its marketing is on the rise in many fields of industry (Limberger et al., 2008).Even so, this natural polymer requires modifications.According to Bemiller (1997), modified starches have better functional properties than natural starches, as they tend to retrograde less, their pastes are more stable during cooling and thawing and more transparent, their gels have good adhesion, improved texture and tend to form films.
Research on new unconventional starch sources and modifications is needed to find starches with rheological properties providing better quality to the final product to meet the demands of the consumer market.
Fruits of the Amazonian regions and the Brazilian Cerrado that have potential as starch sources are still little explored.For example, despite intense pharmacological use of Solanum lycocarpum Saint Hillaire, the wolf's fruit, there is little information on its starch characteristics and chemical modification, therefore, studies are necessary on its technological usefulness.
The fruit of this plant species may contain more than 20 g of starch per 100 g pulp (Junior et al., 2004).These starch granules are rounded shaped and relatively small (16.6 µm), containing 29.3% amylose and their pastes have regular stability, either cold or hot (Mota et al., 2009).These characteristics are comparable with those of rice and cassava starches.
However, the major disadvantage of unconventional starches is their restricted use in industry, because of some undesirable properties.These starches may have organic (lipids, proteins, pigments, etc.) and minerals compounds derived from the extraction process, which when interacting with amylose and amylopectin, can influence their properties and performance (Chan et al., 2010).
Water absorption and solubility, for example, depend on the starch crystalline structure, i.e., inter or intramolecular interaction of hydrogen bonds, which may break in hot water, reducing micelle strength by breaking hydrogen bonds and greatly increasing water absorption, resulting in swelling and solubilization of the starch granule (Swinkels, 1996).
However, starch properties can be improved by chemical modifications.Phosphorylation with sodium tripolyphosphate acid (STPA) is one of the most commonly used chemical modification of starches, as it is a salt of a relatively low cost and a simple process (Batista et al., 2010).Phosphate groups introduced into the starch chains causes repulsion between phosphate groups on adjacent chains and increases hydration.Phosphate groups are covalently bonded to molecules of amylopectin (Noda et al., 2007).The phosphorus content is an important factor in the variation starch functional properties, including gelatinization and retrogradation (Karim et al., 2007).
MATERIALS AND METHODS
Native starch was extracted from unripe S. lycocarpum fruits.Fruits were washed, peeled, chopped and ground in a circular rotor blade mill (MA-580, Marconi, São Paulo, Brazil), with water added with 5 g L -1 sodium metabisulfite (Synth) to prevent browning.The resultant slurry was separated by sieving through 75-250 micron stainless steel screen (Bertel, São Paulo, Brazil), followed by decantation and washing with absolute ethanol (Synth) to remove fats and excess water, drying in oven with air renewal and circulation (Marconi MA-035, São Paulo, Brazil), at 45 °C for 6 h.
The phosphorus (P) content was determined according to Brazil (2005), by spectrophotometry using a UV/visible (SP-2000UV) spectrophotometer at 420 nm.The contents of ash (method 923.03), moisture (method 925.10), ether extract (method 920.39) and crude protein (method 960.52, 5.83 conversion factor) were analyzed according to the methods described by AOAC (2005).Crude fiber was determined according to Brazil (2005).Starch content was determined using the technique described by Cereda et al. (2004).Amylose content was determined by blue value iodometric analysis (McCready & Hassid, 1943).
The shape and size of the starch granules were analyzed by a LEICA EC3 optical microscope (Wetzlar, Germany) and the images analyzed by the software Leica Application Suite v. LAS EZ. 2.0.0 (Leica Microsystems, 2010), which enables clear visualization of shape and estimation of size of the starch granules.
Absolute density (ρ) was determined as described by Leach & Schoch (1964) with some modifications.A 10 ml pycnometer with known mass was used to measure the pycnometer mass with xylene (b), xylene density (d) and the pycnometer mass with xylene and starch (c), using 5 g starch (a) (dry basis).The absolute density was calculated by Equation 1.
The binding capacity in cold water (BCCW) was measured as described by Gilles & Medcalf (1965).A 2.5 g sample was weighed into a centrifuge tube with 40 ml of distilled water and stirred in a Dubnoff pendulum shaker (ET-053, Tecnal Piracicaba, Brazil) for 1 h.Samples were centrifuged at 2200 rpm for 10 min.The supernatant was removed and the tube containing the pellet was weighed.The water bound to the starch was determined by equation 2: Eq 2.
The swelling power (SP) and water solubility index (WSI) were determined according to Schoch & Leach (1964) at temperatures ranging from 60 to 90 °C.
Turbidity and syneresis analyses of starch gel were performed according to Oliveira & Cereda (2003).A suspension of 8% starch in water was heated to obtain a translucent gel, which was distributed into five containers of 100 ml (approximately 20 ml of gel per container), cooled and stored at 4 °C.Turbidity was determined by absorbance at 640 nm in a spectrophotometer.Syneresis was determined as the percentage of water released by the gel in relation to the total mass after centrifugation at 3000 rpm for 15 min, during 5 days.
Viscosity was determined using a Rapid Visco Analyzer 4 (RVA Newport Scientific PTY LTD, Sydney, Australia), according to the manufacturer's recommendations.Suspensions of 2.5 g starch in 25 ml of distilled water were adjusted to 14% moisture and analyzed according to the following time/temperature regime: 50 ºC min -1 , heating from 50 to 95 °C at a 6 °C-1 min rate, held at 95 ° C for 5 min and cooled from 95 to 50 °C at a 6 °C min -1 rate.Viscosity was expressed as centiPoise (cP).From the profiles generated by the RVA, we evaluated the following parameters: maximum peak viscosity, minimum viscosity after peak, breakdown viscosity (difference between the maximum viscosity and minimum viscosity of paste maintained at 95 °C for 5 min), final viscosity and retrogradation (difference between final viscosity and minimum viscosity at 95 °C for 5 min) and pasting temperature (°C).
The calorimetric analysis was performed in a differential scanning calorimeter DSC Q200 (TA Instruments, New Castle, USA), according to Fakirov et. al. (1997).The calorimeter was calibrated using indium standard.For the starch gelatinization temperature, approximately 5 mg of sample of known moisture were placed in a hermetically sealed aluminum crucible.The scanning profile with balance consisted of reading temperatures between 5 and 110 ºC, with heating rate of 10 °C min -1 and nitrogen flow of 50 ml m -1 .The gelatinization enthalpy was calculated using the Universal Analysis software version 4.3A.
The experiment was arranged in a completely randomized design (CRD), with only one factor.Treatments were the phosphorus levels determined, in addition to the native starch, in the different starches prepared with 5, 7 and 11% of STPA.
The statistical analysis of the experimental data was according to Gomes (2009).Measurements of chemical composition (means of five replicates) were expressed as mean ± standard deviation and correlation coefficient (r).The effect of different phosphate levels on the chemical composition was qualitatively determined using one-way analysis of variance (ANOVA) at 5% probability level and significant responses were compared using the Tukey's test at 5% probability level.
Experimental data from other physicochemical properties were designed based on the phosphorus content and, in some cases, adjusted to a non-linear regression exponential model according to equation 3: where y is generic response function, x is the real variable, a, b and n represent the coefficients estimated by the method of least squares, with significance assessed by the t test at 5% probability level.The fit of the nonlinear regression model was evaluated by comparing the standard error of the estimate (SEE) using the mathematical Quasi-Newton method.
A cluster analysis by type of phosphate starch was performed with the means of the variables involved in the physicochemical properties using the procedure Cluster Analysis: Joining (tree clustering) of the Statistica 8.0 software (Statsoft, 2007), which is based on the Euclidean distance.The complete linkage method was selected for grouping.Additionally, the cluster analysis was complemented by the Principal Component Analysis (PCA).
The relationship between ash, starch, amylose, BCCW, breakdown viscosity, retrogradation and variation of gelatinization enthalpy with phosphorus content was obtained with the Pearson correlation coefficient (r), at 5% probability level.The correlation coefficient was interpreted as follows (Callegari-Jacques 2003): -If 0.00 <r <0.30, there is a weak linear correlation; -If 0.30 <r <0.60, there is a moderate linear correlation; -If 0.60 <r <1.00, there is a strong linear correlation.
The statistical analyses and the graphs were performed using the Statistica 8.0 software (Statsoft, 2007).
RESULTS AND DISCUSSION
In this study, the S. lycocarpum native starch showed a high purity (99.31% starch) and low levels of other fractions in its chemical composition (Table 1).Phosphorus was not detected and the content of amylose and amylopectin were 28.79 and 70.52%, respectively, agreeing with the findings of Mota et al. (2009).
Table 1 shows that contents of ether extract, fiber and protein remained constant, indicating that phosphorylation was successful.However, the ANOVA detected differences in moisture content (10.97 to 8.26%), starch (from 99.31 to 95.96%) and amylose (28.79 to 14.05%), which decreased with phosphorus content.
There were significant differences for ash content among the modified starches, increasing approximately 10, 13 and 16 times, when the native starch was phosphorylated up to 0.015, 0.092 and 0.397% phosphorus, respectively (Table 1), showing a strong positive correlation (r = 0.921).These results confirm the observations made by Limberger et al. (2008) with rice starch, who argued that the relationship between ash and phosphorus levels is caused by the introduction of phosphate groups into the starch chains.
Proportionally, the ash content due to the phosphorus introduction was responsible for the decrease in starch and amylose, showing strong inverse correlation (-0.999 <r < -0.975) and, consequently, the increase the proportion of amylopectin (r = 0.969) .
Because the introduction of phosphorus into S. lycocarpum starch changed the chemical composition, it also accounts for the changes in the physico-chemical characteristics, forming products with different properties.
The native starch granules showed a smooth surface with different sizes and shapes (Figure 1).Starch granules were rounded, elliptical, truncate and irregular in shape.In addition to these shapes, swollen granules were observed in phosphorylated starch (Figures 1B,1C and 1D), demonstrating that the native starch undergoes apparent damage after phosphorylation, which can influence paste properties.
The average diameter and density of native starch was 27.3 µm and 1.87 g mL -1 (Figure 2A), respectively.However, after phosphorylation, the diameter increased and the density decreased (up to approximately 42 µm and 1.35 g mL -1 , respectively) because of the swelling of the granules.The binding capacity in cold water of phosphorylated starches was also greater than the native starch (Figure 2B), from an initial BCCW value of 125.3% to 138.7, 153.4 and 175.1% for starches containing 0.015, 0.092 and 0.397%, of phosphorus, respectively.The correlation between BCCW and P was positively strong (r = 0.928).
However, these starches gelatinize when heated in a large amount of water and increase in size and partially solubilize, which can be seen by the swelling power values (Figure 3A) and the water solubility index (Figure 3B) respectively, increasing with temperature.Hoover (2001) explained that this fact occurs because the starch structure breaks, leading to weakening of hydrogen bonds and interaction of water molecules with the hydroxyl groups Rev. Ceres, Viçosa, v. 61, n.4, p. 458-466, jul/ago, 2014
Component (%)
Figure 2. Effect of phosphorus content on diameter, density and binding capacity to cold water (BCCW) of starch extracted from unripe fruits of S. lycocarpum: A) smaller diameter ( ), larger diameter ( ) and density ( ).B) BCCW.The introduction of phosphorus caused the swelling power and solubility index of native starch to increase (Figure 3).Daniel et al. (2006) reported that this phenomenon is due to the ability of phosphate groups to absorb larger quantities of water, that is, they have negative charges that repel each other, thus facilitating penetration and absorption of water (Wang et al., 2003).
Phosphorylated starches showed lower turbidity and syneresis than the native starch because of their higher binding capacity in cold water (Figure 4).According to Limberger et al. (2008), these starches form pastes that can be clearer and prevent retrogradation and hence syneresis, because they restrain greater contact between the amylose molecules.These molecules are solubilized during heating and leave the granule, preventing the formation of micro crystals responsible for retrogradation.
The viscosity profiles (Figure 5) confirm the strong influence of phosphorus introduction on the rheological properties of phosphorylated starches.The pasting temperature of these starches was lower and the viscosity peak was higher (around 95 °C and between 6260 and 6440 cP respectively), while for the native starch, the corresponding values were 70.7 °C and 4407 cP.This viscosity profile was also reported for phosphorylated starches of wheat and corn (Batista et al., 2010) and starches of corn, potato and beans (Chan et al., 2010).Batista et al. (2010) discussed that the reduction in paste temperature was due to starch gelatinization, while the maximum viscosity indicates the presence of intermolecular forces which strengthen the amorphous region of starch granules (Karim et al., 2007).Although it has greater stability to heat and breakdown (breakdown viscosity of 1104 cP), the native starch showed higher retrogradation (535 cP), with final viscosity of 3845 cP, whereas the phosphorylated starches showed higher breakdown viscosity (1705-2556 cP) and lower retrogradation (211-302 cP) with final viscosity between 4170 and 4816 cP.
For Chan et al. (2010), the evident breakdown viscosity in modified starches compared with native starches is probably due to the weak structure of the granules during the chemical modification, which facilitates the breakdown of the granular structure, while the tendency to retrogradation is influenced by the amylose content (Singh et al., 2005), since the amylose inhibits the rearrangement of the granular structure during the cooling of the gelatinized starch paste (Singh et al., 2003).
This may have occurred in this study, however, the breakdown viscosity decreases with the phosphorus content and the relationship between the breakdown viscosity and phosphorus content was strongly negative, with r = -0.826.In the case of retrogradation, it showed a strong negative correlation (r = -0.935)and positive correlation (r = 0.999) with the amylose content.
Starch thermal properties are described in Table 2.In this study, the variation in enthalpy was negatively correlated with phosphorus (r = -0.933),requiring energy of 9.7, 8.5, 8.1 and 6.4 J g -1 for the transition of starch granules with contents of 0, 0.015, 0.092 and 0.397% from the crystalline to the amorphous state, respectively.Bonds with phosphorous may have caused increase in the interaction forces.According to Acquarone & Rao (2003), inter and intra molecular bonds, in random positions in the starch granule, stabilize and strengthen the granule, and this occurs because during phosphorylation a double modification takes place, i.e., a combination of replacement with cross-linking, which hinders retrogradation (Wurzburg, 1986).
The hierarchical clustering dendrogram shows the formation of groups of genotypes with some degree of similarity and the dissimilarity among groups (Figure 6A).For a distance equal to 3, we observe the formation of three groups: group I formed by the native starch with 0% phosphorus; Group II with starches containing 0.015 and 0.092% of phosphorus; and group III with the phosphorylated starch with 0.397% phosphorus.
In the principal components analysis all studied variables contributed to component 1, with correlation greater than 0.70.However, the variables that contributed most to component 2 were phosphorus content and pasting temperature.
The cluster analysis and principal component analysis were in agreement on determining similarity between starches, forming three distinct groups (Figure 6).
Figure 3 .
Figure 3. Variation in swelling power (A) and water solubility index (B) of S. lycocarpum starch as a function of phosphorus content and temperature.
Figure 4 .
Figure 4. Variation in turbidity and syneresis of starch extracted from unripe fruits of S. lycocarpum as a function of phosphorus content.
Table 2 .P
Variation in thermal properties of starch extracted from unripe fruits of S. lycocarpum as a function of phosphorus content initial temperature, T p : peak temperature, T f : final temperature, r r r r rH: variation in enthalpy and r r r r rt: variation in temperature.
Figure 6 .
Figure 6.Cluster analysis and principal component analysis of S. lycocarpum starches as a function of phosphorus content.A) Similarity dendrogram and B) score dispersion graph of principal components (PC).
Table 1 .
Variation in chemical composition (dry basis) of S. lycocarpum starch as a function of phosphorus content* * Means of 5 repetitions ± standard deviation, ** Difference between starch and amylose contents. | v3-fos-license |
2019-03-20T13:04:22.957Z | 2015-03-20T00:00:00.000 | 83969395 | {
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} | pes2o/s2orc | Minor Volatile Compounds Profiles of ‘Aligoté’ Wines Fermented with Different Yeast Strains
The aroma of wine can be classified accordingly to its origin, in varietal aroma, pre-fermentative aroma, fermentative aroma and post-fermentative aroma. Although a number of flavor components are found in the original grape, the dominant and major compounds contributing to white wines are formed during alcoholic fermentation, in concordance with the yeast strain used. In order to highlight the influence of the yeast strain to the aroma composition of wines, wine samples from ‘Aligoté’ grape variety made with 8 different yeast strains were subjected to stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS) analyses. Also, a sensorial analysis of the studied wines was performed by a tasting panel consisting of 15 tasters. 38 minor volatile compounds were quantified by SBSE-GC-MS technique. Different concentration of the same compound and different aroma compounds were identified and quantified in wines obtained with different yeast strains. A wine finger printing was obtained by multivariate data analyses of aroma compounds grouped by chemical families. The analytical and sensorial analysis of the wine samples confirms that there are differences in aroma composition of the wines made with different yeast strains.
Introduction
'Aligoté' (AL) is a white grape used to make dry white wines, with significant plantings all over Eastern European countries including Romania, Russia, Ukraine, Hungary, Moldova and Bulgaria. It is an early-ripening variety and is more frost resistant than its more renowned cousins, meaning that its presence in these cooler sites is ensured for some time to come. 'Aligoté' is able to produce delicate wines when grown on the chalky soils, but will also thrive in sandier soils. In Romania is one of the most common varieties for white wines production (Rotaru, 2009). Even though it is highly cultivated in Romania, there are only few studies upon it.
Aroma is one of the most important parameters responsible for the quality of wines. An important quantity of aroma compounds is formed during the alcoholic fermentation of must, strictly dependent on the yeast strain (Cotea et al., 2009;Estévez, 2004). In this context, a study of the influence of the yeast strain to aroma composition of 'Aligoté' wines was necessary.
Several analytical extraction techniques have been employed to ensure the full characterization of the volatile profiles of grapes and wines, such as solid-phase extraction (SPE), solid-phase micro extraction (SPME), solvent-assisted flavor evaporation (SAFE), dynamic headspace sampling (DHS) and liquid extraction with organic solvents (LLE). Special attention has been given to methods which preclude the use of solvents, such as SPME (Bozalongo et al., 2007) and stir bar sorptive extraction (SBSE) (Marín et al., 2005;Tredoux et al., 2008). SBSE technique has been successfully applied in the last few years to aqueous matrices and wine analyses. A recent review by Castro et al. (2008) contains many references to studies that describe the applications of SBSE for the analysis of wine and oenological products, whereas recent articles have been focused on the characterization of grapes by their content in free and glycosidically bound aroma compounds and their use as markers of wine typicity (Arbulu et al., 2013;Gómez et al., 2012;Pedroza et al., 2010).
The aim of this study was to establish the minor aroma compounds profile of 'Aligoté' wines fermented with eight different commercial yeast strains, using SBSE-GC-MS technique.
Wine samples
Wines obtained from 'Aligoté' grape variety from Vrancea region, Romania, were studied. The wines were produced in the micro winery belonging to the Oenology department of the University of Agricultural Sciences and Veterinary Medicine, Iasi. Healthy ripped grapes of Vitis vinifera cv. 'Aligoté' collected at the industrial maturity were destemmed, crushed, and the must was homogenized and transferred to 9 glass containers in equal quantities, for the alcoholic fermentation. Eight different pure cultures of selected yeasts were added to unsterilized must, the 9-th must being left without inoculums, as a control (AV0). The pure yeast cultures were different strains of S. cerevisiae and they were marked from AV1 to AV8; AV1-AV7 were based on commercial products, while one S. cerevisiae yeast was selected from Iasi vineyard by the Research and Development Station for Viticulture and Wine, Iaşi (AV8). The yeast strains selected are the most frequent yeasts used by the wine makers in this region for dry white wines. The fermentation took place in 25 L glass containers kept in a room at 10 ºC, for 20 days.
General characteristics analyses
The analysis of pH, reducing sugars, titratable acidity and volatile acidity was performed by the official European Union methods (1990). The ethanol content was quantified by oxidation with dichromate according to Crowell and Ough (1979) and measuring the absorbance at 600 nm. The absorbance was measured in a spectrophotometer using 10 mm path length glass cells.
Gas-chromatographic quantification of minor volatile compounds
For the determination of the aroma fraction, stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS) technique was used. The aroma compounds were absorbed on polydimethylsiloxane (PDMS) coated stir bar or Twister, with 0.5 mm film thickness and 10 mm length, at room temperature during 100 min at 1200 rpm. The compounds were transferred to a gas chromatograph coupled to a mass detector and provided with a fused silica capillary column (HP-5MS stationary phase 30 m length, 0.25 mm internal diameter and 0.25 µm film thicknesses). The initial oven temperature was set at 50 ºC (held for 2 min), then raised to 190 ºC at 4 ºC/min and held for 10 min. For mass spectrometry analysis, electron impact mode (EI) at 70 eV was used. The mass range varied from 35 to 550 amu and the detector temperature was 150 ºC. Three replicates have been carried for each analysis.
The identification of volatile compounds was based on comparison of the linear retention indexes (LRI) calculated using the Van der Dool and Kratz's equation (1963) with those reported by specialized literature compilated in the NIST web book of Chemistry and on the matching of mass spectra of the compounds with the reference mass spectra of two libraries (Wiley7N and NIST08) coupled with the ChemStation software. The identification of some chromatographic peaks was also confirmed using pure compounds when available. The quantification was performed by the internal standard method, assuming a response factor equal to one. Relative response for each compound was obtained by using the Total Ion Area of the compound of interest divided by the Total Ion Area of the internal standard.
The characterization of the minor aroma compounds' profiles of wines was based on multivariate statistical analysis using Statgraphics Centurion XVI Software Package. The multivariate statistical methods used in this study were Multiple-Sample Comparison (MSC), Multiple-Variable Analysis (MVA) and Cluster Analysis.
Sensory analysis
The wines were assessed for color, aroma and flavor acceptability by 15 tasters in a panel in accordance with ISO 8586-1:1993. The tasting room was kept at 20 ºC and wines served in tasting glasses certified and coded. Evaluation of the quality of the wines was made using the method according to ISO 4121:2003, with options of desirable (7-9), acceptable (4-6) and undesirable (1-3). The final punctuations were calculated as mean value, taking into account the evaluation of each taster.
Results and discussions
The general characteristics of wines are shown Table 1. It appears that volatile acidity differs between 0.3 and 0.66 g acetic acid/L; ethanol between 10.82 and 12.38% vol, all the wines being semidry to semisweet wines. The final sugar content vary from 6.5 g/L (AV4) to 25.2 g/L (AV2), the yeast strains used for sample AV4 and AV5 being the ones that fermented faster the sugars in our experimental conditions. It has to be mentioned that the fermentation was stopped at all samples after 20 days, and the room temperature where the glass containers were kept during the fermentation was set at 10 ºC, the only variable being the yeast strain.
There have been identified 38 aroma compounds in wines (Tables 2 and 3), out of which 3 (Nerol, Limonene and Terpinolene) were classified as terpenes and nor-isoprenoids, 11 as aldehydes and ketones, 4 as alcohols (3-Methyl-1butanol, Furfuryl alcohol, Hexanol and Phenylethyl alcohol), (Moreno, 2012). This way of grouping the aroma compounds is based on the similarity of structure and functional chemical groups, having as one advantage that the components in the same group have similar aroma descriptors in addition to their similar physical and chemical properties. Terpenes and norisoprenoids usually have floral odor, aldehydes and ketones a herbaceous odor, alcohols show a vinous, sometime 'greenish' odor, benzene compounds have chemical, balsamic odor, while acids have a rancid, dirty, unpleasant odor and esters have a fruit-like odor.
One alcohol (3-Methyl-1-butanol), one acid (Decanoic acid) and two esters (Ethyl octanoate and Ethyl decanoate) registered the greatest values of all the compounds quantified. 3-Methyl-1-butanol have a fusel, alcoholic, pungent, cognac, fruity, banana and molasses odor and registered the highest values at AV3 and AV6 and the lowest values for AV0, AV1 and AV5. Decanoic acid have a metal, mild, fatty, coconut, bay oil odor and registered the highest values at AV5 and AV7 and the lowest values at AV0 and AV1. Ethyl octanoate show odor type as sweet, waxy, fruity and pineapple taste, being predominant in AV4, AV5, AV6 and AV7, with the lowest values in AV0 and AV1, while Ethyl decanoate that shows odor type as waxy, fruity, sweet, apple was predominant in AV4 and AV5 and registered the smallest relative value at AV1 (Table 3). Also, only 15 compounds were found in quantifiable levels in all 9 samples. The other 23 compounds were not found in quantifiable levels in at least one of the samples (Table 3) meaning that the yeast strain had an influence on the minor volatile compounds of wines.
Data obtained by SBSE-GC-MS cannot be considered as an absolute response or content in 'Aligoté' wines because of the limitations of the used Twisters, which are covered with the PDMS adsorbent. This is a non-polar adsorbent and consequently, only the non-polar compounds from the sample are preferentially adsorbed. In this sense, according to Nie and Kleine-Benne (2011) the PDMS Twister does provide a better recovery for non-polar terpene compounds (as alpha-pinene, beta-myrcene, delta-3-carene and d-limonene) then EG-Silicone Twister and conversely, this last Twister gives better recovery for more polar compounds as of some hydroxyterpenes (linalool, 4-terpineol, alpha-terpineol and nerolidol).
Compounds were identified by Linear retention index according to Van der Dool and Kratz (1963) in a HP-5MS capillary column (30 m/0.25 mm/0.25 µm, He) and MS spectrum from Willey and NIST libraries. Superscripts indicates the chemical group where the compound was framed: 1 terpenes and nor-isoprenoids, 2 aldehydes and ketones, 3 alcohols, 4 benzene compounds, 5 acids, 6 esters.
By summing the relative area of each of the individual compounds for the 6 established groups it is possible to establish an effective comparison among the studied wines (Fig. 1). This was made by multivariate data analyses, and the graphic presented in Fig. 1 shows the finger printing obtained this way. This plot can be used to group rows with similar characteristics, or to identify unusual cases. In our case, the sunray plot shows that there are differences among the nine samples, AV0 and AV1 being differentiated from the other samples mainly by their content in aldehydes and ketones, AV2 by its content in terpenes and nor-isoprenoids, AV3 and AV6 by their content in alcohols, AV4, AV5 and AV7 by their 125 content in acids and esters, while AV8 is differentiated mainly by its content in alcohols jointly with acids and esters. Also, a cluster analysis was performed using all the aroma compounds quantified, in order to create 1 cluster from the 27 observations supplied (one triplicate for each of the nine samples studied) (Fig. 3). The clusters are groups of observations with similar characteristics. To form the clusters, the procedure began with each observation in a separate group. Besides, it was combined the two observations which were closest together to form a new group. After recomputing the distance between the groups, the two groups then closest together were combined.
This process was repeated until only 1 group remained. The cluster obtained (Fig. 3) shows that it is possible to establish 9 groups by the largest increase in the Euclidean distances among samples matching with the 9 samples studied.
These results reveal that a good differentiation among the wines based on the aroma profile formed by each yeast strain used is possible. The most similar aroma profiles was registered at AV6 and AV7, with AV1 being the sample that have its aroma profile closest to the control sample. From Fig. 2 one can be concluded that the samples AV6 and AV7 had their aroma profiles similar one to another, the groups formed by these samples being the closest together, meaning that the used yeasts of AV6 and AV7 produced wines with similar characteristics under our experimental conditions. According to the same graph, AV4 and AV8 are also two samples with more or less similar aroma profiles, while AV5 had the most different aroma profile from all the studied samples.
The effect of the yeast strain to the aroma compound profiles of the wines was studied by means of a sensory analysis by a tasting panel. The results (Fig. 2) show that all the samples fermented with selected yeast strains differ comparing with the 127 Fig. 3. Cluster analysis of wines fermented with different yeast strains Fig. 2. Sensorial analysis of wines made with different yeast strains control sample. The most appreciated samples were AV7 and AV8 and the less appreciated fermented sample was AV1.
Conclusions
By SBSE-GC-MS technique 38 aroma compounds have been identified in wines fermented with 8 different yeast strains. Only 15 compounds were found in quantifiable levels in all samples, while the other 23 compounds were missing from at least one of the sample, meaning that the yeast strain had an influence on the minor volatile compounds of wines. A wine-fingerprint based on six aroma compound families was obtained, with samples being differentiated according to the relative area of the six aroma compound families established. A good differentiation between the wines aroma profiles was made using cluster analyses as chemometric tool, AV6 and AV7 having the most similar aroma profiles one to another. The sensory analyses carried out by 15 tasters confirm the analytical results. The most appreciated samples were AV7 and AV8 while the less appreciated fermented sample was AV1. | v3-fos-license |
2018-04-03T00:23:08.597Z | 2004-02-27T00:00:00.000 | 11830787 | {
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} | pes2o/s2orc | Toward Selective Covalent Inactivation of Pathogenic Antibodies
We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys20 residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys20 enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.
Specific antigen recognition by the variable domains underlies the pathogenic effects of certain Abs 1 produced as a result of autoimmune, allergic, and anti-transplant reactions. For instance, Abs found in myasthenia gravis (reviewed in Ref. 1) and hemophilia (reviewed in Ref. 2) bind important epitopes of the acetylcholine receptor and Factor VIII, respectively, that interfere with the biological activity of these proteins by a steric hindrance mechanism. Other Abs utilize their constant region to mediate pathogenic effects, but antigen recognition by Ab variable domains is the stimulus initiating these effects, e.g. Ab recognition of erythrocyte antigens stimulates complement activation by the constant region in autoimmune hemolytic anemia and incompatible blood transfusions. Similarly, allergen recognition by IgE bound to receptors for the constant region on the surface of mast cells stimulates their degranulation. In other diseases, the mechanism of Ab pathogenicity is less clear. For example, Abs to nucleic acids in lupus (reviewed in Ref. 3) and to thyroglobulin in Hashimoto's thyroiditis (reviewed in Ref. 4) are unambiguously disease-associated but additional immune abnormalities are also evident in these diseases and the precise functional effects of the Abs remain debatable. Recently, a novel variable domain mechanism underlying Ab pathogenicity has emerged, viz. the catalytic cleavage of antigens. Hydrolytic catalysts such as Abs to polypeptides (5)(6)(7)(8) and nucleic acids (9) hold the potential of permanent antigen inactivation. Moreover, catalysts are endowed with turnover capability, i.e. a single Ab molecule can hydrolyze multiple antigen molecules, suggesting that such Abs may exert functional effects that are more potent than Abs dependent on stoichiometric antigen recognition.
Abs that catalyze the cleavage of VIP have been identified in patients with autoimmune disease (10). VIP is a 28 amino acid peptide with important biological actions including immunoregulation via actions on T lymphocytes (reviewed in Ref. 11) and control of blood and airflow via actions on the smooth muscle (reviewed in Ref. 12). A model proteolytic Ab interferes with cytokine synthesis by cultured T cells accompanied by depletion of cellular VIP (13), and administration of the Ab to mice interferes with relaxation of airway smooth muscle (14). Proteolytic Abs to VIP appear to utilize a covalent catalytic mechanism reminiscent of serine proteases. This is suggested by studies in which replacement of the active site Ser residue resulted in the loss of catalytic activity (15) and by inhibition of catalysis by haptenic phosphonate diesters (10). These compounds form adducts with the activated nucleophiles of enzymes by virtue of the covalent reactivity of the electrophilic phosphorus atom (reviewed in Ref. 16) and have been developed recently as probes for the active site nucleophiles in Abs displaying serine protease and serine esterase activity (17,18), designated covalently reactive antigen analogs (CRAs).
As in the case of ordinary Abs, traditional noncovalent antigen recognition is hypothesized to underlie the specificity of the proteolytic Abs for VIP. Therefore, CRAs of the VIP sequence represent a potentially specific means to target the Abs by virtue of offering a reaction surface that combines covalent binding to the Ab active site with noncovalent binding at neighboring peptide epitope(s). Here we describe the antigen-specific covalent reaction of monoclonal and polyclonal Abs with a synthetic VIP-CRA compound. Despite positioning of the phosphonate group at a single site, Lys 20 , the covalent reaction resulted in irreversible inhibition of polyclonal Abs that cleave VIP at several peptide bonds located between residues 7 and 22. The results suggest the feasibility of targeted inactivation of individual Ab populations based on their antigenic specificity.
Abs-Monoclonal anti-VIP IgG clone c23.5 and control isotypematched IgG clone UPC10 (IgG2a, , Sigma) were purified from ascites by affinity chromatography on immobilized protein G-Sepharose (23). Polyclonal IgG from the serum of a human subject with chronic obstructive pulmonary disease (designated HS-2 in Ref. 24) was also purified by protein G-Sepharose chromatography. The recombinant light chain of anti-VIP Ab clone c23.5 (GenBank TM accession number L34775) was expressed in bacterial periplasmic extracts and purified by the binding of the His 6 tag to a nickel-affinity column (15). All of the Abs were electrophoretically homogeneous. Protein concentrations were determined with Micro BCA protein assay kit (Pierce).
CRA Adducts-Covalent binding assays were carried out as described previously (17,20). IgG (1 M) was incubated with compound 1 or 3 (10 M) in 10 mM sodium phosphate, 0.137 M NaCl, 2.7 mM KCl, pH 7.4, containing 1 mM CHAPS and 0.1% Me 2 SO (in compound 3 binding experiments) or 0.1% DMF (in compound 1 binding experiments) at 37°C. In some experiments, the reaction was conducted in the presence of human plasma collected in EDTA (pooled from eight healthy blood donors; 1% v/v). Aliquots of the reaction mixtures at 10, 20, 40, 60, 90, and 120 min were boiled in 2% SDS containing 3.3% 2-mercaptoethanol in a water bath (5 min) and then subjected to electrophoresis (4 -20% polyacrylamide gels, Bio-Rad). Following electroblotting onto nitrocellulose membranes (TransBlot, Bio-Rad), biotin-containing adducts were stained with a streptavidin-peroxidase conjugate and a chemiluminescent substrate kit (Supersignal, Pierce). Band density was expressed in arbitrary area units (AAU) determined using a Fluoro-STM Multi-Imager (Bio-Rad), ensuring that the densities were within the linear response range.
Catalysis Assays-Pro-Phe-Arg-AMC (0.2 mM, Peptides International, Louisville, KY) was incubated with Ab (0.8 M) in 96-well plates in 50 mM Tris⅐HCl, 0.1 M glycine, pH 8.0, containing 0.6% Me 2 SO and 0.025% Tween 20 at 37°C, and the release of AMC was determined by fluorometry ( em 470 nm; ex 360 nm, Cary Eclipse spectrometer, Varian, Palo Alto, CA). Preparation and assay of cleavage of [Tyr 10 -125 I]VIP were described previously (24). To determine whether the CRAs inhibit Abs irreversibly, IgG (2 M) was incubated (37°C) with compound 1 or 3 for 16 h in 50 mM Tris⅐HCl, 0.1 M Gly, pH 8.0, containing 2.5% Me 2 SO and 0.025% Tween 20. Unreacted compound 1 or 3 was then removed by chromatography of the reaction mixtures (0.2 ml) on protein G columns as described previously (23) (50 l of settled gel; washed with 0.8 ml of 50 mM Tris⅐HCl, pH 7.4; eluted with 0.2 ml of 0.1 M Gly⅐HCl, pH 2.7; neutralized with 1 M Tris⅐HCl, pH 9). 50-l aliquots of the recovered IgG (and IgG⅐CRA complexes) were incubated with [Tyr 10 -125 I]VIP (86,000 cpm) for 18 h, and peptide cleavage was determined by measuring the radioactivity soluble in trichloroacetic acid. Control IgG samples were incubated without CRA, chromatographed, and analyzed for VIP-cleaving activity in the same way.
RESULTS
VIP-CRA-Important features in design of the VIP-CRA (compound 3 in Fig. 1A) are as follows. (a) Inclusion of the electrophilic phosphonate diester group capable of selective reaction with activated nucleophiles, for example, is found in serine proteases (16). (b) The location of the positively charged amidino group is in proximity to the phosphonate to allow recognition by the model proteolytic IgG clone c23.5, which cleaves peptide bonds preferentially on the C-terminal side of basic amino acids (Arg/Lys) (23,25). (c) The incorporation of these groups is on the side chain of Lys 20 in the sequence of VIP. Hapten CRA 1 contains the phosphonate diester and amidino groups but is devoid of the VIP sequence. Location of the covalently reactive moiety at Lys 20 is based on observations that the Lys 20 -Lys 21 peptide bond is one of the bonds cleaved by monoclonal Ab clone c23.5 (23) and polyclonal human IgG preparations containing Abs to VIP (24). Peptide inhibitors of proteases customarily contain the covalently reactive group located within the peptide backbone or at the peptide termini (e.g. Refs. 26 and 27). In this study, our purpose was to maximize the opportunity for approach of the phosphonate group within covalent binding distance of the nucleophile contained in diverse Ab active sites. For this reason, the phosphonate group was placed at the side chain of Lys 20 using a flexible linker, which allows rotation at several C-C bonds (as opposed to inclusion of the phosphonate within the peptide backbone, which may impose a greater level of conformational constraints on accessibility of this group). VIP-CRA 3 was synthesized by the regioselective on-resin acylation as outlined in Fig. 1B. The VIP sequence was constructed by solid-phase peptide synthesis with standard 9-fluorenylmethoxycarbonyl chemistry with the exception that the 4-methyltrityl group was used for side-chain protection of Lys at position 20 (compound 4a). After selective removal of 4-methyltrityl, peptide resin 4b was acylated with compound 2, which was prepared from diphenyl amino(4-amidinophenyl)methanephosphonate and disuccinimidyl suberate. The resulting peptide resin 4c was treated with anhydrous trifluoroacetic acid to give compound 3, which was purified with HPLC, yielding a single species with the anticipated mass (m/z, 4071.4; calculated value, 4072.0).
Covalent Ab Labeling-Monoclonal Ab c23.5, raised by hyperimmunization with VIP, is characterized by strong recognition of the ground state of VIP (K d 1.9 nM; K m 0.34 nM) made possible by traditional noncovalent Ab paratope-epitope interactions (23). The catalytic site of the Ab is located in the light chain subunit and is composed of a serine protease-like catalytic triad (15). Here, we compared the covalent binding of this Ab by VIP-CRA 3 and hapten CRA 1. The isotype-matched Ab UPC10 (IgG2a, ) served as the control to determine background Ab nucleophilic reactivity independent of noncovalent recognition of VIP. The covalent reaction was visualized by boiling the reaction mixtures followed by denaturing SDS-electrophoresis and detection of biotin-containing adducts ( Fig. 2A, inset). Accumulation of covalent VIP-CRA 3 adducts with the anti-VIP Ab increased linearly as a function of time 2 with the light chain subunit accounting for the majority of the adducts (nominal mass 29 kDa determined by comparison with molecular mass standards). Adducts of VIP-CRA 3 with the control Ab were formed at lower levels. Similarly, hapten CRA 1 reacted with anti-VIP and control Abs slowly compared with the VIP-CRA and there was no preference for covalent binding of the hapten CRA at the light chain subunit. Apparent reaction velocities (V app ) were obtained from the slopes of linear regression curves fitted to the progress data by least square analysis ([Ab-CRA] ϭ V app ϫ t, where [Ab-CRA] represents the intensity of Ab-CRA adduct band in AAU and t is the reaction time). V app values are compiled in Table I. For the anti-VIP Ab, V app of the VIP-CRA 3 reaction with the light chain was 6.6-fold greater than the heavy chain. Hapten CRA 1 V app values for the two subunits of this Ab were nearly equivalent. V app for the reaction of VIP-CRA with the anti-VIP light chain was 66-fold greater than the corresponding reaction with the control Ab light chain. These observations indicate the selective nucleophilic reactivity of the anti-VIP light chain. Inclusion of VIP Step i, solid-phase peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (deprotection, 20% piperidine in DMF (3 min ϫ 2; 20 min ϫ 1); coupling, Fmoc-amino acid (2.5 eq), PyBOP (2.5 eq), 1-hydroxybenzotriazole (2.5 eq), and N,N-diisopropylethylamine (7.5 eq) in DMF (60 min)); step ii, 20% piperidine in DMF (3 min ϫ 2; 20 min ϫ 1); step iii, D-biotin (2.5 eq), PyBOP (2.5 eq), 1-hydroxybenzotriazole (2.5 eq), and N,N-diisopropylethylamine (7.5 eq) in DMF (60 min); step iv, 1% trifluoroacetic acid in CH 2 Cl 2 (5 min ϫ 10); step v, compound 2 (3 eq), 0.1 mM N,N-diisopropylethylamine in DMF (overnight); and step vi, trifluoroacetic acid-ethanedithiol-thioanisole-phenol (90:1:1:8, 2 h). All of the steps were done at room temperature. Protecting groups: Boc, t-butoxycarbonyl; tBu, tert-butyl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Trt, trityl; Mtt, 4-methyltrityl. devoid of the phosphonate group in the reaction mixture inhibited the formation of VIP-CRA 3 adducts with the anti-VIP light chain (Fig. 2B; inhibition in three repeat experiments, 41.0 Ϯ 7%). It may be concluded that selective covalent binding of VIP-CRA 3 by the anti-VIP Ab is made possible by noncovalent interactions due to the presence of the VIP sequence.
Pooled plasma from healthy humans was included in the reaction along with VIPase c23.5 to investigate further the selectivity of the VIP-CRA. As expected, the predominant VIP-CRA 3 adduct appeared at the position of the light chain subunit of the VIPase Ab (Fig. 2C). Little or no reaction of the VIP-CRA with plasma proteins and the control IgG subunits was observed. Similarly, the reaction mixtures of hapten CRA 1 yielded little or no adduct formation with plasma proteins or the exogenously added monoclonal Abs. Faint biotin bands were observed upon prolonged exposure in each of the lanes shown in Fig. 2C at a mass of 67-70 kDa. These bands presumably reflect a low level adduct formation of the hapten-CRA and VIP-CRA with albumin, the major protein present in plasma (see silver-stained electrophoresis lane in Fig. 2C). Covalent reactions of albumin with organophosphorus compounds have been reported previously (28,29).
Diisopropyl fluorophosphate (DFP), a well established serine hydrolase inhibitor, was previously reported to inhibit catalysis by anti-VIP light chain c23.5 (15). In this study, DFP inhibited the covalent VIP-CRA binding to the light chain (Fig. 2D), consistent the presence of a serine protease-like binding site(s).
Inhibition of Catalytic Activity-The cleavage of the model peptide substrate Pro-Phe-Arg-AMC by the recombinant light chain of anti-VIP Ab c23.5 has been reported previously (15). Site-directed mutagenesis studies have suggested that the light chain contains a catalytic triad similar to the active site of serine proteases (15). Here, the progress of Pro-Phe-Arg-AMC cleavage by the light chain was measured fluorimetrically by determining AMC generated due to cleavage at the Arg-AMC amide bond. As expected, a linear increase of AMC fluorescence was evident (Fig. 3A). Inclusion of VIP-CRA 3 in the reaction mixture inhibited the reaction in a time-dependent manner. The deviation of the progress curve from linearity in the presence of VIP-CRA suggests an irreversible inhibition mode (30). Inhibitory potency comparisons using VIP-CRA 3 and hapten Next, we turned to a human polyclonal IgG preparation isolated from a subject with airway disease (designated HS-2 in Ref. 24). Cleavage of VIP by this preparation has been attributed to IgG autoantibodies based on retention of the activity in Fab fragments, adsorption of the activity by IgG binding reagents, and absence of VIP cleavage by control identically purified human IgG preparations. N-terminal sequencing of VIP fragments generated by this IgG has identified the following scissile bonds: Thr 7 -Asp 8 , Arg 14 initially confirmed the ability of the polyclonal IgG preparation to cleave multiple peptide bonds in VIP. Three new radioactive peaks were generated from [Tyr 10 -125 I]VIP by treatment with the IgG (Fig. 4A). The observed radioactive product peaks in Fig. 4A probably represent mixtures of peptide fragments, as the VIP fragments generated by cleavage at the aforestated peptide bonds have previously been noted to elute from the HPLC with similar retention times (24).
To determine whether VIP-CRA 3 is an irreversible inhibitor, aliquots of the IgG treated with varying concentrations of this compound (10,20,40, and 80 M) were subjected to affinity chromatography on protein G to remove the unreacted inhibitor followed by assay of the cleavage of [Tyr 10 -125 I]VIP (Fig. 4B). Control IgG was subjected to an identical incubation without VIP-CRA followed by the chromatographic procedure. Dosedependent inhibition of catalytic activity was evident, and near-complete inhibition of catalysis was observed at VIP-CRA concentrations Ͼ20 M. The observed irreversible inhibition suggests that VIP-CRA forms covalent adducts with the polyclonal Abs, similar to its behavior with the monoclonal Ab examined in the preceding section. Selectivity of the VIP-CRA inhibitory effect was confirmed by comparison with hapten CRA 1. As expected, the VIP-CRA inhibited the cleavage of VIP more potently than the hapten CRA (IC 50 ϭ 7 and 36 M, respectively).
DISCUSSION
The following conclusions may be drawn from these data. (a) Functionally coordinated noncovalent and covalent interactions allowed nucleophilic anti-VIP Abs to form specific and covalent adducts with the VIP-CRAs. (b) The VIP-CRA inhibits each of the reactions involving cleavage of VIP at several peptide bonds, indicating its potential as a universal inhibitor of diverse anti-VIP catalytic Abs. The importance of noncovalent Ab paratope-antigen epitope binding in directing the VIP-CRA to the Ab nucleophile is evident from the following observations: lower reactivity of the anti-VIP monoclonal Ab with the hapten CRA devoid of the VIP sequence; limited reactivity of the irrelevant isotype-matched Ab and plasma proteins with the VIP-CRA; and inhibition of the anti-VIP Ab covalent reaction with the VIP-CRA by VIP devoid of the CRA moiety. Recently, CRA derivatives of other polypeptide antigens (human immunodeficiency virus glycoprotein 120 and epidermal growth factor receptor) have also been reported to form covalent adducts with specific Abs directed to these antigens with only minor levels of reactions evident with Abs directed to irrelevant Abs (31,32). Taken together, these considerations open the route toward permanent inhibition of individual Ab subpopulations based on their antigenic specificity.
The light chain subunit accounted for most of the covalent reactivity of the anti-VIP monoclonal Ab with the VIP-CRA. Reactivity with the hapten CRA serves as an index of Ab nucleophilicity independent of traditional noncovalent forces responsible for Ab-antigen complexation. Hapten CRA reactivities of the anti-VIP heavy and light chain subunits were comparable, suggesting that differences in intrinsic nucleophilic reactivity do not account for rapid formation of adducts of the light chain with the VIP-CRA. It may be concluded that the light chain nucleophile is in the immediate vicinity of the Ab noncovalent binding site and that the noncovalent binding interactions facilitate covalent binding. This statement is consistent with observations that the purified light chain of this Ab is capable of specifically catalyzing the cleavage of VIP (25). Previously, the purified light and heavy chain subunits of the Ab were reported to bind VIP independently determined by a conventional assay for noncovalent Ab-antigen complexes (K d for light chain, heavy chain, and intact IgG, respectively, 10.1, 6.8, and 1.9 nM) (33). In addition to the light chain, the heavy chain subunit appears to contribute noncovalent binding energy for Ab complexation with VIP but the heavy chain nucleophile does not seem to be sufficiently in register with the phosphonate group of the VIP-CRA to participate in the covalent reaction.
Additional evidence for irreversible and specific Ab recognition by the VIP-CRA is available from the catalysis assays. VIP-CRA adducts of the Abs obtained following the removal of unreacted VIP did not display catalytic activity. Catalytic cleavage of Pro-Phe-Arg-AMC by the recombinant light chain of the monoclonal Ab has been documented previously (15). This reaction is characterized by 57.5-fold higher K m than the cleavage of VIP by the light chain and is attributed to crossreactivity of the catalytic site with peptide substrates devoid of an antigenic epitope capable of participating in high affinity noncovalent binding. Pro-Phe-Arg-AMC cleavage by the light chain was inhibited more potently by the VIP-CRA than the hapten CRA. Similarly, the cleavage of VIP by polyclonal human autoantibodies to VIP was inhibited more potently by the VIP-CRA than the hapten-CRA.
Ab diversity poses an interesting challenge in achieving antigen-specific covalent inactivation of pathogenic Abs. Structural differences in the variable domains underlies Ab specificity for individual antigenic epitopes, and even Abs to small molecules presenting a limited surface area can contain structurally distinct binding sites (e.g. Refs. 34 and 35). Catalytic IgG preparations from patients with autoimmune disease cleave several backbone bonds in polypeptide (7, 24) and oligonucleotide (9) antigens. This may be due to the presence of multiple Ab species in polyclonal IgG preparations, each with a distinct scissile bond specificity. We have suggested previously that the nucleophiles enjoy some measure of mobility within Ab active sites that is not subject to restriction when noncovalent binding of Abs and antigens takes place (31,32). To the extent that this hypothesis is valid, Abs with differing peptide bond specificity could react covalently with the VIP-CRA even if the phosphonate group is located somewhat imprecisely in the antigenic epitope. In this study, the placement of the phosphonate on the Lys 20 side chain (as opposed to the peptide backbone) and inclusion of a flexible linker represent attempts to expand further the conformational space available for the covalent reaction. Complete inhibition of catalytic hydrolysis of VIP by polyclonal Abs that cleave several bonds between VIP residues 7 and 22 by the VIP-CRA was evident. Therefore, promising means to obtain antigen-specific covalent inhibition of diverse Abs include the exploitation of intrinsic conformational properties of Ab catalytic sites and the provision of enhanced access to the phosphonate group by manipulating the linker structure. In comparison, if Ab antigen binding is conceived as a rigid body interaction involving inflexible surface contacts, covalent inhibitor design must entail close topographical simulation of the transition state of each scissile bond and individual inhibitors must be developed to effectively inhibit different catalytic Abs. The importance of evaluating conformational factors in inhibitor design is supported by previous reports suggesting a split-site model of catalysis (31,32) in which antigen binding at the noncovalent subsite imposes little or no conformational constraints on the catalytic subsite, allowing the catalytic residue to become positioned in register with alternate peptide bonds as the transition state is formed.
As noted previously, catalytic Abs are proposed to contribute in the pathogenesis of autoimmune disease. Specific covalent inhibitors represent a novel means to help define the precise functional effects of the Abs. Such inhibitors may serve as prototypes for the development of therapeutic agents capable of ameliorating harmful Ab effects. In addition to inactivation of secreted Abs, reagents such as the VIP-CRA may be useful in targeting antigen-specific B cells. The feasibility of this goal is indicated by evidence that CRAs bind covalently to Abs expressed on the surface of B cells as components of the B cell receptor (36). Ab nucleophilicity may be viewed as an indication of their competence in completing the first step in covalent catalysis, i.e. formation of an acyl-Ab reaction intermediate. This is supported by observations that the magnitude of Ab nucleophilic reactivity is correlated with their proteolytic activity (31). A recent study suggests that noncatalytic Abs also contain nucleophiles but are unable to facilitate steps in the catalytic cycle following covalent attack on the antigen, viz. water attack on the acyl-Ab intermediate and product release (31). Regardless of the physiological functions of nucleophiles expressed by noncatalytic Abs, their presence may allow CRA targeting of Ab populations with established pathogenic roles, e.g. anti-factor VIII Abs in hemophilia. | v3-fos-license |
2021-05-11T00:04:04.756Z | 2021-01-13T00:00:00.000 | 234183244 | {
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} | pes2o/s2orc | Comparative Nutritional and Antioxidant Compounds of Organic and Conventional Vegetables during the Main Market Availability Period
: Seven winter and five summer vegetables produced under organic and conventional systems were collected from a supermarket seven times between January and April and between July and October for winter and summer vegetables, respectively, and their ascorbic acid and total phenolic content (compounds with proven antioxidant activity) as well as total antioxidant capacity, soluble solids and nitrates were determined. The results clearly indicated that, from the three factors studied (vegetable species, cropping system and sampling time), vegetable species made the highest contribution to ascorbic acid, phenolics, antioxidant capacity, soluble solids and nitrates. Results for each vegetable species showed that most organic vegetables appear to have lower nitrate content, some have higher phenolics, antioxidant capacity and soluble solids, and only few have higher ascorbic acid compared with conventional vegetables. The significance of the differences in nutritional and antioxidant value between organic and conventional vegetables is questionable, since vegetable species and sampling time can affect their nutritional value to a great or greater extent than the cropping system.
Introduction
Considerable evidence has made known the importance of vegetable consumption in protecting human health from various chronic diseases that have their origin in oxidative stress. This is due to the fact that vegetables are considered one of the main sources of ascorbic acid and antioxidants for human nutrition [1,2]. Based on the eating habits of adult consumers in the European Union, it is estimated that approximately 33% of the daily intake (65-138 mg) of vitamin C comes from the consumption of vegetables, among 21 foods or food groups; in this percentage, juices and other forms of products containing vegetables have not been included [3]. The biological functions of ascorbic acid in man appear to be related to its antioxidant properties [4][5][6]. Phenolic compounds are secondary metabolites in vegetables; their functions in plants are not always known, but some are structural polymers, UV screens, antioxidants and attractants, while others are involved in non-specific defense mechanisms [7]. One of the principal roles that have been proposed as part of the actions of phenolics in man is that of an antioxidant [8,9].
On the other hand, vegetables are also the major dietary source of nitrates, contributing over 80% of the nitrate intake in the European diet, which constitutes a serious threat to man's health [10].
There are several factors affecting the content of nutritional compounds in vegetables, e.g., genetic, environmental and agricultural factors [11], as well as postharvest handling and conditions [12,13]. Of the factors studied, much attention has been paid in the last decades to the cropping systems. Most studies have focused on comparative aspects of quality of organically and conventionally produced vegetables, but as concluded in most recent reviews [11,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32], inconsistent differences in nutritional compounds were detected; only for nitrate and ascorbic acid content were systematic tendencies apparent, with lower and higher levels in organic vegetables, respectively. On the other hand, in most studies, only macronutrients, vitamins or minerals were determined, while regarding antioxidants, data on vegetables are scarce [33] and the published results are contradictory [34].
From the point of view of consumers, the question remains: is there a difference in human nutrition between organically and conventionally produced vegetables? In order to accurately draw any conclusions, it is necessary to continue investigating the effects, if any, that the organic system has on the nutritional compounds of produced vegetables.
There are three ways of undertaking studies to compare conventionally and organically produced vegetables: cultivation tests, surveys and market-orientated supply studies, all having both advantages and disadvantages [35,36]. Taking into consideration the fact that the quality of fresh produce, as seen in the marketplace, can often differ from what might be expected from the produce that was harvested [37], the best way to evaluate differences between organic and conventional vegetables, facing the consumer, is to sample the products as purchased from the market [38], so that all factors which are not only related to the cropping system but which do influence product quality to a large degree are considered. For example, it is well-known [12,13] that most of the vegetables are highly perishable, and postharvest handling and conditions greatly affect their nutritional quality.
However, only a small number of studies have taken the approach of measuring nutritional value of vegetables purchased from the market [38,39]. Conklin and Thompson (1993) reported visible quality characteristics [40], Smith (1993) analyzed a range of minerals [41], Pither and Hall (1990) and Stopes et al. (1998) reported among others results on ascorbic acid and nitrates [42,43], while Faller and Fialho (2010) evaluated polyphenol content and antioxidant capacity of organically and conventionally produced vegetables from retail outlets [44]. No consistent differences between organic and conventional vegetables and a considerable range of values were reported. The most recent survey of consumers showed no significant differences between the sensory attributes of a range of organic and conventional fruits and vegetables available to the Irish consumer [38].
The present work is considered a retail market study which seeks to compare the nutritional quality of vegetables produced under organic and conventional systems. The quality parameters studied included ascorbic acid and total phenolics (compounds with proven antioxidant activity), total antioxidant capacity, soluble solids and nitrates in seven winter and five summer vegetables largely consumed, purchased from the retail market seven times at 15-day intervals during the main market availability period.
Plant Material and Handling
Vegetables included in the study were those that are widely consumed and also were available as certified organic products, e.g., cabbage (Brassica oleracea L. Capitata), carrot (Daucus carota L.), leek (Allium porrum L.), leaf and romaine lettuce (Lactuca sativa L.), potato (Solanum tuberosum L.) and spinach (Spinacea oleracea L.) (winter vegetables) as well as cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), green sweet pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and zucchini (Cucurbita pepo L.) (summer vegetables). Samples were purchased seven times in total, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket in Thessaloniki, Greece.
In each sampling date, the samples were collected in a quantity of 500-1000 g for each of the three replicates for each vegetable, with the exception of cabbage, in which a larger quantity was used (one head per replicate), thoroughly washed with tap water and stored in sealed plastic bags at −30 • C, prior to analysis. After partial thawing, only the edible part of each vegetable was used, based on common household practices (e.g., peeling of carrots and potatoes as well as removal of other non-edible parts such as fruit pedicel and calyx), and then macerated in a Waring blender. The macerated material was used for the determination of ascorbic acid, total soluble phenols, soluble solids, nitrates and antioxidant capacity.
Ascorbic Acid
For the extraction of ascorbic acid, 30 g of the macerated material was homogenized with 50 mL 1% oxalic acid solution in a Polytron (Kinematika GmbH, Eschbach, Germany) and centrifuged at 5000× g for 20 min. The ascorbic acid was measured in the filtrate by using Reflectoquant ascorbic acid test strips and an RQflex portable reflectometer (Merck, Darmstadt, Germany).
Total Soluble Phenols
Total soluble phenols were extracted by homogenizing samples of 10 g macerated material with 20 mL of 95% ethanol in a Polytron (Kinematika GmbH). The pellet, after centrifugation at 5000× g for 20 min, was again extracted with 95% ethanol and then once more with 5% ethanol in the same procedure. The total soluble phenols in the combined supernatants were determined using the Folin-Ciocalteu assay [45]. The standard curve was developed using gallic acid and the results are expressed as mg gallic acid equivalent (GAE) per g fw.
DPPH Radical Scavenging Activity
Radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined using a modified method of Brand-Williams et al. (1995) [46]. Samples of 5 g macerated material were homogenized with 25 mL 95% methanol in a Polytron (Kinematika GmbH) and centrifuged at 5000× g for 10 min. The supernatant was diluted with 95% methanol up to 25 mL, and 50 µL of the extract was added to 2950 µL of 100 µM DPPH methanolic solution in a test tube. The tubes were covered with parafilm, vortexed thoroughly and kept in the dark at room temperature. The reduction in the absorbance of the resulting solution was measured at 517 nm after 30 min. The control solution consisted of 50 µL methanol and 2950 µL DPPH. The standard curve was developed using ascorbic acid and the results are expressed as mg ascorbic acid equivalents antioxidant capacity (AEAC) per 100 g fw.
Soluble Solids
Soluble solid content was measured in the juice of the macerated material using a portable Atago PR-1 refractometer (Atago Co. Ltd., Tokyo, Japan).
Nitrates
For the extraction of nitrates, 10 g of the macerated material was homogenized with 50 mL distilled water in a Polytron (Kinematika GmbH) and centrifuged at 5000× g for 20 min. Nitrates were determined in the filtrate as described by Cataldo et al. (1975) [47].
For each organic to conventional comparison, a percent difference was calculated: (organic − conventional)/conventional × 100.
Data Analysis
Data analyses for both winter and summer vegetables were done by an analysis of variance (ANOVA) using the MSTAT version 4.00/EM (Michigan State University) as a completely randomized design, with three replications. The percent of the total variance for each of the main effects and their interactions were calculated from the sum of squares.
ANOVA for the main effects (vegetable species, farming system and sampling time) and their interactions showed that all three main factors as well as their interactions had a significant effect on the nutritional quality parameters measured for both winter and summer vegetables, but most of the total variance in both winter (60.3, 87.5, 61.3, 70.6 and 61.2% for ascorbic acid, total phenolics, antioxidant capacity, soluble solids and nitrates, respectively) and summer (53.5, 78.6, 62.6, 46.7 and 53.4% for ascorbic acid, total phenolics, antioxidant capacity, soluble solids and nitrates, respectively) vegetables was accounted for by differences between vegetable species. For this reason, ANOVA was performed again for each vegetable species separately.
Ascorbic Acid
Farming system had a significant effect on ascorbic acid content of cabbage, leek, romaine lettuce, cucumber, eggplant, tomato and zucchini but not on the content of carrot, leaf lettuce, potato, spinach and green sweet pepper (Table 1). On the other hand, sampling time significantly affected ascorbic acid content in all vegetables studied, while a significant interaction between farming system and sampling time was also detected for all vegetables with the exception of cabbage. However, most of the total variance for ascorbic acid in all winter and two summer vegetables (eggplant and green sweet pepper) was accounted for by differences between sampling times, while in cucumber, tomato and zucchini, most of the total variance was attributed to the farming system × sampling time interaction. Table 1. Analysis of variance for ascorbic acid of seven winter and five summer vegetable species produced under two cropping systems (organic and conventional) and purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket. DF, degrees of freedom; MS, mean square; %TV, % of total variance; ns, not significant effect. * Significant effect at the 0.05 level; ** significant effect at the 0.01 level; *** significant effect at the 0.001 level.
Source of
Among the vegetables studied, spinach from winter vegetables and green sweet pepper from summer vegetables had the highest ascorbic acid content with 32.1 and 17.8 mg/100 g fw, respectively, as an average of the seven sampling times and the two cropping systems. For winter vegetables, as an average of the both cropping systems, the highest ascorbic acid content was found in cabbage, carrot, romaine lettuce and potato from middle of January to middle of February, while for leaf lettuce, leek and spinach, it was found from the end of January to middle of February (data not shown).
As an average of the seven sampling times, organic cabbage, leek and zucchini had higher ascorbic acid content by 64, 46 and 29%, respectively, than the conventional ones, while organic cucumber, tomato, romaine lettuce and eggplant had lower ascorbic acid content by 33, 26, 20 and 17%, respectively, than the conventional ones ( Table 2). Table 2. Ascorbic acid, total phenolics, antioxidant capacity, soluble solids and nitrates of seven winter and five summer organically produced vegetable species as % of those produced conventionally. Samples were purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket. Data are presented as an average of the seven sampling times.
Phenolics
Farming system had a significant effect on phenolic content of cabbage, carrot, leek, romaine lettuce, spinach, cucumber, tomato and zucchini but not on the content of leaf lettuce, potato, eggplant and green sweet pepper (Table 3). On the other hand, sampling time significantly affected phenolic content in all vegetables studied, while a significant interaction between farming system and sampling time was also detected for all vegetables studied. However, most of the total variance for phenolics only in three vegetables (cabbage, spinach and cucumber) was accounted for by differences between farming system; in six vegetables (carrot, leaf and romaine lettuce, eggplant, green sweet pepper and zucchini) this was accounted for by differences between sampling times; and in three vegetables (leek, potato and tomato), most of the total variance was attributed to the farming system × sampling time interaction. Table 3. Analysis of variance for total phenolics of seven winter and five summer vegetable species produced under two cropping systems (organic and conventional) and purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket. Among the vegetables studied, spinach and green sweet pepper had the highest phenolic content with 112 and 80 mg gallic acid equivalents/100 g fw, respectively, as an average of the seven sampling times and the two cropping systems (data not shown). No clear tendency in the phenolic content was observed throughout the sampling period.
Source of
As an average of the seven sampling times, organic spinach, leek, tomato, cucumber and zucchini had higher phenolic content by 37, 34, 29, 20 and 16%, respectively, than the conventional ones, while organic romaine lettuce, carrot and cabbage had lower phenolic content by 16, 15 and 12%, respectively, than the conventional ones (Table 2).
Antioxidant Capacity
Farming system had a significant effect on antioxidant capacity of cabbage, carrot, leek, romaine lettuce, spinach, eggplant, green sweet pepper, tomato and zucchini but not on the capacity of leaf lettuce, potato and cucumber (Table 4). On the other hand, sampling time significantly affected antioxidant capacity in all vegetables studied, while a significant interaction between farming system and sampling time was also detected for all vegetables studied with the exception of carrot, romaine lettuce, cucumber and eggplant. However, most of the total variance for antioxidant capacity was accounted for by differences between sampling times in all vegetables, with the exception of spinach and tomato, in which most of the total variance was attributed to the farming system × sampling time interaction. Table 4. Analysis of variance for antioxidant capacity of seven winter and five summer vegetable species produced under two cropping systems (organic and conventional) and purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket. Among the vegetables studied, spinach and tomato had the greatest antioxidant capacity with 27.7 and 20.7 mg ascorbic acid equivalents/100 g fw, respectively, as an average of the seven sampling times and the two cropping systems. As an average of the both cropping systems, the highest antioxidant capacity was found in winter vegetables from the beginning of March to middle of April, while in summer vegetables (with the exception of sweet pepper) from the end of January to middle of February (data not shown).
Source of
As an average of the seven sampling times, organic tomato, spinach, leek, zucchini and romaine lettuce had higher antioxidant capacity by 67, 45, 33, 13 and 13%, respectively, than the conventional ones, while organic carrot, green sweet pepper, eggplant and cabbage had lower capacity by 23, 20, 20 and 17%, respectively, than the conventional ones ( Table 2).
Soluble Solids
Farming system had a significant effect on soluble solids content of cabbage, carrot, leek, leaf lettuce, potato, spinach, cucumber and zucchini but not on the content of romaine lettuce, eggplant, green sweet pepper and tomato (Table 5). On the other hand, sampling time significantly affected soluble solids content in all vegetables studied, while a significant interaction between farming system and sampling time was also detected for all vegetables studied, with the exception of green sweet pepper. However, most of the total variance for soluble solids was accounted for by differences between sampling times in all vegetables, with the exception of carrot, potato, cucumber and tomato, in which most of the total variance was attributed to the farming system × sampling time interaction. Table 5. Analysis of variance for soluble solids of seven winter and five summer vegetable species produced under two cropping systems (organic and conventional) and purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket. Among the vegetables studied, leek from winter vegetables and zucchini from summer vegetables had the highest soluble solids content with 9.44 and 4.63%, respectively, as an average of the seven sampling times and the two cropping systems (data not shown). No clear tendency in the soluble solids content was observed throughout the sampling period.
Source of
As an average of the seven sampling times, organic leaf lettuce, spinach, leek, zucchini, cucumber and carrot had higher soluble solids content by 30,21,17,8,8 and 5%, respectively, than the conventional ones, while organic cabbage and potato had lower soluble solids content by 11 and 9%, respectively, than the conventional ones (Table 2).
Nitrates
Farming system had a significant effect on nitrate content of carrot, leek, leaf and romaine lettuce, spinach, cucumber, eggplant, green sweet pepper and tomato but not on the content of cabbage, potato and zucchini (Table 6). On the other hand, sampling time significantly affected nitrate content in all vegetables studied, while a significant interaction between farming system and sampling time was also detected for all vegetables studied, with the exception of cabbage. However, most of the total variance for ascorbic acid in all winter vegetables, with the exception of carrot, and in three summer vegetables (eggplant, tomato and zucchini) was accounted for by differences between sampling times, while in carrot, cucumber and green sweet pepper, this was accounted for by differences between farming systems.
Among the vegetables studied, romaine lettuce from winter vegetables and zucchini from summer vegetables had the highest nitrate content with 330 and 193 mg/kg fw, respectively, as an average of the seven sampling times and the two cropping systems (data not shown). No clear tendency in the nitrate content was observed throughout the sampling period.
As an average of the seven sampling times, organic green sweet pepper, cucumber, eggplant, spinach, romaine lettuce, leaf lettuce, leek and tomato had lower nitrate content by 46,40,30,30,25,16,15 and 14%, respectively, than the conventional ones, while only organic carrot had higher nitrates by 74%, than the conventional one (Table 2). Table 6. Analysis of variance for nitrates of seven winter and five summer vegetable species produced under two cropping systems (organic and conventional) and purchased at seven sampling times, every 15 days, between January and April and between July and October for winter and summer vegetables, respectively, from a supermarket.
Discussion
The present study evaluated the levels of the main nutritional and antioxidant compounds which are actually available in vegetables when the consumers purchase them from the retail market. Three main factors were considered: vegetable species, cropping system and sampling time. The results clearly indicated that from the three factors studied, the vegetable species had the highest contribution in ascorbic acid, phenolics, antioxidant capacity, soluble solids and nitrates.
On the other hand, results for each of the vegetable species, when examined separately, showed that although cropping system affected the measured level of nutritional and antioxidant compounds in most of the vegetables studied, the highest contribution was found only in phenolic content of cabbage, spinach and cucumber, as well as in nitrate content of carrot, cucumber and green sweet pepper (Tables 1 and 3 -6).
Based on the comparison of the farming system, only nitrates had an apparent consistent tendency, with lower levels in organic vegetables (Table 2). This was in accordance with the conclusions of the recent reviews [11,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. As an average, organic farming lead to an approximately 22% reduction in the intake of nitrates by humans from winter leafy vegetables. It should be mentioned that the major sources for nitrates in Western diets are potatoes and leafy winter vegetables, the first because they are consumed in the largest quantity and the latter due to its high nitrate content [10].
Throughout the sampling period, the nitrate content for the samples analyzed did not exceed a level of 480 mg/kg fw. More important, the highest measured content of 477, 473 and 328 mg/kg fw for conventional leaf and romaine lettuce and spinach, respectively, were lower (one-eighth) than the specified maximum limits for these vegetables by the European Commission Regulation. Overall, the levels of nitrate content of vegetables found in this study were very similar to values that have been reported in the USA. In contrast, the corresponding values, especially for leafy vegetables, were much lower than values reported in Northwestern Europe [48]. This is in agreement with values found in previous surveys for nitrate content in leafy vegetables in Greece [49].
Such differences in nitrate content between organic and conventional vegetables may be attributed mainly to cultivation practices, since no appreciable changes for nitrate content in vegetables have been reported under normal postharvest handling conditions [50]. It is well-known than high nitrogen availably in the soil results in nitrate accumulation [10]. In the conventional systems, higher fertilization rates are usually applied with readily available mineral nutrients when compared with organic systems, in which organic fertilizers release nutrients more slowly than mineral fertilizers.
When it comes to secondary metabolites such as ascorbic acid and phenolics, which are the most abundant antioxidants in fruit and vegetables [51], inconsistent differences were found between vegetables produced under the two cropping systems examined (Table 2). For example, for potatoes, no significant differences were detected between cropping systems. Potatoes are considered the major source of both ascorbic acid [4] and total phenolics [52] in the European diet.
Accumulation of ascorbic acid is increased whenever nitrogen available in the soil is low [53]; thus it should be expected that organic vegetables produced under low nitrogen availability would contain higher ascorbic acid levels. However, this was not confirmed by the results of our study with vegetables purchased from the market. The differences in the ascorbic acid content found in our study may be attributed mainly to other factors than the cultivation practices; it is well-known [53] that postharvest handling and conditions significantly affect its content in fruit and vegetables. Temperature management during postharvest handling and operations is the most important factor to maintain ascorbic acid in vegetables; its loss is accelerated at higher temperatures and with longer storage durations.
It has been reported [54] that organic foods had elevated antioxidant levels in about 85% of the cases studied and that these levels were on average about 30% higher compared to foods produced conventionally. The collected data for phenolics compounds from 15 studies showed that their content in organic crops relative to those in conventional crops was in the range of −57 to +732% [11]. However, for vegetables produced under similar environmental conditions, inconsistent results have been reported. According to a study [55], both green and red sweet pepper fruit harvested from plants grown with the organic method showed significantly higher (about 42 and 27%, respectively) content of total phenolics compared with fruit from plants grown with the conventional method. On the contrary, two varieties of sweet peppers in a three-year study [56] and bell pepper fruits supplied by 24 commercial greenhouses during two consecutive growing seasons [57] did not display any differences due to cropping system when harvested at both green and red maturity stages. Moreover, it was reported that overall differences between harvesting times or between years were far greater than those due to the cropping system [57]. On the other hand, no differences were detected in the levels of individual and total phenolics in leaf lettuce and collards when they were cultivated under organic or conventional practices [34].
Few statistical differences were observed for polyphenol content and antioxidant capacity of six vegetables (potato, broccoli, onion, carrot, tomato and white cabbage) purchased from three different local markets in Rio de Janeiro, Brazil. Both nutritional parameters tended to be higher in organic vegetables [44]. In some organic vegetables (leek, romaine lettuce, spinach, tomato and zucchini), we also found higher antioxidant capacity by 13-67%, while in some others (cabbage, carrot, eggplant and green sweet pepper), it was lower by 17-23% when compared with conventional vegetables ( Table 2).
In our study, most of the organic vegetables (carrot, leek, leaf lettuce, spinach, cucumber and zucchini) contained more soluble solids than the conventional ones ( Table 2). Higher content of dry matter (the biggest part of which is soluble solids) has been reported for organic vegetables [35] that may be associated with better storage quality, resulting in less extensive decay [11]. Moreover, reduced water content may lead to a higher concentration of plant compounds and thus to a better taste in tomato [58].
Conclusions
The results clearly indicated that from the three factors studied (vegetable species, cropping system and sampling time), vegetable species had the highest contribution on ascorbic acid, phenolics, antioxidant capacity, soluble solids and nitrates. Results for each vegetable species showed that most of the organic vegetables appear to have lower nitrate content, some have higher phenolics, antioxidant capacity and soluble solids, and only a few have higher ascorbic acid compared with conventional vegetables. Therefore, the suspected differences between vegetables from the two cropping systems are not sufficiently consistent, and dietary importance is expected to cause a difference in nutritional value. Vegetable species and sampling time can affect their nutritional value to a great or greater extent than cropping system. Moreover, it should be emphasized that it is difficult to guarantee that the choice of organic rather than conventional vegetables will result in a higher concentration of bioactive compounds, since the cultivar would also play a crucial role in this respect, in addition to the vegetable species. | v3-fos-license |
2020-10-28T19:20:51.704Z | 2020-10-19T00:00:00.000 | 226354552 | {
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} | pes2o/s2orc | The influence of environmental condition on the creation of organic compounds in Pinus sylvestris L. rhizosphere, roots and needles
Studied organic molecules in Pinus sylvestris L. seem to have acted as a safety net for metal transport, chelation and sequestration, allowing adaptation and growth under highly polluted conditions. Pinus sylvestris L. is known for its ability to survive in areas of highly elevated metal pollution, such as flotation tailings. The aim of the study was to estimate the content of selected organic molecules (including aliphatic low molecular weight organic acids (ALMWOAs), phenolic compounds and terpenes) and the physiological mechanisms underlying differences in metal/metalloid tolerance of P. sylvestris growing in unpolluted (soil) and polluted (flotation tailings) areas. The dominant ALMWOAs in rhizosphere soil extracts were citric acid followed by malic and oxalic acids, whereas in flotation tailings malic and oxalic acids. In roots and needles, the content of ALMOWAs was significantly higher in P. sylvestris L. tissue from flotation tailings in comparison to soil. Phenolic compounds were detected only in roots and needles, with a generally higher content of nearly all detected compounds from flotation tailings. The composition of roots did not contain all the compounds detected in needles. The profile of needles additionally contained four hydroxybenzoic, protocatechuic and salicylic acids. In pine needles, 24 volatile terpenes were identified in total. The content of these compounds in pine needles from the polluted area was markedly different from the unpolluted area. The dominant volatile monoterpenes in P. sylvestris L. needles from the unpolluted area was three carene, while in pine needles from the polluted area monoterpenes α-pinene was dominant.
Introduction
Soil contamination with toxic trace elements is widespread and has a significant impact on such specific ecosystem functions as found in soil (Huang et al. 2016). It is particularly Communicated by E. Magel .
* Zuzanna Magdziak [email protected]; [email protected] associated with anthropogenic activities which currently pose a serious problem for ecological equilibrium, adversely affecting the maintenance of environments and proving destructive for flora and fauna (Rocha et al. 2016). Several species of woody plants, such as Salix and Populus (Tlustoš et al. 2007;Vamerali et al. 2009;, Quercus robur L. and Acer platanoides L. (Budzyńska et al. 2017a), Ulmus laevis Pall (Budzyńska et al. 2017b) and Betula pendula Roth. (Mleczek et al. 2016), have been shown to have a high potential for phytoextraction, uptake and accumulation of metals present in the soil matrix and can be a suitable choice for dendroremediation of extremely polluted areas. Pinus sylvestris L. (P. sylvestris) is a common woody species that can be used for restoring degraded soil ecosystems because of its adaptive capabilities (Placek et al. 2016). According to , P. sylvestris may survive in areas with high levels of metal pollution, such as flotation tailings. In studied specimens of P. sylvestris originating from polluted areas a significantly lower rate of phytoextration of selected elements was recorded in comparison with unpolluted areas ). Nevertheless, the P. sylvestris was able to survive, grow and develop. Differences in biomass and phytoextraction ability clearly demonstrate the influence of environmental conditions of growth, but at the same time indicate the importance of physiological changes in the studied plants. In the light of the diverse potential of the studied plants and limited literature data on the creation and exudation by woody plant roots of different organic compounds (aliphatic low molecular weight organic acids (ALMWOAs), enzymes, amino acids, phenolic compounds, simple and complex sugars, vitamins, purines, proteins and flavonoids) into the rhizosphere (Ryan et al. 2001;Adeleke et al. 2017;Magdziak et al. 2017), we investigated the effects of flotation tailings in organic biomolecule activity in the rhizosphere. The rhizosphere is the most important soil zone, where fundamental processes occur that are responsible for plant functioning. Any change in the soil chemistry, including metal/metalloid concentration, can impact the cycling of carbon and other nutrients (Huang et al. 2016). The above molecules, especially ALM-WOAs and phenolic compounds, are essential factors for nutrient acquisition (Dinh et al. 2017). They are present in the most abundance and are most reactive with metals (Koo et al. 2010); they play a role in alleviation of anaerobic stress in roots as well as mineral weathering (Adeleke et al. 2017). They additionally influence several soil processes, e.g. sorption and desorption (Wang et al. 2015), oxidation and reduction (Blaylock and James 1994) and precipitation and dissolution (Zhou et al. 2007). Moreover, in some of the previously mentioned studies, ALMWOAs and phenolic compounds have been found in plant tissues (mainly, roots and leaves) (Drzewiecka et al. 2017;Magdziak et al. 2017), where next to the rhizosphere, plants use ALMWOAs to transport, sequestrate and prevent cytoplasmic precipitation of toxic elements in cells, or in the case of phenolic compounds, in participation in adaptation and detoxification mechanisms mainly related to their structure and antioxidant properties (Ivanov et al. 2012;Jiang et al. 2017a, b;Benbettaieb et al. 2018). Plant cells also produce many other groups of chemical compounds. Extractives include, e.g. essential oils, which are multicomponent mixtures of mono-, sesqui-and diterpene compounds or phenylpropane derivatives (phenolics). They take various forms: hydrocarbons, alcohols, aldehydes, ketones, esters or ethers. Furthermore, essential oils may contain sulfur and nitrogen substances of coumarins. The percentage shares of individual components vary and depend on many factors, i.e., plant ontogenesis, geographical region, growing and harvest conditions, storage method and preparation for further processing (Gonçalves et al. 2003;Silva et al. 2003;Dob et al. 2005;Bakkali et al. 2008;Gilles et al. 2010;Stefanakis et al. 2013).
It is known that the chemical composition of oils depends on many factors (Grochowski 1990;Głowacki 1994). Unfavorable growth conditions, such as soil or air pollutions lead to a biochemical tree response to stress (including changes in the composition of essential oils) (Chojnacki and Cichy 1995). However, up to now, no studies have been able to show a clear relationship between the composition of the oils and toxic metal at high content level in the substrate on which the trees grow (Kainulainen et al. 1992;Supuka and Berta 1998;Fuksman 2002).
More studies are necessary to understand the mechanisms triggered by woody plants. Among toxic metals described in literature data, cadmium (Cd), lead (Pb) and zinc (Zn) are often presented as metal pollution stress which activates defense mechanisms related to changes in the creation and exudation of organic molecules. On the other hand, there is limited data on the response of woody species to their exposure to highly toxic elements, such as arsenic (As), mercury (Hg) or thallium (Tl).
For this reason, the present study extends the research carried out by , where P. sylvestris trees were grown in soil and flotation tailings characterised by specific chemical properties. The aim of the study was to estimate the content of selected organic molecules and physiological mechanisms underlying differences in metal/metalloid tolerance in P. sylvestris growing on soil (unpolluted area) and extremely contaminated flotation tailings (polluted area). The investigation focused on an evaluation of changes in aliphatic ALMWOAs, phenolic compound content in the rhizosphere and/or roots, as well as ALMWOAs, phenolic compounds and terpenes in needles. Such a determination is essential to verify how organic molecules might influence an increase in bioavailability and the accumulation of metals/metalloids in P. sylvestris organs. The above molecules were chosen as biochemical parameters of plant reaction to elevated concentrations of toxic elements in flotation tailings due to their accumulation, chelation, regulation and translocation as a probable mechanism of their detoxication as well as an antioxidant function (Viehweger 2014;Dinh et al. 2017).
Characteristics of P. sylvestris L. specimens and area description
The experimental materials were five specimens of 9-yearold P. sylvestris L. collected from two different experimental areas; an unpolluted area with soil characterised by an element concentration similar to the geochemical background of Polish soils and an area polluted by tailings from copper ore flotation processes. The trees had grown in Dystric Arenosols (unpolluted area); in a mixed forest in the Greater Poland province (mid-west region of Poland) and in Spolic Technosols (polluted area) in a flotation tailings disposal area in the Lower Silesian province (southwest region of Poland). They were not only characterised by similar height, but also by the size of their upper branches. From each experimental area five specimens of P. sylvestris L. were collected and analyzed. A detailed description of this experiment and characteristics of the mineral composition of the experimental areas was published in our previous study ).
Sample collection
In accordance with the method of Hammer and Keller (2002), the root zone from the unpolluted area (soil) and the polluted area (flotation tailings) was sampled from the surroundings of the P. sylvestris L. plant roots. Shaking and gentle cleansing by hand detached the soil attached to the roots. Samples of the root zone as well as soft roots (unheated) were separately preserved in polyethylene bags. Needles were collected from both the bottom and top of P. sylvestris L. trees and immediately placed in a portable refrigerator. Secured samples were then transported to the laboratory.
Preparation of rhizosphere, roots and needle samples of P. sylvestris L. for ALMWOAs and phenolic compound analysis
Environmental subsamples collected from the two significantly different environmental surfaces were carefully cleaned of the residue of roots and any other extraneous materials, homogenised, dried at room temperature, sieved by a nylon fibre sieve (< 1 mm) and stored for subsequent analysis.
Roots were immersed in 0.01 M HCl cold solution in order to eliminate trace elements adsorbed at the root surface (Adeniji et al. 2010), washed with cold deionised water and gently dried on a filter paper to remove excess water. Samples of P. sylvestris L. roots and needles (~ 1.0 g), ground to powder in a mortar chilled using liquid nitrogen were collected in 50 mL centrifuge tubes and stored frozen (− 80 °C) until analysis. The extraction method for ALMWOAs and phenolic compounds analysis in all the studied matrices was presented in detail by Magdziak et al. (2017) and Gąsecka et al. (2017). The obtained solutions were evaporated to dryness and stored frozen (− 80 °C) until analysis.
Samples prepared from the rhizosphere, roots and needles before HPLC analysis were dissolved in 1 mL of deionised water, centrifuged and filtered through filters of 0.22 μm immediately prior to chromatographic analysis. For the determination of ALMWOAs and phenolic compounds 10 μL of liquor was injected onto the HPLC column C 18 according to the method presented by Magdziak et al. (2017) and UPLC phenolic compounds analyses as described by Gąsecka et al. (2017).
Preparation of samples of P. sylvestris L. needles for terpenes analysis
Until analyses the needles were stored in a freezer at a temperature of − 18 °C after which they were cut manually to approx. 1-2 mm in length. Samples of 0.1 g were weighed and transferred to 15 mL glass vials equipped with a silicone-Teflon septum. In order to confirm the equilibrium of compounds in the headspace over the material each sample was subjected to a 10-min preincubation at a temperature of 40 °C in a water bath.
Microextraction on CAR/PDMS (carboxen/polydimethylsiloxane) fibre was run after the needles were placed into the vial through the septum. Absorption lasted for 10 min and, similarly to preincubation, it was run at 40 °C. Thermal desorption was run after the needles were introduced to the gas chromatograph injector heated to 230 °C. Desorption time was 5 min.
The GC oven temperature was set at 50 °C for 4 min and then programmed to 240 °C for 5 min at a rate of 10 °C/min, using He as a carrier gas (60 kPa). The injector and detector temperatures were maintained at 220 °C and 250 °C, respectively.
Volatile compounds from pine needles were analyzed by solid phase microextraction (SPME). The assay principle consists in the sorption of microscopic amounts of organic compounds in a thin, cylindrical layer of the stationary phase, which covers glass or quartz fibres. In analytical practice the headspace technique (HS-SPME) is most commonly applied, in which the fibre is placed in the headspace over the tested sample for a specific period of time, after which the fibre is placed within a needle. Sorption of volatile compounds depends on their affinity to the stationary phase. Silicone phases are the most frequently used group. The next stage consists in the desorption of the analyte to the gaseous phase which takes place after the fibre is placed in the injection port of a gas chromatograph (Lord and Pawliszyn 2000).
Chromatographic analysis was run in a GC/MS TRACE 1300 gas mass chromatograph (Thermo-Scientific) equipped with a DB 5 column (30 m × 0.25 mm i.d., film thickness 0.25 μm), a mass spectrometer with a single quadrupole and an ionization voltage of 70 eV, m/z scan range 35-350 Da. Qualitative analysis was based on a comparison of retention times and indices with NIST mass spectra libraries and other corresponding data (Adams 2007). Quantities of individual VOC components were calculated as relative concentrations (peak area percentages).
Statistical analysis
Statistical analysis was done using STATISTICA 10 and consisted of ANOVA followed by the post hoc Tukey's test. For the comparison of values characterising the studied material from two independent areas, the significances (*P < 0.05, **P < 0.01, ***P < 0.001) between control and treated plants were determined using a Student's Test. Principal Component Analysis, PCA, was performed to present the relationships between independent variables (the content of separately ALMWOAs, phenolic compounds and terpenes). To determine if the average content of individual ALMWOAs and phenolic compounds in two variants: root vs needle on the bottom and root vs needle on the top are equal, a t test with Welch correction for unequal variance (t test () in R) was used. These analyzes were performed independently for plants from unpolluted and polluted areas.
Results
ALMWOAs and concentration of phenolic compounds in rhizosphere zone of P. sylvestris L.
Concentration of ALMWOAs was lower in the rhizosphere zone than in roots and needles ( Table 1). The profile and concentration of acids were strictly dependent on the condition of P. sylvestris growth.
The total amount of identified organic acids in the rhizosphere zone of both soil and flotation tailings was almost the same. However, in the case of profile, the dominant acids in rhizosphere soil extracts from the unpolluted area were citric acid (~ 41.9 μg kg −1 dry weight (DW)) followed by malic and oxalic acids (~ 16.8 and 16.7 μg kg −1 DW, respectively), whereas from the flotation tailings root zone, malic and oxalic acids (~ 35.6 and 46.2 μg kg −1 DW, respectively). The rest of the acids were found at lower concentration and fumaric acid in both root zones was below the limit of detection. Phenolic compounds in the rhizosphere of P. sylvestris L. were also below the detection limit.
ALMWOAs content in roots and needles of P. sylvestris L.
Six ALMWOAs were identified in the root and needles extracts (acetic, citric, fumaric, malic, oxalic and succinic) of P. sylvestris L. plants. As shown in Table 1, the content of organic acids in the roots of pine had a tendency to increase. Among the detected acids, acetic, citric, malic and oxalic predominated in roots (Table 1). The amount of the aforementioned acids was significantly higher in P. sylvestris L. roots from flotation tailings in comparison to the unpolluted area. It should be noted that a significant increase was observed for citric and malic acids (~ 5.2 and ~ 8.5 times more, respectively, in comparison to control plants from soil), while, particularly in the case of acetic and oxalic acids, the situation was opposite (reduction was observed more than ~ 2 and 1.6 times, respectively). Fumaric acid was present in roots from unpolluted soil, while in roots of trees from flotation tailings it was below the limit of detection. The sum of ALMWOAs content in fine root samples increased ~ 2.4 times for plants harvested from the polluted area.
The dominant ALMWOAs present in P. sylvestris L. needles from the bottom and top of the control plants were acetic and oxalic (Table 1). The content of these acids decreased in whole needles of pine trees grown in the polluted area in comparison with the control plants. The exception were needles collected from the bottom in pine growing on flotation tailings, where the content of oxalic acid increased almost ~ 1.6 times. In the P. sylvestris L. needles collected from flotation tailings, the dominant acids were citric, malic and succinic. However, their content exhibited several differences related especially to the height of needles (bottom or top) and the area conditions of growth and accounted for approximately 14-37% of the total acid content. A significant effect on the citric acid creation in the bottom and top needles was noted, where their content increased in needles sampled from P. sylvestris L. growing on flotation tailings (more than ~ 3.5 times). In the case of malic and succinic acids, their content also increased significantly, but only in needles from the bottom (~ 5.9 and ~ 4 times higher, respectively). The content of these acids in needles from the top increased slightly (~ 1.18 and ~ 1.8, respectively). The lowest content of all the analyzed acids was observed for fumaric acid. In the case of needles collected from the bottom, its content increased in comparison to the control plants, where its content significantly decreased (~ twofold) in needles collected from the top.
More, for a graphical presentation of the obtained results and relationships between ALMWOAs determined in roots and needles of plants collected from unpolluted and polluted areas, a Principal Component Analysis (PCA) was performed. In the analysis of ALMWOAs, 64.75% (45.70 + 19.05) of variability was explained, what good reflects of observed relationships. PCA analysis allowed to show the high concentration of malonic acid in the rhizosphere of P. sylvestri from the unpolluted area ( Fig. 1).
Fig. 1
Principal component analysis for low molecular weight organic acids contents in rhizosphere and studied Pinus sylvestris L. organs (U Unpolluted, P Polluted). ALMWOAs: 1-acetic; 2-citric; 3-fumaric; 4-malic; 5-malonic; 6oxalic; 7-succinic; 8-sum Moreover, the higher content of malonic, succinic and sum of ALMWOAs in needles on the bottom and needles on the top, than in the same tissues of P. silvestris collected from the unpolluted area was observed. It is worth to underline a high content of citric acid in roots of plants from polluted than the unpolluted area was also recorded.
Besides, the content of ALMWOAs in roots was compared with their content in needles (both on the bottom and the top, separately) for plants collected from the unpolluted and polluted area. As it is shown in Table 2, significant differences were especially found for plants growing at the unpolluted area for almost all detectable ALMWOAs, but at different levels of significance.
However, no significant differences were found only for acetic acid determined in the root and the needle on the bottom and also citric acid and the sum of ALMWOAs in the root and the needle on the top, for plants collected from the polluted area.
The dominant was p-coumaric acid. Gallic and vanilic acids were also abundant. The content of the acids ranged from 10.5 to 79.9 μg g −1 DW. The content of phenolic compounds found in the contaminated area was higher; the Acetic * *** n.s *** Citric * *** ** n.s Fumaric *** *** * * Malic *** *** * ** Malonic nd nd nd nd Oxalic *** *** *** ** Succinic *** *** * ** Sum *** *** *** n.s increases were significant for caffeic, chlorogenic, ferulic, p-coumaric and sinapic acids and apigenin, katechin, kaempferol, quercetin, rutin and vitexin. The profile of needles at the bottom included phenolic compounds from roots enriched by 4-HBA, protocatechuic and salicylic acids. The content of phenolic compounds exceeded 1 μg g −1 DW for nearly all components (besides kaempferol and naringenin) up to ~ 31 μg g −1 DW. Among phenolic compounds protocatechuic acid > siringic acids > chlorogenic acid > sinapic > luteonin > quercetin > rutin were dominant and exceeded 10 μg g −1 DW. However, a significant increase in the content of phenolic compounds in the flotation tailings area was noted for 4-HBA, caffeic, ferulic, p-coumaric, protocatechiuc, salicylic (only in contaminated area), sinapic, syringic and t-cinnamic acids as well as apigenin, luteolin, naringenin and quercetin. The phenolic composition of top needles was similar to those at the bottom with the exception of 4-HBA and chlorogenic acid, which did not occur in the unpolluted area. The content of all the detected compounds was above 1 μg g −1 DW, but did not exceed 45 μg g −1 DW. A significantly higher content of phenolic compounds was detected in the flotation tailings, excluding apigenin. A reduction of the content of ferulic and galic acids was noted in the polluted area. The PCA analyses were also performed for phenolic compounds to show similarities and differences in their content in the studied trees of P. sylvestris, where 83.95% (66.59 + 17.36) of variability was explained. Dominant phenolic compounds in roots of P. silvestris from polluted and unpolluted areas were p-coumaric acid and kaempferol, while in case of needles on the bottom from both areas acids: protocatechuic, chlorogenic, synapic and also luteolin, quercetin and rutin (Fig. 2). Additionally, needles from the polluted area showed higher content of the majority of phenolic compounds than those from the unpolluted area.
Significant differences between the content of majority phenolic compounds determined in roots and needles (on the bottom and the top, separately) of plants collected from the unpolluted and polluted area were observed. According to data in Table 4, significant differences were found in almost all cases besides for sinapic acid in the roots and the needles on the top of plants from the unpolluted and p-cumaric acid for root vs. needle on the bottom from polluted area. Table 5, the characteristics of terpenes emitted from the needles of pines growing in diverse forest area types are shown.
A total of 24 compounds were identified, present in pine needles collected from the polluted and unpolluted area. However, significant differences were found in terms of amounts of individual terpenes (Fig. 4).
In pine needles from the polluted area α-pinene was the dominant compound. In needles collected from tree tops its mean content was 27.9%, while in needles sampled from the middle sections of trees it was 23.3%. In terms of the amounts emitted from these needles 3-carene ranked second. In needles collected both from the tops and middle sections of trees growing in the contaminated area the levels of this compound were very similar, amounting to 16.3 and 16.7%, respectively. Other monoterpenes were dominant in needles of pines growing in the unpolluted area. In needles from both the central and apical parts of trees 3-cerene was emitted in the greatest amounts, at 27.2 and 24.4%, respectively. In turn, α-pinene ranked second in terms of the emitted amounts among monoterpenes emitted from pine needles from the unpolluted area. The levels were 17.2% from needles in the apical parts and 16.7% from needles in the middle sections of trees. Differences between emissions of α-pinene from needles of pines growing in the polluted and unpolluted areas amounted to approx. 10% (27.9 vs. 17.2% and 16.9 vs. 23.3%). There are significant differences between the content of dominant terpenes, as confirmed by statistical analysis (Table 5). Greater amounts of β-pinene were also emitted by needles of pines from the polluted area (2.93 and 3.31%) in comparison to those from the unpolluted area (2.53 and 2.64%). The emission of bornyl acetate from pine needles from the polluted area (tree top-1.45% and middle sections-1.95%) was almost threefold greater than the emission of this compound from pine needles from the unpolluted area (tree top-0.59% and middle sections-0.67%).
In case of relationships between terpenes determined in P. sylvestris needles (on the bottom and the top) collected from studied areas, the PCA analysis 94.86% (66.75 + 28.11) of variability was explained, what showed a good reflect of observed relationships. From the unpolluted area, the higher content of 3-carene, 2-hexenal, 4(10)thujene, ς-terpinene and cubedol than in needles from the polluted area was observed (Fig. 3). An opposite situation for β-pinene, boryl acetate, germacrene D, camphene and tricyclene was recorded (Fig. 4).
Discussion
Different conditions of growing trees affect not only their growth and development but also the diversity of the amount and composition of biologically active compounds (ALMWOAs, phenolic compounds, terpenes, proteins, sugars) secreted to the rhizosphere, as well as created and accumulated in the roots or leaves or/and needles of trees (Viehweger 2014). In turn, the tree species is a decisive factor in the dendroremediation process and one that determines the essence of the plant's response to metals. Therefore, our research focused on the analysis of organic acids, phenolic compounds and terpenes as a response to the high content of metals in post-flotation wastes. Such studies are necessary, but what is most important for P. sylvestris is really new.
The presented work showed that P. sylvestris responded differently, a fact which was strictly dependent on the physicochemical parameters of the soil where P. sylvestris grew. Metal/metaloid (As, Hg or Tl) present in flotation tailings were the main metals influencing the exudation of ALM-WOAs by P. sylvestris roots; metal stress having been frequently reported to influence the exudation of ALMWOAs (Qin et al. 2007;Mucha et al. 2010;Meier et al. 2012). These elements have been previously shown to affect the exudation of oxalic, malonic and formic acids and were found as dominant in Ulmus leavis Pall. ) and oxalic, malic and succinic acids in Pteris vittata (P. vittata) (Das et al. 2017). Acids exudation has been frequently considered to be a resistance and detoxifying mechanism against metal contamination. This stimulation might therefore have been triggered to reduce metal toxicity by, for instance, complexing their ions. However, in the case of P. sylvestris, the significantly higher concentration of metals in flotation tailings inhibited the exudation of oxalic and malic acids, but highly induced citric acid secretion. The total concentrations of the analyzed acids were lower in comparison to the rhizosphere from the controlled area. In the case of oxalic acid a possible explanation of its decrease in concentration is exudation accompanied by plant uptake of the remaining ALMWOAs present in solution (Rocha et al. 2016), and in the case of malic acid being released, P. sylvestris roots will have taken up the metal-malic complexes formed during their growth. Nevertheless, an increased amount of citric acid in the rhizosphere could plays a detoxifying role at the level of the root zone, as well as increasing its amount in the root at the cellular level. Such a situation is highly probable according to literature data, but also in accordance with the results obtained from the analysis of acids in the roots. Metal-ALMWOAs complexes have been found in soil plant tissue (Walker and Welch 1987;Rellán-Álvarez et al. 2010;Rocha et al. 2016) and ALMWOAs are well-known for their ability to form complexes with metal ions, being involved in several processes such as metal tolerance, metal transport through xylem and metal sequestration in vacuoles (Clemens 2001). Therefore, in the presented study, ALMWOAs were determined not only in the root zone, but also in roots, thereby supporting the possible explanations for the content of the studied acids.
Woody plants can tolerate high metal pollution, utilising different defence mechanisms ). The phenomenon of rhizodesposition has been shown to be a plant defence mechanism against metal toxicity (Miyasaka et al. 1991;Ryan et al. 2001;Pinto et al. 2008) and ALMOWAs obtained in roots could be the main molecules for decreasing metal toxicity in P. sylvestris roots ). Moreover, the higher content of ALM-WOAs obtained in pine roots may indicate possibilities of compartmentation of metals by the studied ALMWOAs in roots and their probable role as ligands, confirming previous research Potdukhe et al. 2018). However, it should also be noted that concentration of ALMWOAs in the rhizosphere as well as in roots is strictly correlated, hence it may be simultaneously assumed that their ability to tolerate metal is also correlated. What is also important in the dendroremediation process is that pines have been shown to uptake increasing metal content in their roots as exposure to metal concentration increases , with more than two times the content of ALMWOAs in roots in comparison to the control.
These data have also been extended with respect to the content of acids in the pine needles. Regardless of the height from which the needles were removed (the bottom or the top part of the tree) the same profile of analyzed acids was found, although with a significant difference in the content. In the case of needles collected from the bottom, significantly higher concentrations of ALMWOAs were determined as compared to needles growing at the top of the studied P. sylvestris. In addition, the needles from the bottom part of the pines growing on the flotation tailings were characterised by a significantly increased content of citric, fumaric, malic, oxalic and succinic acids, while the needles from the top part showed a significant increase of citric acid only. In studies previously carried out and described by it was also found that needles from the bottom part accumulate significantly higher concentrations of metals present in the floatation tailings, and from the top part, significantly less. Therefore, the marked increase in the total acid content within the needles from the bottom part of P. sylvestris (more than 2.5 times), could be a specific pine response to stress and may be a potential mechanism of detoxification of accumulated metals (Ma et al. 1997;Meier et al. 2012). The obtained results could also confirm previous literature reports on woody plants where a significant increase in the content of metals in the above-ground parts of studied woody plants was correlated with an increased content of metals/metalloids in the substrate . The studied ALMWOAs could maintain a balance in metal homeostasis and keep the toxicity within physiological limits in whole plants. ALMWOAs could also be used as chelates to transport, sequestrate and protect the pine from the negative effects of their growth and development (Kutrowska and Szelag 2014), which may be indicated by significant differences in the number of marked acids in the roots and their amount in the needles.
Changes in the quantity and content of phenolics in different plant tissues are reported under metal stress because they play an important role in defense and adaptive mechanisms (Martinez et al. 2016;Pradas del Real et al. 2017;Ullah et al. 2018;Drzewiecka et al. 2018). The study on P. sylvestris shows the quantitative and qualitative changes in roots and needles. Moreover significant differences in nerealy all phenolic matabolites content were detected between needles and roots in polluted and unpolluted area. The phenolic compounds detected in P. sylvestris roots occurred predominantly in small amounts not exceeding 10 μg g −1 DW. Only three components (gallic acid, p-coumaric acid and vanillic acid) archived a content > 10 μg g −1 DW. The dominant acids were p-coumaric and gallic acid. However, only the changes in p-coumaric acid in roots from different localizations were significant. Data concerning phenolic compounds in roots under metal stress are rare. Most authors have focused on total phenolic contents, pointing out the changes that occur under metal stress. A hydroponic experiment with R. communis confirmed the increase of phenolic compounds in roots and leaves under Cd and iron (Fe)-stress; moreover, a correlation between phenolics and Cd was also confirmed (Ullah et al. 2018). Additionally, a lower content of phenolics in roots than in leaves has been recorded, although no profiling of the phenolics was conducted (Ullah et al. 2018). The induction of total phenolics in roots has also been noted in Phaseolus vulgaris under Cu treatements.
The significant increase of phenolic compounds in P. sylvestris from the contaminated area confirmed their contribution in defence mechanisms. Owing to their chemical structure, phenolic compounds are able to directly scavenge reactive oxygen species ROS, chelate ions of transition metals and suppress lipid peroxidation, making the diffusion of harmful radicals and peroxidative reactions difficult (Arora et al. 2000;Michalak 2006;Younis et al. 2018). ROS are generated via Fenton-Haber-Weiss reactions; however, some metals can induce the expression of enzymes such as lipoxygenases, which indirectly generate oxidation of polyunsaturated fatty acids (Kováčik et al. 2014). According to mechanisms proposed by Jiang et al. (2017a, b) for mangrove tissues and Cd-stress, the action of polyphenols may act in the apoplast and cytoplasm and include adsorption and lignification. Enhanced lignification is a natural barrier for metal ions which immobilise metals in the cell wall (Uraguchi et al. 2006). According to Jiang et al. (2017a, b), phenolic compounds are able to affect the bioactivity and translocation of toxic metals in plant tissues because the ability to chelate metals is associated with the presence of carboxyl and hydroxyl groups in the structure (Michalak 2006) and further translocation from cytoplasm to vacuole. Pradas del Real et al. (2017) pointed out that the increase in the contents of some phenolic compounds (quercetin-3-O-(2,6-di-O-rhamnosyl-galactoside and apiin) in the roots of Silene vulgaris under chrome (Cr)-stress was related to a defense mechanism based on free radical scavenging and chelation of Cr. The confirmed changes in the content of phenolic compounds in P. sylvestris roots in the contaminated area suggested that acids are important compounds in defence mechanisms (caffeic, chlorogenic, ferulic, p-coumaric and sinapic) and some flavonoids (apigenin, katechin, kaempferol rutin, quercetin and vitaxin). In P. sylvestris roots under Zn stress phenolic changes caused the opposite reaction, related to enhanced lignification (Ivanov et al. 2012). The results of our study pointed to a significant increase of phenolic compounds, suggesting they have a prevailing role in the defence mechanism connected with antioxidant mechanisms rather than a structural role associated with lignification, although the precursors of lignin are present in roots (caffeic, p-coumaric, chlorogenic, ferulic and sinapic acids). A very surprising fact is that gallic acid is one of the most dominant compounds in roots, but changes in its content are not significant, even though gallic acid is known to be a strong antioxidant with the ability to directly scavenge ROS.
The study of López-Orenes et al. (2018) on Pinus halepensis (P. halepensis) growing on mine tailings suggests that phenolics could participate in the defense mechanism as chelating ligands playing a role in the detoxification and accumulation of metals and metalloids.
The phenolic composition of needles depends on their position in the crown, contrary to observations of Norway spruce (Kopačková et al. 2015). This could be a result of the age of the needle as demonstrated by (Kopačková et al. 2015). Little is known about the phenolic composition of needles. Roitto et al. (2005) found flavonoids in needles of P. sylvestris and the content alternated in their responses to metal content (Ni and Cu) in soil. The results of our study confirmed a wide range of phenolic acids and flavonoids in needles on the bottom and the top with significant changes in the content of most of these components, especially in the top needles. The significant enhancement of phenolic acids and flavonoids pointed to their significant role in defense mechanisms, most likely associated with their ability to scavenge ROS (e.g. gallic acid, caffeic acid, p-coumaric acid (Benbettaieb et al. 2018) and functioning as ligands, protocatechiuc acid (Kováčik et al. 2010). In needles salicylic acid is present and the increase in its content in the contaminated area was observed as a result of its function in response mechanisms to environmental stressors, including metal stress (Arasimowicz-Jelonek et al. 2014;Drzewiecka et al. 2018). The reduction in ferulic and galic acids was probably connected with lignification, as also confirmed by (Ivanov et al. 2012).
Differing growth conditions for trees affect not only their growth and development but also result in varied amounts and composition of essential oils in needles and other plant parts. Both nutrients and pollutants found in the substrate influence the quantities and quality of the byproducts produced by plants (essential oils, tannins, resins, waxes) (Sembratowicz et al. 2008;Kupcinskiene et al. 2008;Sandre et al. 2014). Werner et al. (2004) recorded terpenoid contents in the roots of P. sylvestris seedlings growing in a substrate contaminated with heavy metals and stated that in the case of polluted substrate roots showed significantly lower terpenoid levels. Kupcinskiene et al. (2008) reported that the most heavily polluted environments trigger an increase in the concentrations of sabinene and β-pinene in needles of pines growing in the vicinity of industrial facilities. Along transects an increase was observed in the amount of some diterpenes and a decrease in the components of the shorter chain essential oils. In this study, lower amounts of certain diterpenes were recorded, i.e. ς-muurolene, cadina-1(10),4-diene and cubedol in needles of pines growing in the heavily contaminated environment. Judzentiene et al. (2006) reported the dominance of two basic monoterpenes (α-pinene and 3-carene) in oils from needles of pines growing in an environment with elevated ammonia levels in the air. However, it was stated in that study that the contents of a terpene, 3-carene (22.7-33.7%), exceeded those of α-pinene (19.8-35.0%). In contrast, needles from the polluted area showed an opposite relationship as the amounts of α-pinene (23.23-26.57%) exceeded those of 3-carene (16.57-16.89%). Similarly, to the authors of this study, Dziri and Hosni (2012) also observed an increase in the contents of β-pinene (29.5% and 22.0%) in needles of P. halepensis from a polluted area in comparison to control samples (11.6%). Supuka et al. (1997) recorded lesser amounts of terpenes in essential oils from needles collected in the area of Nitra than in oils from needles sampled in the arboretum. Moreover, like the findings of this study, analyses of the material collected in Nitra revealed an increase in β-pinene contents in needles from an urban area in comparison to needles collected from an unpolluted area. Urban conditions caused a changed proportion of α-pinene in relation to the following terpenes: α-phelandrene, 1.8-cineole, terpineol, citral and carvone (Supuka and Berta 1998). These changes discovered in the contents of terpenes in needles of Pinus strobus L. have shown that the group of secondary metabolites is an important biochemical marker of the environmental impact on woody plants. An increase in α-pinene contents in pine needles from an area polluted by dusts emitted by a cement plant was reported by Hosni et al. (2014). In the control samples those authors recorded 48.1% of this compound, while in samples from the polluted area it was 62.2% and 63.8%, respectively.
All the cited literature data are concerned with chemical changes in essential oils of plants growing in contaminated environments. Thus, the results of the performed experiment are consistent with the data published by other authors. According to Oviasogie et al. (2009) production of essential oil is an indicator of plant adaptation to habitat conditions and helps plants to easily adjust to environmental stress conditions, among others: drought, intense radiation, high temperature, high heavy metal contents. Natural selection favours the survival of plants with a higher concentration of essential oils which enhance their adaptive value (Stevovič et al. 2011). This plant defense mechanism caused an increase in the content of essential oils in the examined needles of P. sylvestris L. growing in a polluted area. However, the role of selected monoterpenes, especially α-pinene, in this mechanism requires further research.
Another interesting aspect related to monoterpens is their role in biotic interactions and soil processes. Ludley et al. (2009) analyzed the monoterpene content and distribution in the litters and roots of three species of conifer: Picea abies, Picea sitchensis and Pinus sylvestris. They showed an environmentally relevant content of α-or ß-pinene sufficient to increase mycorrhizal colonization of Picea abies root tips and to decrease the respiration rate of two species of saprotrophic fungi, in relatively natural substrata. Smolander et al. (2006) investigated the impact of monoterpens on soil microbes. According to authors there were differences in the response of both microbial biomass C and N to different monoterpenes, α-and ß-pinene being the most inhibitory whereas myrcene inhibited only microbial biomass N in the presence of arginine. Generally, they stated that monoterpens have negative effects on soil N transformations but may serve as a carbon and energy source for some soil microbes.
Conclusion
The obtained results reveal how the growth environment has a significant influence on the content of organic compounds in the rhizosphere, roots and needles. In the case of the concentration of ALMWOAs secreted into soil and into highly toxic flotation tailings, the root zone was similar. However, in roots and needles their value was significantly higher in tissue from the polluted area. Such an observation indicates that ALMWOAs may be one of the most important factors in P. sylvestris species, contributing to metal/ metalloid detoxification, involved in several processes such as metal tolerance, metal transport through xylem and metal sequestration in vacuoles. Moreover, the changes in the phenolic profile of roots and needles point to a major role in the defence mechanism associated with antioxidant mechanisms based on the scavenging ROS, functioning as ligands rather than having a structural role connected with lignification, although the precursors of lignin are present in roots (caffeic, p-coumaric, chlorogenic, ferulic and sinapic acids. The analysis of the results of volatile terpenes in pine needles growing in substrate contaminated with sludge and uncontaminated soil indicates a different dominance of the main monoterpenes. The contaminated soil had a significant effect on the reduction of 3-carene and the increase of α-pinene in pine needles.
Author contribution statement PG, MM and AM chose experimental areas and designed the study; WJC, BM, TK, PN and MM selected appropriate research material for testing; ZM, MG, BW, MZ analysed samples and took part in writing the manuscript, MG, AMP performed the statistical analysis; PN methodology. All authors contributed to writing the final version of the manuscript. | v3-fos-license |
2019-08-29T11:22:42.867Z | 2019-08-28T00:00:00.000 | 201661548 | {
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} | pes2o/s2orc | Prim-O-glucosylcimifugin enhances the antitumour effect of PD-1 inhibition by targeting myeloid-derived suppressor cells
Background Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that play an important role in immune evasion, PD-1/PD-L1 inhibitor tolerance and tumour progression. Therefore, MDSCs are potential targets for cancer immunotherapy. In this study, we screened an effective polymorphonuclear MDSC (PMN-MDSC) inhibitor from the Traditional Chinese Medicine Library and evaluated its synergistic antitumour effects with PD-1 inhibitor. Methods In the present study, we found that PMN-MDSCs accumulate heavily in the spleen and bone marrow of melanoma (B16-F10) tumour-bearing mice. Then, we determined the top 10 key proteins in the upregulated KEGG pathways of PMN-MDSCs in tumour-bearing mice through proteomics and Cytoscape analysis. The key proteins were then used as targets for the screening of PMN-MDSC inhibitors from the traditional Chinese Medicine Library (20000 compounds) through molecular docking and weight calculation of the docking score. Finally, the inhibitory effect of the inhibitor was verified through proteomics and metabolomics analysis in vitro and melanoma (B16-F10) and triple-negative breast cancer (4 T1) mouse tumour models in vivo. Results Traditional Chinese medicine saposhnikovia root extract Prim-O-glucosylcimifugin (POG) could bind well to the target proteins and inhibit the proliferation, metabolism and immunosuppressive ability of PMN-MDSCs by inhibiting arginine metabolism and the tricarboxylic acid cycle (TCA cycle). POG could also increase CD8 T-lymphocyte infiltration in the tumours and enhance the antitumour effect of PD-1 inhibitor in B16-F10 and 4 T1 mouse tumour models. Conclusions POG was successfully screened from the traditional Chinese Medicine library as a PMN-MDSC inhibitor. POG exhibited a good synergistic antitumour effect with PD-1 inhibitor. This study provided a potential option for enhancing the efficacy of PD-1 inhibitors in clinical applications. Electronic supplementary material The online version of this article (10.1186/s40425-019-0676-z) contains supplementary material, which is available to authorized users.
In the present study, we successfully screened prim-Oglucosylcimifugin (POG) as a PMN-MDSC inhibitor from the traditional Chinese Medicine library. In vitro and in vivo experiments showed that POG could inhibit the proliferation, metabolism and immunosuppressive ability of PMN-MDSCs, improve the tumour immunosuppressive microenvironment and generate a synergistic effect with PD-1 inhibitors in B16-F10 and 4 T1 mouse tumour models. This finding suggested that POG is a sensitiser for PD-1 inhibitors.
Materials and methods
Tissue processing and flow cytometry Bone marrow cells were flushed from the femurs and tibias with PBS with a syringe. The spleen samples were processed through mechanical dissociation, and tumour tissues were processed into single-cell suspensions by dissociating the tissues enzymatically for 1 h with 1 mg/ml type I collagenase (Sigma-Aldrich) in the presence of 50 units/mL DNase (Sigma-Aldrich). The cells were lysed with red blood cell lysis buffer and filtered with a 100 μm membrane, further washed with 1% BSA in PBS and blocked by non-specific staining with Fc Block (anti-mouse CD16/32 mAb; BD Biosciences). The samples were then stained with fluorescenceconjugated antibodies against the surface markers CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, eBioscience), Ly6C (clone HK1.4, eBioscience), Ly6G (clone 1A8-Ly6g, eBioscience), CD3 (clone 145-2C11, eBioscience) and CD8 (clone 53-6.7, eBioscience) and detected using flow cytometry (LSR BD Fortessa).
PMN-MDSC isolation and proteomic analysis
Bone marrow cells were harvested from naive C57BL6 mice and B16-F10 tumour-bearing mice and then processed into single-cell suspensions. Naive PMN-MDSCs and B16-F10 tumour-bearing PMN-MDSCs were sorted through flow cytometry. The sorted naive PMN-MDSCs and B16-F10 tumour-bearing PMN-MDSCs were then prepared for proteomics analysis. A fold change of more than 2 was defined as significantly different. Gene ontology (GO) analysis and KEGG enrichment analysis were performed using the DAVID database [19,20]. Protein-protein interaction networks were analysed with the STRING database [21].
Screening PMN-MDSC inhibitors by molecular docking and weight calculation of docking scores
To screen the natural inhibitors of PMN-MDSCs, we performed Cytoscape analysis on the proteins in the upregulated KEGG pathways of the PMN-MDSCs in B16-F10 tumour-bearing mice, and the top 10 key proteins were obtained according to the degree in the protein-protein interaction networks analysed with Cytoscape. The natural inhibitors of PMN-MDSCs were then screened from the traditional Chinese Medicine library (20000 compounds) by targeting the top 10 key proteins with molecular docking. The structures of the traditional Chinese medicines were downloaded from TCM Database@Taiwan (http://tcm. cmu.edu.tw/) [22,23] and showed in Additional file 1. The compounds with docking score absolute values of more than 4 for all the targets were selected as candidate compounds. Finally, the weight calculation of the candidate compounds was performed according to the degree of the top 10 key proteins in Cytoscape analysis and the compound docking scores with the top 10 key proteins by using the formula proteins degree × the compound docking scores. The effects of the first five compounds on PMN-MDSCs were verified in vitro. All results are expressed as the mean ± SD.
In vivo experiment B16-F10 cells and 4 T1 cells were purchased from KeyGen Biotech (Nanjing, China). The cells were cultured with RPMI 1640 (HyClone) with 10% foetal bovine serum (HyClone). Female C57BL/6 and BALB/C mice (6 weeks old) were purchased from the Animal Centre of the Academy of Military Medical Sciences (Beijing, China) and maintained in a temperature-controlled room with a 12 h/ 12 h light/dark schedule. All animal experiments conformed to the guidelines of the Animal Ethics Committee of the Tianjin International Joint Academy of Biotechnology and Medicine. To establish the B16-F10 tumour model, we resuspended 4 × 10 5 cells in 0.1 mL PBS, and the suspensions were subcutaneously injected into the right lateral flank of the C57BL/6 mice. After the tumour sizes reached 120-180 mm 3 , the animals were randomly assigned to six groups (n = 6): control, POG, 1H-indole-3-carboxylic acid, tetrahydrofolate, okanin and 6-methoxy-2-benzoxazolinone groups. The control group only received the vehicle (5% DMSO in 20% hydroxypropyl beta-cyclodextrin buffer). The POG (Push bio-technology, PS00838), 1H-indole-3carboxylic acid (SIGMA-ALDRICH, 284734), tetrahydrofolate (SIGMA-ALDRICH, T3125), okanin (YUANYE, JO51 5750) and 6-methoxy-2-benzoxazolinone (SIGMA-ALDRI CH, 543551) treatments were performed by intraperitoneal injection (100 mg/kg/day) for 14 days. To evaluate the dose dependence of POG, we randomly assigned the animals when the tumour sizes reached 120-180 mm 3 to three groups (n = 6), namely, control, POG-low and POG-high groups, which were administered vehicle (5% DMSO in 20% hydroxypropyl beta-cyclodextrin buffer) or 100 or 200 mg/kg/day POG intraperitoneally for 14 days. Tumour volume was measured every 3 days. Tumour volume was calculated as length × width 2 / 2.
Cell apoptosis assay
To determine the cytotoxic effect of POG on PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells, we sorted PMN-MDSCs and CD8 T-lymphocytes from the bone marrow and spleen of B16-F10 tumour-bearing mice, and the cells were cultured in MDSCs and T-lymphocyte media, respectively. The PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells were then divided into three groups: control, POG (50 μM) and POG (100 μM) groups. After 48 h, the cells were stained with an Annexin V/PI apoptosis detection kit (KeyGen Biotech, China) and analysed by flow cytometry after the cells were incubated in the dark for 30 min. All results are expressed as the mean ± SD.
Cell proliferation assay
To determine the effect of POG on PMN-MDSCs, CD8 Tlymphocytes and B16-F10 cells, we sorted PMN-MDSCs and CD8 T-lymphocytes from the bone marrow and spleen of the B16-F10 tumour-bearing mice, and the cells were cultured in MDSCs and T-lymphocyte media, respectively. PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells were stained with carboxyfluorescein succinimidyl ester (CFSE; Sigma), and PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells were divided into three groups, namely, control, POG (50 μM) and POG (100 μM) groups. After 48 h POG treatment, the CFSE dilution was determined using flow cytometry analysis [24,25]. All the results are expressed as the mean ± SD.
Proteomic and metabolomic analysis
To determine the effect of POG on PMN-MDSCs, we sorted PMN-MDSCs from the bone marrow of B16-F10 tumour-bearing mice, and the cells were cultured in MDSC medium. The PMN-MDSCs were then divided into two groups, namely, the control and POG (100 μM) groups. After 48 h, the cells were detected by proteomics analysis and UHPLC-QE-MS non-target metabolomics analysis. A fold change of more than 2 or 1.5 is defined as significantly different.
Western blot analysis
PMN-MDSCs were sorted from the bone marrow of the B16-F10 tumour-bearing mice, cultured in MDSC culture medium, and divided into three groups, namely, control, POG (50 μM) and POG (100 μM) groups. After 48 h, the cells were harvested, and the effect of POG on the expression of iNOS and Arg-1 in the PMN-MDSCs was determined by Western blot analysis. The cells were then washed with PBS and lysed in ice-cold lysis buffer with protease inhibitor cocktail (Sigma) for 30 min. The lysates were separated through SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked and incubated with primary antibody Arg-1 (Affinity Bioreagents, USA) and iNOS (Affinity Bioreagents, USA). The membranes were incubated with the second antibody (Santa Cruz Biotechnology, USA). GAPDH was used as the loading control. Protein expression was detected with an enhanced chemiluminescence detection kit (Millipore, USA). Densitometric analysis was performed with ImageJ software. All results are expressed as the mean ± SD.
ARG-1, ROS and NO measurements
PMN-MDSCs were sorted from the bone marrow of the B16-F10 tumour-bearing mice, cultured in MDSC culture medium, and divided into three groups, namely, control, POG (50 μM) and POG (100 μM) groups. After 48 h, the cells were harvested. ARG1 activity, ROS and NO were detected by using an ARG1 activity assay kit (Abcam), DCFDA (Invitrogen) and a Griess reagent system (Promega) in accordance with the manufacturer's instructions. All results are expressed as the mean ± SD.
T-lymphocyte proliferation assay
T-lymphocytes sorted from the spleens of the B16-F10 tumour-bearing mice were cultured in T-lymphocyte medium and stained with CFSE (Sigma). After the cells were co-cultured with PMN-MDSCs or M-MDSCs for 48 h, the cells were stained for surface markers with CD8 antibody (clone 53-6.7, eBioscience). The CFSE dilution in CD8 Tlymphocytes was determined through flow cytometry analysis [24,25]. All results are expressed as the mean ± SD.
IFN-γ production assays
T-lymphocytes sorted from mouse spleens were cultured in T-lymphocyte medium with or without POG. After 48 h, supernatant IFN-γ levels were quantified by ELISA (eBioscience) in accordance with the manufacturer's instructions. All the results are expressed as the mean ± SD.
Effect of POG combined with PD-1 inhibitor in vivo
The B16-F10 tumour model was established by using the method described above. To establish the 4 T1 tumour model, we injected the resuspended 4 × 10 5 4 T1 cells in 0.1 mL PBS into the fourth pair of the mammary fat pad of BALB/C mice. When the tumour volumes of B16-F10 and 4 T1 tumour-bearing mice reached 120-180 mm 3 , the mice were randomly distributed into the following groups (n = 6): control, POG, anti-PD-1 (Bio X Cell RPM1-14, rat IgG2a) and a combination of POG and anti-PD-1 groups. The control group was treated with the vehicle alone (5% DMSO in 20% hydroxypropyl beta-cyclodextrin buffer).
Statistical analysis
All statistical analyses were performed with GraphPad Prism7 software for Windows. Statistically significant differences were calculated by using Student's t-test. Overall survival analysis was performed by using the Kaplan-Meier method with the log-rank test, and a p value of < 0.05 was considered statistically significant.
Results
More PMN-MDSCs accumulated in B16-F10 tumourbearing mice than in naive mice When the tumour volume reached 1000 mm 3 , the naive mice and B16-F10 tumour-bearing mice were sacrificed, and the proportion of MDSCs in the spleen and bone marrow samples was measured. The results showed that the proportion of MDSCs in the spleen and bone marrow samples of the B16-F10 tumour-bearing mice considerably increased relative to the proportion in the naive mice. The CD11b + Ly-6G + Ly-6C low PMN-MDSC population in the bone marrow and spleen samples of the B16-F10 tumourbearing mice increased more significantly than the CD11b + Ly-6G − Ly-6C high M-MDSC population ( Fig. 1a-b). We sorted naive PMN-MDSCs, B16-F10 tumour-bearing PMN-MDSCs, naive M-MDSCs and B16-F10 tumourbearing PMN-MDSCs and then co-cultured these cells with CD8 T-lymphocytes at 4:1, 2:1, 1:1 and 1:2. The results of T-lymphocyte proliferation experiments showed that the ability of PMN-MDSCs to inhibit CD8 T-lymphocyte proliferation is stronger than that of M-MDSCs in B16-F10 tumour-bearing mice (Fig. 1c-d).
Differentially expressed genes of PMN-MDSCs in tumourbearing mice are mainly enriched in proliferation and metabolism-related pathways The PMN-MDSCs sorted from the bone marrow of the naive and B16-F10 tumour-bearing mice were collected for proteomic analysis and analysed by the DAVID database. The results of GO analysis showed that the upregulated genes of PMN-MDSCs in tumour-bearing mice were enriched in the function of proliferation and metabolism compared with PMN-MDSCs in naive mice. The enhanced functions included cell cycle, cell division, metabolic process-related biological processes (Fig. 2a) and oxidoreductase activity, NADH dehydrogenase activity and electron carrier activity-related molecule function (Fig. 2c). The upregulated genes associated with the cell cycle, cell division and metabolic process in the B16-F10 tumour-bearing PMN-MDSCs are shown in Fig. 2b. The upregulated genes associated with oxidoreductase, NADH dehydrogenase and electron carrier activities in the B16-F10 tumour-bearing PMN-MDSCs are shown in Fig. 2d. The KEGG analysis showed that the upregulated genes of PMN-MDSCs in B16-F10 tumour-bearing mice were enriched in cell proliferation and metabolic pathways, such as the metabolic pathways, tricarboxylic acid cycle (TCA cycle) and DNA replication (Fig. 2e). Furthermore, we analysed the proteinprotein interaction of the upregulated differential genes of B16-F10 tumour-bearing PMN-MDSCs by using the STRING database. The results showed that the upregulated genes were mainly related to cell metabolism (Fig. 2f). Based on the results of proteomic analysis, we found that the major enhancement pathways of the PMN-MDSCs in B16-F10 tumour-bearing mice were related to proliferation and metabolism. We then screen the key proteins in these pathways and the inhibitors that repressed these pathways by targeting the key proteins. We performed Cytoscape analysis of the proteins in the upregulated KEGG pathways and then ranked the top key 10 proteins in these pathways in accordance with the degree level in the Cytoscape analysis (Fig. 3a). We then screened natural inhibitors of MDSCs from the traditional Chinese Medicine library by targeting the top 10 key proteins with molecular docking. The compounds with a docking score absolute value with all 10 key proteins of more than 4 were selected as candidate inhibitors (Fig. 3b-c). The structure of 10 candidate inhibitors could be found in the Appendix. We then performed weight calculations of candidate inhibitors to sort the candidate inhibitors (Fig. 3d). Furthermore, we verified the inhibitory activities of the top 5 candidate inhibitors, namely, POG, 1H-indole-3-carboxylic acid [26], tetrahydrofolate [27], okanin [28] and 6-methoxy-2-benzoxazolinone [29], on PMN-MDSCs in vitro and in vivo. In vitro, bone marrow cells from the B16-F10 tumour-bearing mice were treated with the vehicle control and 100 μM of the top 5 compounds. After 48 h, we evaluated the percentages of (Fig. 3e). In vivo, a B16-F10 subcutaneous tumour model in C57BL6 mice was established for the evaluation of the antitumour effects of the top five compounds. We found that POG exhibited the best antitumour effect at a dose of 100 mg/kg and reduced the proportion of PMN-MDSCs in the bone marrow, spleen and CD45 + cells in tumours (Fig. 3f-h). POG also increased the number of CD8 T-lymphocytes in the spleens and CD45 + cells in tumour samples at a dose of 100 mg/kg. (Fig. 3i). e Inhibitory effect of the top 5 compounds on PMN-MDSCs (CD11b + Ly6G + Ly6C low ) in vitro. f The tumour growth curves of B16-F10 tumourbearing mice after the top 5 compound treatments (n = 6). g Body weight of B16-F10 tumour-bearing mice after the top 5 compound treatments (n = 6). h Relative proportion of PMN-MDSCs (CD11b + Ly6G + Ly6C low ) in bone marrow, spleen and CD45 + cells from tumours of control and top 5 compound-treated B16-F10 tumour-bearing mice (n = 6). i Relative proportion of CD8 T-lymphocytes (CD3 + CD8 + ) in spleens and CD45 + cells from tumours of control and top 5 compound-treated B16-F10 tumour-bearing mice (n = 6). The pooled data from three independent experiments are shown. All data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001
POG inhibits the proliferation and metabolism of PMN-MDSCs in vitro
To verify the inhibitory effect of POG on PMN-MDSCs, we evaluated the effects of POG on apoptosis and proliferation of PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells. The results showed that POG exhibited no cytotoxic effect on PMN-MDSCs, CD8 T-lymphocytes and B16-F10 cells. However, POG could specifically inhibit the proliferation of PMN-MDSCs (Fig. 4a-b). To detect the key cellular signalling pathways affected by POG, we performed proteomics and metabolomics analysis. Proteomic profile changes in the POG-treated PMN-MDSCs were analysed. Consistent with the results of the upregulated proteins of B16-F10 tumour-bearing PMN-MDSCs, the results of GO analysis showed that the functions of cell proliferation, oxidation-reduction process, nucleoside metabolic process-related biological processes (Fig. 4c), NADH dehydrogenase activity, oxidoreductase activity The pooled data from three independent experiments are shown. All data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 and ATP binding-related molecule function of the PMN-MDSCs were downregulated after POG treatment (Fig. 4d). KEGG analysis results showed that after POG treatment, the RNA polymerase, biosynthesis of amino acids and metabolic pathways of the PMN-MDSCs were downregulated (Fig. 4e). GSEA analysis also revealed that POG mainly inhibits the cell cycle of PMN-MDSCs (Fig. 4f). Furthermore, we analysed the protein interaction in the downregulated genes after POG treatment with the STRING database. The results indicated that the downregulated genes after POG treatment were mainly related to cell metabolism (Fig. 4g). These findings showed that POG could inhibit the proliferation and metabolism of PMN-MDSCs. The metabolomics results showed that POG mainly inhibited arginine and proline metabolism and the citrate cycle in the PMN-MDSCs. Through i Effect of POG on the IFN-γ content in CD8 T-lymphocytes. The pooled data from three independent experiments are shown. All data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 pathway analysis, we found that after POG treatment, the metabolic pathways of arginine into ornithine and citrulline regulated by ARG-1 and iNOS were downregulated, and the metabolism of citrulline and ornithine further affected the TCA cycle ( Fig. 5a-b).
POG inhibits the immunosuppressive capacity of PMN-MDSCs without affecting the function of CD8 Tlymphocytes in vitro
To verify the inhibitory effect of POG on arginine metabolism in B16-F10 tumour-bearing PMN-MDSCs, we used qRT-PCR and Western blot analysis to examine the effect of POG on the expression of iNOS and Arg-1 in PMN-MDSCs. The results showed that POG decreased the expression of Arg-1 and iNOS in PMN-MDSCs (Fig. 5c-d). We then examined the ARG1 activity, ROS and NO levels of the PMN-MDSCs after POG treatment. The findings revealed that POG inhibited ARG1 activity, the production of ROS and the production of NO in PMN-MDSCs ( Fig. 5e-g). To evaluate the effect of POG on the immunosuppressive capacity of PMN-MDSCs, we co-cultured the control and POG-treated PMN-MDSCs with CD8 T-lymphocytes at 1:1 for 48 h to detect the proliferation of CD8 T-lymphocytes. The results indicated that POG inhibited the inhibitory activity of the PMN-MDSCs on T-lymphocyte proliferation (Fig. 5h). To evaluate the effect of POG on CD8 T-lymphocyte function, we co-cultured CD8 T-lymphocytes with POG in T-lymphocyte medium for 48 h to examine the production of IFN-γ in T-lymphocytes. The results showed that POG did not influence the production of IFN-γ in CD8 T-lymphocytes (Fig. 5i).
POG exerts a dose-dependent antitumour effect and improves the immunosuppressive microenvironment of tumours
We established the B16-F10 subcutaneous tumour model in C57BL6 mice to evaluate the dose-dependent effect of POG on B16-F10 primary tumour growth and the tumour immunosuppressive microenvironment. The results showed that POG resulted in significant inhibition of tumour growth dose-dependently, and 200 mg/kg exerted no significant effect on the body weight of mice (Fig. 6a-c). To investigate the dose-dependent effect of POG on the immunosuppressive microenvironment, the proportion of PMN-MDSCs and CD8 Tlymphocytes in the spleens, bone marrow and tumours of mice in the control group and the POG-treated group was compared. The results showed that the proportion of PMN-MDSCs in bone marrow, spleen and CD45 + cells from tumours was reduced, and the proportions of CD8 T-lymphocytes in spleens and CD45 + cells from tumours were increased dose-dependently after treatment with POG ( Fig. 6d-e). To investigate the effect of POG on the immunosuppressive ability of PMN-MDSCs, we co-cultured PMN-MDSCs sorted from the bone marrow and tumours of the control and POG-treated B16-F10 tumour-bearing mice with CD8 T-lymphocytes at 1:1. CD8 T-lymphocyte proliferation was examined after 48 h. The results showed that the immunosuppressive ability of PMN-MDSCs from the bone marrow and tumours in the POG-treated group considerably decreased relative to that of the control group in a dose-dependent manner (Fig. 6f-g). To assess the effect of POG on CD8 T-lymphocyte proliferation and function, we sorted the CD8 T-lymphocytes from the spleens of the control and POG-treated B16-F10 tumour-bearing mice. After 48 h, we examined the proliferation and IFN-γ production ability of spleen CD8 T-lymphocytes. The results showed that POG did not affect the proliferation and IFN-γ production ability of the spleen CD8 T-lymphocytes ( Fig. 6h-i). These results showed that POG selectively inhibited the proliferation and immunosuppression of PMN-MDSCs and improved the immunosuppressive microenvironment of B16-F10 tumour-bearing mice, thereby inhibiting tumour growth in vivo in a dose-dependent manner.
POG enhances the antitumour effect of PD-1 inhibitor in B16-F10 and 4 T1 mouse tumour models Given that POG reduced PMN-MDSCs in the bone marrow and tumours and increased CD8 T-lymphocytes in the spleens and tumours of the B16-F10 tumourbearing mice, we hypothesised that POG enhances the antitumour effect of the PD-1 inhibitor. We established mouse B16-F10 subcutaneous and 4 T1 in situ tumour models. The results showed that the combination of POG and PD-1 mAb group showed better antitumour effects than did the POG and PD-1 mAb groups. The combination index [30] of POG (100 mg/kg) and POG (200 mg/kg) with PD-1 mAb was 1.27 and 1.32 in the B16-F10 tumour model and 1.23 and 1.21 in the 4 T1 tumour model, respectively (Fig. 7a-d). The combination group also showed the best ability to prolong survival time of B16-F10 and 4 T1 tumour-bearing mice compared with the other groups ( Fig. 7e-h). These results indicated that POG and PD-1 inhibitors exhibited synergistic antitumour effects.
Discussion
MDSCs comprise a highly immunosuppressive population of tumour-infiltrating immature myeloid cells that contribute to tumour immune escape by inhibiting cytotoxic T-lymphocyte proliferation and driving T regulatory cell induction [31,32]. MDSCs penetrate the entire tumour and are correlated with tumour size and malignancy. Therefore, targeting MDSCs is an important therapeutic strategy for tumour immunotherapy.
In the present study, we found that PMN-MDSCs heavily accumulated in the spleens and bone marrow of B16-F10 tumour-bearing mice, and the proliferation, metabolism and immunosuppression of B16-F10 tumour-bearing PMN-MDSCs increased. We selected the top 10 key proteins, namely, Eprs, Gart, Umps, Paics, Atp5o, Hadha, Dld, Mrpl4, Rpl8 and Mrpl13, in the upregulated KEGG pathways of B16-F10 tumour-bearing PMN-MDSCs as targets to screen the natural inhibitors of PMN-MDSCs from the traditional Chinese Medicine library (20000 compounds). 6 POG exerts a dose-dependent antitumour effect and improves the immunosuppressive microenvironment of tumours. a Tumour growth curves of B16-F10 tumour-bearing mice after POG treatment (n = 6). b Representative tumour images of control and POG-treated B16-F10 tumour-bearing mice (n = 6). c Body weight of B16-F10 tumour-bearing mice after POG treatment (n = 6). d Dotplots of live, CD45 + CD11b + cells in the tumours of control and POG-treated B16-F10 tumour-bearing mice (left panels) and proportion of PMN-MDSCs (CD11b + Ly6G + Ly6C low ) in bone marrow, spleen and CD45 + cells from tumours of control and POG-treated B16-F10 tumour-bearing mice (n = 6) (right charts). e Dotplots of live, CD45 + cells in the tumours of control and POG-treated B16-F10 tumour-bearing mice (left panels) and proportion of CD8 T-lymphocytes (CD3 + CD8 + ) in spleens and CD45 + cells from tumours of control and POG-treated B16-F10 tumour-bearing mice (n = 6) (right charts). f-g Ability of PMN-MDSCs sorted from bone marrow (f) or tumours (g) of control and POG-treated B16-F10 tumour-bearing mice to inhibit CD8 Tlymphocyte proliferation (n = 6). h Proliferation of CD8 T-lymphocytes sorted from the spleens of control and POG-treated B16-F10 tumourbearing mice (n = 6). i IFN-γ content of CD8 T-lymphocytes sorted from the spleens of control and POG-treated B16-F10 tumour-bearing mice (n = 6). The pooled data from three independent experiments are shown. All data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 The top 10 key proteins are mainly RNA and ATP binding proteins involved in protein translation, amino acid metabolism and ATP synthesis. Among these proteins, Eprs is an ATP binding protein involved in the metabolism of L-glutamate and L-proline, Dld is an E3 component of the three alpha-ketoacid dehydrogenase complexes with electron transfer activity, and Atp5po participates in the synthesis of ATP [33,34].
Finally, we found that POG could bind well to the key proteins in these pathways, inhibit B16-F10 primary tumour growth and improve the immunosuppressive microenvironment of B16-F10 tumour-bearing mice. POG is a chromone extracted from Saposhnikovia root [35]. POG has been reported to inhibit the production of TNFα, IL-1β and IL-6 in Raw 264.7 cells by inhibiting the activation of MAPK and NF-κB signalling pathways and reducing serum TNFα, IL-1β and IL-6 in vivo [36,37]. In addition, POG could dose-dependently inhibit the expression of iNOS, COX-2 and PGE2 by suppressing the activation of JAK2/STAT3 signalling in vitro and in vivo [37,38].
Mechanically, POG reduces the content of ornithine and citrulline in PMN-MDSCs by inhibiting the expression of Arg-1 and iNOS, which further inhibits polyamine production and the TCA cycle and ultimately inhibits the proliferation, metabolism and immunosuppressive ability of cells [39,40]. As mentioned above, MDSCs might have partly limited immune checkpoint inhibitors, and combination therapies increase the response rates of PD-1/PD-L1 inhibitors [41][42][43]. In the present study, we found that POG treatment enhanced the effect of anti-PD-1 immune The tumour growth curves of 4 T1 tumour-bearing mice after POG and anti-PD-1 antibody (alone or in combination) treatment (n = 6). c-d The tumour growth curves of 4 T1 tumour-bearing mice after POG and anti-PD-1 antibody (alone or in combination) treatment (n = 6). e-f Survival rate of 4 T1 tumour-bearing mice with POG and anti-PD-1 antibody (alone or in combination) treatment (n = 6) g-h Survival rate of B16-F10 tumour-bearing mice with POG and anti-PD-1 antibody (alone or in combination) treatment (n = 6). The pooled data from three independent experiments are shown. All data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 | v3-fos-license |
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} | pes2o/s2orc | Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia
Preeclampsia is a maternal hypertensive disorder that affects up to 1 out of 12 pregnancies worldwide. It is characterized by proteinuria, endothelial dysfunction, and elevated levels of the soluble form of the vascular endothelial growth factor receptor-1 (VEGFR-1, known as sFlt-1). sFlt-1 effects are mediated in part by decreasing VEGF signaling. The direct effects of sFlt-1 on cellular metabolism and bioenergetics in preeclampsia, have not been established. The goal of this study was to evaluate whether sFlt-1 causes mitochondrial dysfunction leading to disruption of normal functioning in endothelial and placental cells in preeclampsia. Endothelial cells (ECs) and first-trimester trophoblast (HTR-8/SVneo) were treated with serum from preeclamptic women rich in sFlt-1 or with the recombinant protein. sFlt-1, dose-dependently inhibited ECs respiration and acidification rates indicating a metabolic phenotype switch enhancing glycolytic flux. HTR-8/SVneo displayed a strong basal glycolytic metabolism, remaining less sensitive to sFlt-1-induced mitochondrial impairment. Moreover, results obtained in ECs exposed to serum from preeclamptic subjects demonstrated that increased sFlt-1 leads to metabolic perturbations accountable for mitochondrial dysfunction observed in preeclampsia. sFlt-1 exacerbated mitochondrial reactive oxygen species (ROS) formation and mitochondrial membrane potential dissipation in ECs and trophoblasts exposed to serum from preeclamptic women. Forcing oxidative metabolism by culturing cells in galactose media, further sensitized cells to sFlt-1. This approach let us establish that sFlt-1 targets mitochondrial function in ECs. Effects of sFlt-1 on HTR-8/SVneo cells metabolism were amplified in galactose, demonstrating that sFlt-1 only target cells that rely mainly on oxidative metabolism. Together, our results establish the early metabolic perturbations induced by sFlt-1 and the resulting endothelial and mitochondrial dysfunction in preeclampsia.
Preeclampsia is a maternal hypertensive disorder that affects up to 1 out of 12 pregnancies worldwide. It is characterized by proteinuria, endothelial dysfunction, and elevated levels of the soluble form of the vascular endothelial growth factor receptor-1 (VEGFR-1, known as sFlt-1). sFlt-1 effects are mediated in part by decreasing VEGF signaling. The direct effects of sFlt-1 on cellular metabolism and bioenergetics in preeclampsia, have not been established. The goal of this study was to evaluate whether sFlt-1 causes mitochondrial dysfunction leading to disruption of normal functioning in endothelial and placental cells in preeclampsia. Endothelial cells (ECs) and first-trimester trophoblast (HTR-8/SVneo) were treated with serum from preeclamptic women rich in sFlt-1 or with the recombinant protein. sFlt-1, dose-dependently inhibited ECs respiration and acidification rates indicating a metabolic phenotype switch enhancing glycolytic flux. HTR-8/SVneo displayed a strong basal glycolytic metabolism, remaining less sensitive to sFlt-1-induced mitochondrial impairment. Moreover, results obtained in ECs exposed to serum from preeclamptic subjects demonstrated that increased sFlt-1 leads to metabolic perturbations accountable for mitochondrial dysfunction observed in preeclampsia. sFlt-1 exacerbated mitochondrial reactive oxygen species (ROS) formation and mitochondrial membrane potential dissipation in ECs and trophoblasts exposed to serum from preeclamptic women. Forcing oxidative metabolism by culturing cells in galactose media, further sensitized cells to sFlt-1. This approach let us establish that sFlt-1 targets mitochondrial function in ECs. Effects of sFlt-1 on HTR-8/SVneo cells metabolism were amplified in galactose, demonstrating that sFlt-1 only target cells that rely mainly on oxidative metabolism. Together, our results establish the early metabolic perturbations induced by sFlt-1 and the resulting endothelial and mitochondrial dysfunction in preeclampsia.
In normal pregnancies, uterine blood flow increases to allow adequate perfusion of the placental intervillous space and physiological oxidative stress (Chaiworapongsa et al., 2014). In PE, a prolonged hypoxic placental microenvironment, due to a reduction in placental perfusion and oxygen availability, results in exacerbated oxidative stress (Chaiworapongsa et al., 2014). Hypoxia triggers several cellular responses, including increased placental angiogenesis (Zamudio, 2003), cell survival and metabolic adaptations (Illsley et al., 1984), established in developmental biology as "placental metabolic reprogramming" (Illsley et al., 2010;Jose et al., 2011).
Mitochondrial activity is essential in pregnancy because it sustains the metabolic activity of the placenta throughout gestation (LaMarca et al., 2008). Recently, a potential association between increased soluble anti-angiogenic factors levels and mitochondrial dysfunction has been suggested (Jiang et al., 2015). Exogenous administration of sFlt-1 in pregnant mice have shown to induce placental mitochondrial swelling, oxidative stress and apoptosis in trophoblasts (Jiang et al., 2015). In addition, preeclamptic plasma mediators induced deleterious effects on mitochondrial function of human umbilical vein endothelial cells (HUVEC) (McCarthy and Kenny, 2016). Together, findings suggest that mitochondrial function plays an important role in the onset of PE. Nevertheless, the role of early dysregulated sFlt-1 levels in PE, to induce perturbations in the mitochondrial oxygen consumption and bioenergetics in endothelium and placenta, has not been established.
Here, we report the effects of PE serum on mitochondrial oxygen consumption and metabolism in ECs and first-trimester extravillous trophoblasts (HTR-8/SVneo). As early elevated circulating levels of sFlt-1 are known to be implicated in the development of the disease (Maynard et al., 2003;Levine et al., 2004Levine et al., , 2006, we also established the effects of increasing levels of exogenous sFlt-1 on mitochondrial function. We demonstrate that PE serum significantly affects mitochondrial maximal respiration and spare respiratory capacity of ECs and trophoblast, enhancing a metabolic glycolytic phenotype. In addition, PE serum-induced mitochondrial reactive oxygen species (mtROS) formation. These effects were partially abrogated by exogenous VEGF. Finally, sFlt-1 treatment caused a dose-dependent loss of mitochondrial oxygen consumption in ECs and trophoblasts, affecting mitochondrial maximal respirations and spare respiratory capacities, and, inducing a metabolic phenotype switch to glycolysis, only in ECs. Our results provide novel insights on the differential metabolic perturbations exerted by sFlt-1 in the endothelium and placenta and their overall role in the development of PE.
Subjects
Antecubital blood samples were collected from preeclamptic (PE) (n = 23) and normotensive women (NOR) (n = 23), before cesarean delivery. Subjects were recruited from the Maternal Fetal Units of Fundación Cardiovascular de Colombia (FCV), Floridablanca, Colombia and Clínica Materno Infantil San Luis (CMISL), Bucaramanga, Colombia, using protocols approved by hospital respective Ethics Committees. Ten non-pregnant subjects were recruited as controls (CTL
Cell Viability Assays
MTT assay was performed as cell viability assays. Cells were seeded at a density of 1 × 10 4 cells/well on 96-well plates and cultured for 24 h. Then, cells were treated with 50 ng/mL of sFlt-1 recombinant protein (Novoprotein Scientific, Summit, NJ) for another 24 h. Cells were cultured in glucose and galactose supplemented media. After treatments, cells were exposed to 100 µL of MTT solution (5 mg/mL). Two hours later, formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Absorbance was measured in a plate reader at 570 nm using a Varioskan Flash multimodal plate reader (Thermo Fisher Scientific, Vantaa, Finland).
Mitochondrial Oxygen Consumption
Mitochondrial bioenergetics was assessed using an XFe24 Extracellular Flux Analyzer (Agilent Seahorse, Billerica, MA, USA). ECs and HTR-8/SVneo were seeded in V7 Seahorse micro-well plates at 3.5-4.0 × 10 4 cells/well in 100 µL standard growth media. Cells were treated with sFlt-1 recombinant protein (Novoprotein Scientific) and 2% serum from recruited women, respectively, and incubated at 37 • C and 5% CO 2 for 24 h. Following treatments, culture media was changed to a non-buffered DMEM media, to allow temperature and pH equilibrium. Initially, oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured simultaneously three times to establish a baseline rate. Then, to evaluate mitochondrial function, oligomycin (1 mM) (Sigma Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 mM) (Sigma Aldrich) and a mixture of rotenone and antimycin A (Rot/AntA) (1 mM) (Cayman Chemicals) were injected into each well sequentially, with intervals of 3-5 min of mixing between the injections, to respectively inhibit the ATP synthase, uncouple oxidative phosphorylation, and estimate non-mitochondrial respiration. Cellular respiratory control ratio (RCR) was obtained as the ratio of basal and oligomycin-inhibited (basal RCR) and FCCP-stimulated and oligomycin-inhibited mitochondrial respiratory rates (maximal RCR) (Brand and Nicholls, 2011;Dranka et al., 2011). OCR and ECAR measurements were
Mitochondrial ROS Formation
Mitochondrial ROS production (mtROS) was evaluated by fluorescence microscopy, using the fluorescent probe MitoSOX Red (Invitrogen, Oregon, USA). Briefly, ECs and trophoblast cells were cultured in 24-well plates, exposed to serum from recruited women and exogenous sFlt-1 (50 ng/mL), respectively, for 24 h. Cells were washed twice with HBSS and incubated with 5 µM MitoSOX Red probe in HBSS for 15 min at 37 • C in 5% CO 2 , protected from light. Then, cells were washed again with HBSS and the red fluorescence emitted at 580 nm was analyzed, using a Nikon Eclipse Ti-S inverted microscope and NiS Elements software.
Mitochondrial Membrane Potential
Changes in mitochondrial membrane potential ( m) were performed using the JC-1 fluorescent dye by fluorescence microscopy (Invitrogen, Oregon, USA). JC-1 accumulates within the mitochondria and forms aggregates that emit red-orange fluorescence (Ex: 550/Em: 600 nm). When the m is dissipated, cells emit green fluorescence (Ex: 485/Em: 535 nm). Briefly, ECs and HTR-8/SVneo cells were cultured in 24-well plates at densities of 5.0 × 10 4 cells/well. Treatments with sFlt-1 or serum were applied for 24 h. Then, cells were washed twice with sterile Phosphate Buffered Saline (PBS) at 37 • C, followed by incubation with 5 µM of JC-1 in culture media for 30 min at 37 • C, protected from light. The red/green fluorescence was evaluated using a Nikon Eclipse Ti-S inverted microscope and NiS Elements software.
Statistical Analyses
Each test was performed in three independent experiments. Data were plotted as means ± SEM. Statistical analysis was performed using Student T-test and ANOVA with Bonferroni's post hoc test, using Stata 8 statistical software (StataCorp, TX, USA). Values of p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.
In Vitro Correlation Model of PE: Role of VEGF in Mitochondrial Bioenergetics
Early metabolic perturbations are considered the hallmark of common human diseases (DeBerardinis and Thompson, 2012). To understand the metabolic basis that would accompany the onset of PE, we assessed the cellular bioenergetics profile of ECs and HTR-8/SVneo cells, exposed to serum from pregnant and not pregnant women. For these purposes, we established a case-control study in where pregnant preeclamptic (PE), normotensive (NOR), and non-pregnant women (CTL), were recruited (Supplementary Table 1). Circulating sFlt-1 levels were determined in recruited women, to corroborate preeclamptic conditions. As reported previously, sFlt-1 levels were increased in PE women (Maynard et al., 2003;Levine et al., 2004) by several orders of magnitude in comparison with NOR and CTL patients (Supplementary Figure 2). Then, serum from all groups was used to replace FBS at 2% in culture media, to establish an in vitro correlation model (Rodgers et al., 1988;Maynard et al., 2003) of PE. Next, the effects in mitochondrial metabolism and bioenergetics were assessed, using Agilent-Seahorse technology (Dranka et al., 2011) as described in methods sections.
As evidenced in Figure 1A respiratory traces show that treatment of ECs with serum from PE women induced a profound change in mitochondrial respiration rates. Maximal respiration and spare respiratory capacity rates were calculated after FCCP administration. FCCP induces the collapse of the mitochondrial membrane potential, leading to the determination of maximum OCR (Brand and Nicholls, 2011;Dranka et al., 2011). Administration of CTL and NOR serum did not affect respiration rates ( Figure 1A). As observed in cell respiratory control ratios (RCR) (State 3/State 4), PE serum significantly affected mitochondrial respiration profile, suggesting mitochondrial dysfunction, associated with a low substrate oxidation capacity ( Figure 1B).
Interestingly, when we assessed the cell energy phenotype of ECs, we found a slight increase in basal OCR in ECs treated with NOR and PE serum. However, when cells were challenged with FCCP to uncouple mitochondria, only CTL and NOR treated cells showed an increase in both OCR and ECAR. ECs treated with PE serum exhibited a drastic drop in OCR below basal levels, with a non-significant increase in glycolytic function ( Figure 1C). This suggested that ECs under PE conditions (modeled with serum from women with PE), are not going to be able to cope with physiological challenges that will require an increase in energy utilization via mitochondria.
To establish the effects of PE serum in placenta, we used HTR-8/SVneo cells, which is an immortalized first trimester EVT cell line, as our model. Since early perturbations of sFlt-1 will be developing before the 20th-week of gestation, a trophoblast cell line that resembles the metabolic profile from those early stages was used. Results obtained after treatment with serum from PE women showed a reduction in maximal respiration in comparison with NOR and CTL serum of about 30% ( Figure 1D). As in ECs, PE serum depleted the spare respiratory capacity rates. RCR determinations identified reduced oxidative phosphorylation in NOR serum-treated trophoblasts, along with an enhanced glycolytic response ( Figure 1E). As observed in ECs, PE serum induced a significant decrease in OCR parameters, consistent with a weak coupling of respiration for ATP synthesis and enhanced ability to activate glycolytic pathways (Figures 1D-F).
Together, these results suggest that under full manifestation of PE conditions, the combination of several PE-key players, along with upregulated sFlt-1 (Maynard et al., 2003;Levine et al., 2004Levine et al., , 2006Tosun et al., 2010;Vitoratos et al., 2010), will induce an insightful effect in the energetic phenotype of both, ECs and trophoblast, that will not allow them to metabolically respond to harsh conditions.
Role of VEGF in Mitochondrial Function in PE
Once the impact of PE serum was established in ECs and trophoblasts, it was imperative to verify the implications of sFlt-1 in these observations. Although sFlt-1 levels in PE serum were higher than in NOR and CTL, other factors like proinflammatory molecules (i.e., TNF-α and IL-6), are known to be present in serum as described extensively in the literature (Tosun et al., 2010;Vitoratos et al., 2010), contributing with sFlt-1 to the effects observed.
To address the role of sFlt-1 and VEGF bioavailabilities in the early metabolic perturbations in PE, VEGF (20 ng/mL) was administered to cells exposed to serum. VEGF administration should discriminate the antagonizing effects of sFlt-1 in serum from other possible factors. ECs and trophoblasts were treated with serum alone, and serum containing VEGF. As shown in (Figure 2A), VEGF administration to ECs treated with NOR serum did not affect maximal respiration or spare respiratory capacity. However, PE serum impaired the maximal respiration and spare respiratory capacity of ECs ( Figure 1A) and these effects were partially reverted by VEGF (Figure 2A). This suggests that reduced VEGF bioavailability, due to sFlt-1 up-regulation, affects mitochondrial function (Figures 2A,B). VEGF co-treatment significantly improved maximal respiration rates, resulting in increased RCR when compared to ECs treated with PE serum (Figures 2A,B). Still, this improvement was not enough to reach the levels detected in cells treated with NOR serum. This scenario was similar in trophoblasts. PE serum caused a significant decrease in the maximal respiration rate and the addition of VEGF was not able to restore these values ( Figure 2C). Then, the dramatic decrease in the spare respiratory capacity and RCR of cells treated with PE serum was partially restored by VEGF (Figures 2C,D). These observations suggested that sFlt-1 might be responsible for antagonizing the homeostatic activity of VEGF, having a direct impact on mitochondrial function and bioenergetics. However, it is expected that other factors in PE serum (and to a lesser extent in NOR and CTL women) are also contributing to the effects observed.
Next, we examined the effects on ECs and trophoblasts of PE serum alone and with VEGF, in mitochondrial function, by assessing the production of mtROS with Mito-SOX fluorescence and m with JC-1 dye. In ECs, treatment with PE serum caused a drastic increase in mtROS in comparison with CTL and NOR serum. VEGF was only able to reduce mtROS generation by approximately 30% in comparison with ECs treated with PE serum (Figure 3A). In contrast, treatment in trophoblasts caused a significant increase as compared to CTL serum of 33% with NOR serum treatment and 71% increase in mtROS with PE serum (Figure 3B). VEGF decreased mtROS production about 30%, as in trophoblasts treated with NOR serum. PE Scale: 100 µm. Data are presented as means ± SEM. (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, vs. CTL exposed cells. #P < 0.05, vs. PE serum exposed cells, ANOVA (Bonferroni's post hoc test).
serum also caused changes in the m in both ECs and HTR-8/SVneo cells. Treatment with PE serum caused a drop in m of about 27% in comparison to ECs treated with CTL serum (Figure 3C). Administration of VEGF was not able to restore the m as evidenced in the 8% increase in m in treated ECs. Similar results were obtained in treated trophoblasts ( Figure 3D). Cells treated with PE serum experienced a drop in m of 28% in comparison with CTL serum. Treatment with PE serum and VEGF was only able to restore the drop in mitochondrial membrane potential with a 7% increase. In contrast, the effects of VEGF almost recover the changes in m to similar levels of cells treated with NOR serum.
sFlt-1 Induces a Metabolic Phenotype Switch to Glycolysis in ECs, but Not in Trophoblasts
Circulating levels of sFlt-1 are known to increase drastically prior the onset of clinical signs of PE (Maynard et al., 2003;Levine et al., 2004Levine et al., , 2006. To assess the effects of sFlt-1 on the mitochondrial bioenergetics of ECs and HTR-8/SVneo, cells were treated with sFlt-1 (0-50 ng/mL) for 24 h and subjected to OCR and ECAR measurements. As shown in Figure 4A, sFlt-1 decreased basal and ATP-dependent OCR in a dose-dependent manner in ECs. Also, significant changes in FCCP-stimulated OCR were shown in sFlt-1-treated cells, indicating that mitochondrial spare respiratory capacity was also affected (Figure 4A). These evidence demonstrate that sFlt-1-induced angiogenic imbalance, reduce the capacity of the endothelium to respond to changes in energy demand coupled to mitochondrial respiration (Supplementary Figure 3A). The analysis of OCR versus ECAR in sFlt-1-treated ECs showed an inhibition of the mitochondrial respiration, with a dosedependent ability to switch to glycolysis ( Figure 4B). In addition, the glycolytic response increased more than 5-fold in 50 ng/mL sFlt-1-exposed cells, suggesting a metabolic phenotype switch to glycolysis in ECs, after treatment with sFlt-1 ( Figure 4C and Supplementary Figure 3B). Regarding HTR-8/SVneo cells, cells demonstrated a higher basal OCR with an overall reduced FCCP-dependent OCR, in comparison to ECs, displaying high glycolytic rates and glycolytic reserves even at basal conditions (Figures 4E,F). Response to sFlt-1 treatment showed a reduction in mitochondrial respiration without increases in the glycolytic flux exerted by 50 ng/mL of sFlt-1 (Figures 4D-F).
Results obtained clearly state that in both, ECs and HTR-8/SVneo, sFlt-1 is acting as a disruptor directly into mitochondria. Nonetheless, based on the differential cell energy phenotypes of ECs and HTR-8/SVneo, the effects are interestingly dissimilar in both cell lines.
sFlt-1 Acts as a Mitochondrial Bioenergetics Disruptor in ECs and HTR-8/SVneo
To further confirm the effects of sFlt-1 as a mitochondrial bioenergetics disruptor, mitochondrial metabolism (OXPHOS) was forced by growing cells in galactose media (Robinson et al., 1992;Marroquin et al., 2007). This approach forces cells to exclusively rely on mitochondrial metabolism of glutamine for their ATP energy requirements (Reitzer et al., 1979). Reliance on OXPHOS for ATP production, sensitize cells to mitochondrial disruptors (Marroquin et al., 2007;Dott et al., 2014).
ECs and HTR-8/SVneo exposed to galactose presented slower growth rates in comparison to cells cultured in glucose media ( Figure 5A). ECs, cultured in galactose, did not evidence changes in their viability or proliferations rates in comparison to cells exposed to glucose (Figure 5B and Supplementary Figure 4A). Nonetheless, when ECs were exposed to sFlt-1 in galactose media, they had significant reduction in the proliferation rate and viability of about 20% in comparison to non-treated cells (Figure 5B and Supplementary Figure 4A).
Divergently, the culture of HTR-8/SVneo cells in galactose, evidenced their dependence on glycolysis as a main source of energy, as described previously (Figures 4E,F). By forcing OXPHOS in HTR-8/SVneo cells, we confirmed diminished proliferation and viability rates of about 50% in comparison to cells grown in glucose (Figure 5C and Supplementary Figure 4B). These observations are consistent with HTR-8/SVneo evolutionary metabolism of a first-trimester trophoblast, previously defined to be contingent mainly on non-oxidative pathways to support its energetic demands (Illsley et al., 2010). Thus, treatment of HTR-8/SVneo cells with sFlt-1 in galactose media evidenced a reduced viability of about 40% in comparison to controls in galactose ( Figure 5C and Supplementary Figure 4B). Together, these results evidenced that sFlt-1, as a mitochondrial disruptor, affects more dramatically cells that rely on mitochondria machinery for energy proposes, rather than those with a basal glycolytic dependence for their metabolism.
To further corroborate the impact of sFlt-1 over the mitochondrial function and ROS formation, we evaluated the production of mtROS and m in ECs and trophoblasts, as measured by fluorescence microscopy. As shown in Figure 6A, using fluorescent probe MitoSOX Red, we evidenced that sFlt-1 (50 ng/mL) significantly increased mtROS formation in ECs 1.5-fold, but these effects were not observed in trophoblasts ( Figure 6B). Then, the evaluation of the m using JC-1 fluorescent dye, established that doses of 50 ng/mL of exogenous sFlt-1, reduced m in ECs by 20% (Figure 6C). Again, no significant changes were found in trophoblast cells ( Figure 6D). These results confirm our previous observations FIGURE 5 | sFlt-1 acts as a mitochondrial bioenergetics disruptor in preeclampsia. (A) Morphological changes in endothelial cells (ECs) and trophoblasts cultured in glucose and galactose media (40X). (B) Cell viability of ECs and (C) trophoblasts cultured in glucose and galactose media and exposed to exogenous 50 ng/mL of sFlt-1 during 24 h. Scale: 100 µm. Data are presented as means ± SEM. (n = 3), **P < 0.01, ***P < 0.001, vs. galactose exposed cells. # P < 0.05, vs. glucose-exposed cells. Student T-test. (C) sFlt-1 dissipated the mitochondrial membrane potential ( m) measured by fluorescent microscopy using JC-1 fluorescent probe in ECs, but not in (D) trophoblasts. Cells were exposed to sFlt-1 (50 ng/mL) for 24 h. Data is presented as means ± SEM. (n = 3), *P < 0.05 vs. untreated controls. Student T-test.
regarding the role of sFlt-1 to induce alterations in mitochondrial bioenergetics in ECs and its impact on the overall mitochondrial function, as previously demonstrated in vivo (Jiang et al., 2015).
DISCUSSION
PE remains as the leading cause of maternal and neonatal deaths worldwide (Duley, 2009). Within the last decade, several reports opened a novel window for understanding the pathogenesis of the disease, by describing the role of circulating anti-angiogenic factors like sFlt-1, in the development and early detection of the disease (Maynard et al., 2003;Levine et al., 2004;Young et al., 2010;Perni et al., 2012;Verlohren et al., 2012). sFlt-1 has been found to provoke endothelial dysfunction (Powe et al., 2011;Sánchez-Aranguren et al., 2014), hypertension (LaMarca et al., 2008 and proteinuria (Maynard and Karumanchi, 2011), demonstrating its culprit role in the onset of PE (Roberts and Rajakumar, 2009). The present study demonstrates the striking effects of sFlt-1 and serum from PE women in the overall cell energy metabolism, mitochondrial bioenergetics and mitochondrial dysfunction, linked to oxidative stress in ECs and first trimester EVT (HTR-8/SVneo cells). Early metabolic perturbations are known to be the hallmark of a range of human pathologies (DeBerardinis and Thompson, 2012). Then, the study of cellular energy metabolism arrives a novel strategy for understanding the etiology of diseases and potentially to develop novel treatments targeting these alterations in energy metabolism (Gohil et al., 2010).
sFlt-1, Increased in PE Serum, Induce Metabolic Perturbations in Preeclampsia
By the aid of the Seahorse-Agilent Extracellular Flux Analyzer, we evaluated the metabolic perturbations preceding the onset of PE. In order to establish a model that resemble PE conditions, ECs and HTR-8/Svneo cells were treated with serum from pregnant women, normotensive and preeclamptic, based on current diagnostic criteria (American College Obstetricians Gynecologist Task Force on Hypertension in Pregnancy, 2013). ELISA analyses showed increased sFlt-1 levels in serum from PE women by several orders of magnitude, in comparison with NOR and CTL serum levels, as reported previously (Maynard et al., 2003;Levine et al., 2004Levine et al., , 2006. In a normal pregnancy, circulating sFlt-1 levels increase with gestation age (Maynard et al., 2005). However, in PE, due to unknown mechanisms, sFlt-1 is dramatically upregulated (Fan et al., 2014), resulting in increased circulating levels of sFlt-1 prior the onset on the clinical signs of PE (Maynard et al., 2003;Levine et al., 2004Levine et al., , 2006. Here, we have demonstrated and described the alterations in the mitochondrial bioenergetics profiles induced by sFlt-1 increased levels, in both cell lines tested. Our results are in accordance with recent work showing that incubation of HUVEC with 3% plasma from PE women, significantly reduced the overall OCR, measured by a fluorescence-based approach, when compared to cells treated with serum from uncomplicated and non-pregnant women (McCarthy and Kenny, 2016). In addition, our results demonstrate the culprit role of sFlt-1 in the onset of metabolic alterations in PE. sFlt-1 might be responsible for antagonizing the homeostatic activity of VEGF, having an impact at the mitochondrial level. However, it is likely that other molecules, existing in the maternal serum of PE women, could be potentiating the effects observed.
We also showed that mtROS are increased in trophoblasts exposed to NOR serum in comparison to cells exposed to CTL serum. This fact is consistent with the increased oxidative stress status at the intrauterine level, observed by others in normal pregnancies (Chaiworapongsa et al., 2014). PE serum, in ECs and trophoblasts, led to a significant increase in mtROS production in comparison to cells exposed to NOR serum. The augmented mitochondrial oxidative stress, evidenced an alteration in mitochondrial function induced by elevated sFlt-1 levels, present in PE serum. It is likely that increased mtROS production would be directly related to the effects of sFlt-1 on mitochondrial bioenergetics. As it has been reported previously in vivo, sFlt-1 induces ROS formation in placental vessels and trophoblasts (Jiang et al., 2015). Potential mechanisms of increased mtROS formation have been associated to a reverse electron transport through complex I, in the mitochondrial electron transport chain, in response to an elevated NADH/NAD + ratio (Murphy, 2009). Our observations suggest that dysregulated sFlt-1 levels during pregnancy, induce metabolic perturbations and oxidative stress that might contribute to vascular endothelial dysfunction in PE.
sFlt-1 Induce a Metabolic Phenotype Switch to Glycolysis in Endothelial Cells
To better understand the metabolic bases that would accompany the onset of PE, we studied the role of increasing concentrations of sFlt-1, as a potential early metabolic disturber. We showed a dose-dependent loss of mitochondrial function in ECs treated with sFlt-1. Treatment with increasing concentrations of sFlt-1, evidenced a metabolic phenotype switch from OXPHOS to an enhanced glycolytic cellular response, not been establish before. Our data suggest that in PE, increasing sFlt-1 levels could result in loss of mitochondrial function early in gestation, leading to impaired bioenergetics profiles. Cell energy metabolism varies in tissue of different origins (Benard et al., 2006). Therefore, cell metabolism can be adapted according to the microenvironment surrounding cells. In evidence, other authors have reported that various growth conditions may alter metabolism, contributing to a greater cell dependence on glycolysis (Jose et al., 2011). Our observations of an enhanced glycolytic metabolism, along with an impaired oxygen consumption, suggested a metabolic reprogramming process, as described in tumor biology studies (Jose et al., 2011). These observations illustrate how ECs can alternate OXPHOS to glycolytic pathways under sFlt-1induced stressful conditions. Nonetheless, the potential effects derived from the prolongation of a aerobic glycolytic metabolism (Warburg effect) in ECs, remains to be determined. Enhanced glycolysis linked to a reduced OXPHOS could markedly affect ECs capabilities to switch from a quiescent metabolic state to an energetic phenotype during angiogenesis.
Regarding HTR-8/SVneo cells, we found, as reported before (Illsley et al., 2010), that they have a functional mitochondrial machinery. In contrast to ECs, sFlt-1 treatment in trophoblasts, causes a non-significant mild drop in OCR. Since the cell basal energetic profile is glycolytic, the overall energetic phenotype after sFlt-1 treatment was not impaired. Studies performed on term isolated trophoblasts have shown that both, syncytium and cytotrophoblast cells, exhibit high reserve respiratory capacity (Maloyan et al., 2012) when compared with our results. This suggests that trophoblast cells, in early stages of gestation, are metabolically programmed to overlap and compensate for the effects of metabolic disruptors. Based on these facts, we presumed that in later stages of pregnancy, when placental function reaches its inevitable end, the metabolic profile of trophoblasts changes as their biological functions terminate. These suggest that cells isolated from full-term placentas may not be the most appropriate approach to study the effects of early metabolic perturbations.
sFlt-1 Acts a Mitochondrial Disruptor
Our results demonstrate a dose-dependent alteration in the mitochondrial bioenergetics, suggesting that sFlt-1 is acting as mitochondrial disruptor. To demonstrate the ability of sFlt-1 to act as a mitochondrial disruptor that drives energy metabolism from OXPHOS to glycolysis, we forced ATP dependence on OXPHOS, by culturing cells in galactose media. Since oxidation of galactose to pyruvate through glycolysis yields no net production of ATP, cells are more sensitive to mitochondrial toxicants, than when grown in glucose (Marroquin et al., 2007). First, culture of ECs in galactose did not involve changes in proliferation rates or viabilities, consistent with their metabolic flexibility to switch from ATP predominantly generated by OXPHOS, to glycolysis as their main energy source (Vallerie and Bornfeldt, 2015). sFlt-1 impaired ECs viability and proliferation rates under OXPHOS dependence, demonstrating that cells that rely mainly in mitochondrial metabolism are highly sensitive to sFlt-1. Results were markedly drastic in trophoblasts. Galactose media enhanced the effects of sFlt-1, decreasing cell viability and proliferation rates of about 40%. In both cell lines tested, culture in galactose media overblown sFlt-1 effects, demonstrating its role as a mitochondrial disrupter, effects that are not basally appreciated in glucose media. Previously, culture in galactose has been employed to identify mitochondrial toxicants (Dott et al., 2014) and molecules that target cellular metabolic FIGURE 7 | Schematic view of the effects of sFlt-1 dysregulation over cellular metabolism and bioenergetics in PE. Dysregulated VEGF signaling due to up-regulation of sFlt-1 levels in preeclampsia leads to reduced activation of VEGF receptors Flt-1 and Flk-1/KDR, respectively. Effects of dysregulated VEGF bioavailability affect mitochondrial oxygen consumption (OCR), inducing a metabolic phenotype switch enhancing glycolytic response (ECAR) in endothelial cells, but not in trophoblasts. sFlt-1 due to loss of mitochondrial bioenergetics increase oxidative stress in mitochondria (mtROS). Together, these events lead to mitochondrial dysfunction, that would result in vascular dysfunction and the onset of preeclampsia.
The increased mtROS production and decreased mitochondrial respiration, coupled with a higher glycolytic capacity of ECs exposed to sFlt-1, evidenced oxidative stress and a metabolic phenotype switch to compensate the detrimental effects of sFlt-1 and mediators present in serum from PE women, over the endothelium. Our results demonstrated that exogenous sFlt-1 induce mtROS formation and a drop in the mitochondrial membrane potential in ECs, but not in trophoblasts. This suggested that sFlt-1 plays a key role in metabolic modulation and reprogramming in endothelium during pregnancy. Whereas, when sFlt-1 levels increase drastically, their role is balanced toward an anti-angiogenic state that leads to metabolic impairment, vascular dysfunction, and PE. sFlt-1 seems to be an important linker between mitochondrial dysfunction, oxidative stress and endothelial dysfunction (Figure 7).
Here we have demonstrated that in trophoblast and endothelial cells, disruption of the finely-tuned VEGF signaling by sFlt-1 affects mitochondrial function and metabolism in preeclampsia. As Figure 7 reviews, sFlt-1 strongly impairs mitochondrial metabolism and bioenergetics, increasing mROS and inducing a metabolic switch to glycolysis in ECs. These findings are very important because they confirm the differential responses of sFlt-1 in both ECs and trophoblasts that are directly related in the development of the disease, from the maternal circulation and placenta, respectively. In addition, results obtained in ECs have strong implications in the maternal hypertension events that are the hallmark of PE. Various reports have revealed the direct implication of VEGF signaling in the regulation of mitochondrial function and angiogenesis (Wang et al., 2011;Guo et al., 2017;Kim et al., 2017). However, the exact mechanisms on how VEGF and downstream events regulate mitochondrial function are still unknown. Nevertheless, the role of the PI3k/Akt/mTOR pathways and eNOS and NO production in relation to high levels of sFlt-1, VEGF signaling, and mitochondrial function is currently under study. Establishing the clear role of sFlt-1/VEGF signaling in PE is key for developing novel strategies for preventing or treating this multifactorial disease.
ACKNOWLEDGMENTS
We will like to thank the staff from FCV and CMISL for their support during patient recruitment. We are also grateful to Drs. Balaraman Kalyanaraman and Jacek Zielonka from the Free Radical Research Center from the Department of Biophysics of the Medical College of Wisconsin for their help and guidance. | v3-fos-license |
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} | pes2o/s2orc | Coupling Aggressive Mass Removal with Microbial Reductive Dechlorination for Remediation of DNAPL Source Zones: A Review and Assessment
The infiltration of dense non-aqueous-phase liquids (DNAPLs) into the saturated subsurface typically produces a highly contaminated zone that serves as a long-term source of dissolved-phase groundwater contamination. Applications of aggressive physical–chemical technologies to such source zones may remove > 90% of the contaminant mass under favorable conditions. The remaining contaminant mass, however, can create a rebounding of aqueous-phase concentrations within the treated zone. Stimulation of microbial reductive dechlorination within the source zone after aggressive mass removal has recently been proposed as a promising staged-treatment remediation technology for transforming the remaining contaminant mass. This article reviews available laboratory and field evidence that supports the development of a treatment strategy that combines aggressive source-zone removal technologies with subsequent promotion of sustained microbial reductive dechlorination. Physical–chemical source-zone treatment technologies compatible with posttreatment stimulation of microbial activity are identified, and studies examining the requirements and controls (i.e., limits) of reductive dechlorination of chlorinated ethenes are investigated. Illustrative calculations are presented to explore the potential effects of source-zone management alternatives. Results suggest that, for the favorable conditions assumed in these calculations (i.e., statistical homogeneity of aquifer properties, known source-zone DNAPL distribution, and successful bioenhancement in the source zone), source longevity may be reduced by as much as an order of magnitude when physical–chemical source-zone treatment is coupled with reductive dechlorination.
Widespread use of chlorinated solvents in dry cleaning and metal degreasing operations over the last century has resulted in extensive groundwater contamination by compounds such as tetrachloroethene (PCE) and trichloroethene (TCE). When released into the subsurface as dense non-aqueous-phase liquids (DNAPLs), chlorinated solvents tend to migrate downward through the unsaturated zone and can penetrate the water table because of their higher density (Mercer and Cohen 1990). During DNAPL migration, hysteretic capillary forces cause retention of a portion of the liquid within the pores as discontinuous globules or ganglia [Lenhard et al. 1989;Schwille 1988; U.S. Environmental Protection Agency (U.S. EPA) 1990]. Substantial DNAPL volumes can also be retained because of the presence of nonuniform soil texture, which may result in DNAPL pooling (i.e., zones of DNAPL at much higher saturation) above layers or lenses of lower-permeability media (Dekker and Abriola 2000;Essaid and Hess 1993;Saenton et al. 2002;Schwille 1988). The resulting distribution of DNAPL is, thus, typically complex and nonuniform ( Figure 1). Entrapped DNAPL mass tends to dissolve slowly into flowing water, serving as a long-term source of groundwater contamination (Mackay and Cherry 1989;Schwille 1988). The implementation of conventional pump-and-treat remediation for such DNAPL source zones has been ineffective in reducing contaminant concentrations to regulatory end points in acceptable time frames (MacDonald and Kavanaugh 1994;Travis and Doty 1990;U.S. EPA 1996).
A number of innovative technologies have been developed to enhance contaminant removal from DNAPL source zones [National Research Council (NRC) 1994, 1999. Although these technologies are capable of substantial mass removal under favorable conditions, some DNAPL will likely remain within the porous medium even when treatment is most effective (Fountain et al. 1995;Sale and McWhorter 2001). This remaining contaminant mass can continue to serve as a source of down-gradient contamination, and thus further source-zone treatment or containment may be required. Despite a number of successful fieldscale demonstrations of aggressive source-zone treatment technologies, skepticism and concern remain that application of such technologies may not substantially reduce risk and could potentially worsen site conditions (e.g., through mobilization and redistribution of DNAPL, enhanced transport of metals, elimination of microbial activity, or increased aqueous-phase concentration of contaminants after treatment) (Cherry et al. 1997;Oostrom et al. 1999). From this perspective, some authors have suggested that source containment (i.e., treatment or mitigation of down-gradient contamination emanating from DNAPL source zones) is preferable to aggressive physical-chemical source-zone treatment (Cherry et al. 1997;Freeze 2000;Freeze and McWhorter 1997;Kent and Mosquera 2001). Freeze (2000) advocates a new remediation paradigm in which only source containment is implemented because of the technical impracticability of removing sufficient DNAPL mass to reduce contaminant concentrations to drinking water standards. In contrast, guidelines put forth by the Interstate Technology and Regulatory Cooperation work group, a team composed of state and federal regulators, call for aggressive source-zone remediation (Jackson 2001). The latter recommendation is based in part on the contention that mass removal from a source zone, even if incomplete, will result in a) a reduction in mass flux, Environmental Health Perspectives • VOLUME 113 | NUMBER 4 | April 2005 465 This article is based on a presentation at the conference "Bioremediation and Biodegradation: Current Advances in Reducing Toxicity, Exposure and Environmental Consequences" (http://www-apps.niehs.nih.gov/sbrp/ bioremediation.html) held 9-12 June 2002 in Pacific Grove, California, and sponsored by the NIEHS Superfund Basic Research Program. The overall focus of this conference was on exploring the research interfaces of toxicity reduction, exposure assessment, and evaluation of environmental consequences in the context of using state-of-the-art approaches to bioremediation and biodegradation. The Superfund Basic Research Program has a legacy of supporting research conferences designed to integrate the broad spectrum of disciplines related to hazardous substances. b) a reduction in source longevity, c) a reduction in risk, and d) a potential enhancement in posttreatment biodegradation (Jawitz et al. 2000;Londergan et al. 2001;Martel et al. 1998;Rao et al. 2002;Yang and McCarty 2003). Recent analytical and numerical modeling investigations suggest that partial sourcezone removal may result in significant (several orders of magnitude) reductions in posttreatment contaminant mass flux (Lemke and Abriola 2003;Rao et al. 2002;Rao and Jawitz 2003). Although a reduction in mass flux may not eliminate the need for further treatment, it could reduce concentrations to levels where microbial transformation of the dissolvedphase chlorinated solvents becomes feasible (Adamson et al. 2003;Nielsen and Keasling 1999;Sung et al. 2003;Yang and McCarty 2000). Biostimulation of source-zone microbial dechlorination activity may achieve attenuation of contaminant mass flux to levels that achieve regulatory compliance (i.e., a flux averaged concentration) at a down-gradient well.
Thus, combination of physical-chemical source-zone treatment and posttreatment bioremediation may be an attractive remediation alternative, resulting in reduced source longevity and contaminant mass flux (de Blanc et al. 1997;Rao et al. 2002;Zoller 1998;Zoller and Rubin 2001). Coupling a physical-chemical remediation process that removes significant contaminant mass with a bioremediation "polishing step" to control the contaminant mass flux emanating from remaining DNAPL may provide a synergism that cannot be obtained with existing remediation strategies. Such a staged treatment approach could leverage initial high removal efficiencies of physical-chemical source-zone treatment methods to minimize time to site closure. This sequential treatment approach should not be confused with natural attenuation, a remediation approach generally associated with bioremediation of low contaminant concentrations in a groundwater plume (Wiedemeier et al. 1999), nor should it be confused with the recent work on source-zone bioremediation, which relies solely on biotic processes to transform source-zone contamination (e.g., Adamson et al. 2003).
Observations from longer term monitoring at sites where innovative flushing technologies have been implemented suggest that tailoring physical-chemical treatment to enhance posttreatment bioremediation efforts is feasible (Mravik et al. 2003;Ramsburg et al. 2004). Application of such a staged treatment methodology, however, would require a thorough understanding of both physical-chemical treatment technologies and source-zone bioremediation. Our objective in this article is to review and integrate knowledge gained from recent demonstrations of field-scale sourcezone remediation with that from laboratory investigations of solvent biotransformation to assess the potential promise of technology coupling. This work differs from published reviews of specific technologies (e.g., Bradley 2003;Henry et al. 2003) in its focus on the influence of physical-chemical treatment technologies on posttreatment microbial reductive dechlorination. A technology assessment is provided and recommendations for future work are presented. Although some observations may be generally applicable to any DNAPL site, the focus herein is on sites where source-zone contamination mainly comprises chlorinated solvents (e.g., PCE, TCE).
Chlorinated Ethene Biodegradation
The degradation of chlorinated ethenes in microcosms and the detection of degradation products at contaminated groundwater sites in the 1980s inspired researchers to investigate biotic and abiotic transformation processes (McCarty and Semprini 1994;Vogel and McCarty 1985;Vogel et al. 1987). As early as 1980, researchers identified links between microbial metabolism and the destruction of chlorinated hydrocarbons (Higgins et al. 1980). As more work was completed, researchers recognized that oxidation or reduction of chlorinated hydrocarbons under different redox conditions is feasible (Table 1). The following discussion briefly reviews microbial dechlorination processes that can occur in the subsurface and identifies those processes that are most promising for stimulation in a source zone after active mass removal. For a more thorough discussion and review of chloroethene biodegradation, see Bradley (2003), Holliger (1995), Janssen et al. (2001), Semprini (1997Semprini ( , 2001, or Smidt and de Vos (2004).
Although oxidation of chlorinated hydrocarbons in both aerobic and anaerobic environments has been demonstrated (Bradley et al. 1998;Bradley and Chappelle 1996;Coleman et al. 2002aColeman et al. , 2002bHartmans et al. 1985;Hartmans and deBont 1992;Singh et al. 2004;Verce et al. 2000Verce et al. , 2001, aerobic metabolic oxidation is a productive pathway only for removal of lesser chlorinated ethenes [i.e., cis-dichloroethene (cis-DCE) and vinyl chloride (VC)]. No organisms that grow aerobically with PCE or TCE as a carbon source have been identified. In anoxic environments the metabolic oxidation of chloroethenes is still poorly understood. Although the mineralization of cis-DCE and VC under iron-and manganese-reducing conditions has been demonstrated (Bradley et al. 1998;Bradley and Chapelle 1996), the relevance of this process for bioremediation has yet to be established. Co-metabolism is an alternative nonmetabolic process that has been shown to transform contaminants in both aerobic and anaerobic environments (Anderson and McCarty 1997;Chauhan et al. 1998;Ensign et al. 1992;Hopkins et al. 1993;Ryoo et al. 2001;Shim et al. 2001). Aerobic cometabolism can act on all chloroethenes Shim et al. 2001); however, the need for a primary substrate such as methane or toluene, and the fact that the degradation of the target compounds can only be indirectly controlled are major drawbacks of this approach. Anaerobic co-metabolic reductive dechlorination of PCE has been observed under methanogenic Boyd 1988a, 1988b), acetogenic (Terzenbach and Blaut 1994), and sulfidogenic conditions (Cole et al. 1995). However, because of low rates and incomplete dechlorination, this process is least likely to contribute to detoxification of contaminated subsurface environments. More recently, chlororespiration, a process in which chlorinated compounds serve as a metabolic electron acceptor for energy generation, has been demonstrated (Holliger et al. 1998;Löffler et al. 1996Löffler et al. , 1999Smidt and de Vos 2004). The metabolic reductive dechlorination pathway (chlororespiration) is a strict anaerobic process that requires an electron donor (i.e., source of reducing equivalents). The chlororespiratory pathway is promising in that it can lead to efficient dechlorination to ethene and achieve complete detoxification (He et al. 2003a(He et al. , 2003b. The ability to use chloroethenes as energyyielding electron acceptors is distributed among several bacterial groups, including different subdivisions of the proteobacteria, the gram-positive bacteria, and the Chloroflexi (formerly green nonsulfur bacteria). Organisms capable of metabolic reductive dechlorination (i.e., chlororespiration) have been isolated from contaminated and pristine sites (Smidt and de Vos 2004). These populations are generally strict anaerobes, with only Enterobacter strain MS-1 exhibiting facultative metabolism (Sharma and McCarty 1996). Bacterial populations capable of gaining energy from reductive dechlorination of chloroethenes have been classified into a number of phylogenetic groups, including Dehalobacter, Sulfurospirillum, Desulfuromonas, Desulfitobacterium, Clostridium, and Dehalococcoides (Bradley 2003;Löffler et al. 2003;Smidt and de Vos 2004). This broad range of organisms capable of chlororespiration is encouraging for posttreatment bioremediation; however, most of these organisms are incapable of complete dechlorination of chloroethenes to ethene Major et al. 2003). At many sites, DCEs (primarily cis-DCE) and, in some cases, VC accumulate. Cupples et al. (2004) recently demonstrated dechlorination of cis-DCE and VC, but they identified a minimum threshold chlorinated contaminant concentration below which dechlorination could not be sustained. There is an apparent link between the presence of members of the Dehalococcoides group and complete dechlorination (i.e., ethene formation) (Cupples et al. 2003;He et al. 2003aHe et al. , 2003bHendrickson et al. 2002;Maymo-Gatell et al. 1997Ritalahti et al. 2001). Dehalococcoides ethenogenes strain 195 was the first isolate described to dechlorinate PCE to ethene, but the last dechlorination step, VC to ethene, was cometabolic and slow (Maymo-Gatell et al. 1997). A major breakthrough was the isolation of Dehalococcoides species strain BAV1, the first isolate capable of using all DCE isomers and VC as growth-supporting electron acceptors (He et al. 2003a(He et al. , 2003b. Although it was originally believed biotransformation processes could not occur near a chlorinated solvent source zone because of the toxicity of high contaminant concentrations associated with the presence of NAPL (Abelson 1990;Bouwer 1994;Robertson and Alexander 1996), recent chlororespiration investigations have been performed in the presence of non-aqueous-phase PCE (Adamson et al. 2004;Carr et al. 2000;Cope and Hughes 2001;Dennis et al. 2003;Nielsen and Keasling 1999;Sung et al. 2003;McCarty 2000, 2002). Nielsen and Keasling (1999) demonstrated complete reductive dechlorination (e.g., ethene formation) at saturated PCE concentrations in batch systems with a dechlorinating consortium. Most reducing equivalents from the electron donor (glucose) were consumed in reductive dechlorination, probably due to the inhibition of other microbial processes by the high chloroethene concentrations. Yang and McCarty (2000) also reported degradation of PCE in batch systems where concentrations of PCE approached the aqueous solubility limit. Although dechlorination stalled at cis-DCE, incomplete dechlorination could still be beneficial for source-zone bioremediation because a) dissolution rates are enhanced 3-fold McCarty 2002, 2003) to 6-fold (Cope and Hughes 2001) and b) cis-DCE and VC are more accessible to aerobic degradation in down-gradient aerobic zones (Coleman et al. 2002a(Coleman et al. , 2002b. In column studies, a nonuniform distribution of NAPL and organisms resulted in significant competition for reducing equivalents and bioclogging due to excessive microbial growth of nondechlorinating biomass (Yang and McCarty 2002). Competition and bioclogging may be controlled by slow-release electron donors. However, application of a simplified numerical model suggested that under electron-donor-limiting conditions, a biofilm develops around the NAPL, reducing dissolution and increasing the difficulty of supplying sufficient electron donor (Chu et al. 2003). Partitioning of lesser chlorinated ethenes (TCE, cis-DCE, VC) into PCE-DNAPL and decreases in pH due to the release of HCl have also been observed and may affect the dechlorination of the lesser chlorinated ethenes (Adamson et al. 2004;Cope and Hughes 2001).
These findings have important ramifications for source-zone bioremediation, as well as posttreatment biopolishing. Although a variety of organisms are capable of PCE-to-cis-DCE dechlorination, complete detoxification requires the presence and activity of Dehalococcoides populations Ritalahti et al. 2001). Contaminant removal and plume containment after bioaugmentation with Dehalococcoides-containing cultures have been demonstrated in the field (Ellis et al. 2000;Lendvay et al. 2003;Major et al. 2002), and recent results suggest that bioaugmentation is also a viable approach for initiation of reductive dechlorination in PCE source zones (Adamson et al. 2003). These findings suggest that combined bioaugmentation strategies that a) initiate the reductive dechlorination process in source zones (Adamson et al. 2003) after physicalchemical treatment and b) establish bioreactive barriers for treatment of dissolved contaminants down-gradient (Lendvay et al. 2003) are promising remediation approaches that warrant further exploration.
To sustain the reductive dechlorination process, a source of reducing equivalents (i.e., an electron donor) must be provided. Chlororespiring populations depend on the activity of fermentative organisms to convert (complex) organic materials into suitable electron donors (e.g., hydrogen or acetate) (DiStefano et al. 1992;He et al. 2002). A variety of substrates including pentanol, ethanol, lactate, propionate, butyrate, and oleate have been shown to produce suitable electron donors (e.g., acetate, hydrogen) to support chlororespiring populations (Carr and Hughes 1998;Fennell and Gossett 1998;He et al. 2002;McCarty 1998, 2002). Alternative amendment strategies that supply slow-release, nonsoluble substrates for example, olive oil, chitin, polylactate esters [e.g., Hydrogen Release Compound (HRC; Regenesis Bioremediation Products, San Clemente, CA)], have also been successfully, used (Koenigsberg and Farone 1999;Yang and MacCarty 2002). Chlororespiring populations are highly competitive hydrogen users and outcompete methanogens, acetogens, and sulfate-reducing populations for this electron donor (Löffler et al. 1999). Thus, substrates that result in slow release (or production) of Article | Mass removal and dechlorination for source-zone remediation Environmental Health Perspectives • VOLUME 113 | NUMBER 4 | April 2005 hydrogen are advantageous because most reducing equivalents are directed toward the process of interest (Ballapragada et al. 1997;Fennell et al. 1997;Fennell and Gossett 1998;He et al. 2002;Smatlak et al. 1996). It should be noted that any approach that increases the flux of hydrogen in a subsurface environment will also result in an increased flux of acetate, which has been implicated as a relevant source of low concentrations of hydrogen through syntrophic oxidation (He et al. 2002;Schink 1997).
Physical-Chemical Treatment of Chlorinated Solvent Source Zones
Over the past decade, a number of innovative technologies have been developed that show promise for recovering a large fraction of the DNAPL mass at a given site (e.g., Brusseau et al. 1999;Stroo et al. 2003). Although the number of field-scale demonstrations of these technologies is growing, more standardization of assessment and reporting of results are necessary before larger-scale implementations can be considered sound practice (NRC 1997). Furthermore, the lack of consensus pertaining to the potential benefits of partial source-zone removal (e.g., Rao et al. 2002;Rao and Jawitz 2003;Sale and McWhorter 2001) points to the need for a better understanding of the long-term influence of physical-chemical treatment on contaminant fluxes, plume development, and enhanced microbial activity.
Given that innovative source-zone removal technologies have been extensively documented (e.g., NRC 1994NRC , 1997NRC , 1999, this article provides only a brief summary of selected approaches including air sparging, chemical oxidation, thermal treatment, co-solvent flushing, and surfactant-enhanced aquifer remediation (SEAR). Application of any of these treatment technologies would require detailed site characterization, a well-delineated source zone, and, in most cases, efficient contact between injected fluids and DNAPL. The discussion below focuses on assessing the potential for coupling each technology with microbial reductive dechlorination.
Air sparging. A source-zone remediation technology that has been implemented at many DNAPL-contaminated sites is air sparging (NRC 1997; for more detailed descriptions and reviews of air sparging technologies, see Brown 1997;Hinchee 1994;Johnson et al. 1993;Reddy et al. 1995;Suthersan 1996). Air is injected below the water table to volatilize or strip contaminants from groundwater ( Figure 2). The vapor-phase contaminant rises into the unsaturated zone, where it can then be extracted with a soil vapor extraction system (Johnson et al. 1993). Typically, design of these systems is empirical and based upon two primary assumptions: a) the gas phase will contact the nonaqueous phase, resulting in direct mass transfer from the DNAPL to the vapor phase, and b) the gas phase will strip dissolved contaminants from the aqueous phase (Suthersan 1997;Unger et al. 1995).
Although air sparging may be applied to reduce DNAPL mass (Unger et al. 1995), concerns remain that the introduction of air to a source zone may increase the extent of contamination through lateral and vertical spreading of NAPL (Blanford et al. 1999;Henry et al. 2003). Air sparging has been reported to stimulate aerobic microbial processes, including co-metabolism of chlorinated ethenes, as long as a suitable primary substrate is present Johnson et al. 1993;Raes et al. 2002). Sustained enhanced aerobic biodegradation, however, may be problematic because aerobic degradation of unsaturated chlorinated solvents is limited at the high contaminant concentrations commonly found within DNAPL source zones (Alvarez-Cohen and McCarty 1991). The implementation of the aerobic co-metabolic process has been successfully demonstrated for TCE removal under field conditions ); however, the requirement for a primary substrate (e.g., toluene) remains problematic. Although lower-chlorinated ethenes (e.g., cis-DCE and VC) are amenable to growthlinked microbial degradation under aerobic conditions, a metabolic process capable of oxidizing PCE and TCE has yet to be identified . For these reasons, it is unlikely that stimulation of reductive dechlorination after air sparging is a viable approach.
Chemical oxidation. In situ chemical oxidation (ISCO) was developed to transform contaminants into benign products (i.e., CO 2 and salts) [for mechanistic descriptions of ISCO technologies, see NRC (1999) and Siegrist et al. 2001]. A common form of this technology involves the injection of hydrogen peroxide (∼10 to 50% by weight) in conjunction with an iron catalyst (e.g., ferrous sulfate), which forms highly reactive hydroxyl radicals (OH • ) via Fenton's chemistry. The hydroxyl radicals are strong oxidants and react rapidly with surrounding molecules. Solutions of hydrogen peroxide, without catalyst, have been introduced into the subsurface (Oberle and Schroder 2000) to reduce iron catalyst requirements and the need for pH adjustments. However, hydrogen peroxide at ambient temperature and pressure is a relatively poor oxidizing agent for chlorinated solvents. When hydrogen peroxide solutions are injected alone (i.e., without an iron catalyst), reductions in contaminant concentrations are frequently the result of volatilization or stripping, which occurs because of increased temperature and O 2 production as the hydrogen peroxide decomposes (Oberle and Schroder 2000). Permanganate, in the form of either sodium permanganate or potassium permanganate, offers an attractive alternative to Fenton's chemistry because it does not rely on the formation and transport of short-lived OH • radicals. The use of permanganate, however, results in the formation of manganese dioxide, which may precipitate and reduce aquifer permeability (Dai and Reitsma 2002;Li and Schwartz 2003;Siegrist et al. 2001). The potential for permeability reduction, as well as increased metal mobility, that may accompany use of chemical oxidants depends upon site-specific geochemical conditions. Thus, as with all source-zone treatment technologies, thorough site characterization is required to mitigate potential adverse effects (Crimi and Siegrist 2003;Siegrist et al. 2001).
Application of chemical oxidation to DNAPL source zones ( Figure 3) has produced mixed results (Siegrist et al. 2001;Urynowicz and Siegrist 2000). Still, some evidence suggests that permanganate oxidation of DNAPLs may be plausible if delivery of chemical oxidants to DNAPL mass can be improved (Nelson et al. 2001;Schnarr et al. 1998;West et al. 1998) and MnO 2 crusting of the DNAPL avoided (Dai and Reitsma 2002;Li and Schwartz 2003;Siegrist et al. 2001). These issues notwithstanding, the fate of microorganisms through the oxidation process remains unclear (Bassel and Nelson 2000;Kastner et al. 2000). Although a limited number of studies indicate that both aerobic and anaerobic populations may rebound after treatment with relatively low concentrations (< 2% weight) of oxidants (e.g., Allen and Reardon 2000), the posttreatment environment may have pH levels that are unfavorable for microbial activity depending upon site conditions (Kastner et al. 2000;Siegrist et al. 2001). Additionally, permanganate residuals in the source zone or oxygen produced during treatment is likely to maintain oxidative conditions, which prohibit reductive dechlorination of chloroethenes.
Thermal treatment. Thermal treatment techniques include steam (or hot water) flooding, resistive heating (e.g., three-or six-phase heating), conductive heating (e.g., thermal blankets), or some combination thereof [for more detailed descriptions of several thermal technologies, see Falta (2000); NRC (1999); Udell (1997)]. Of these technologies, steam flushing is frequently employed for treatment of sites contaminated with NAPL (Figure 4).
Laboratory and field tests have demonstrated the robustness of steam flushing (Udell 1997). There are, however, two drawbacks limiting widespread implementation: a) energy demands contribute significantly to project costs (Henry et al. 2003) and b) the potential for NAPL mobilization (Davis and Heron 1998;Falta 2000). During steam flushing, DNAPL mobilization occurs through a reduction in capillary forces at the condensation front and may become problematic if the recondensed organic liquid phase escapes hydraulic control and contaminates pristine regions of the subsurface. Thus, recent work has focused on designs that reduce the potential for downward migration of DNAPLs during steam flooding (Kaslusky and Udell 2002). Lesser understood impacts of steam treatment include the potential formation of intermediates or byproducts during thermal degradation (Cai and Guengerich 1999;Davis and Heron 1998;Kline et al. 1978;McKinney et al. 1955), and effects of steam and high temperatures on the microbial community (Davis 1998;Richardson et al. 2002).
Long-term monitoring efforts provide limited evidence that microbial activity may rebound after field-scale steam treatment (Smith et al. 1998. Richardson et al. (2002) found that mesophilic bacterial and archaeal populations survived steam treatment in laboratory studies using soils collected from contaminated sites. In their study microbial activity was only detectable after periods of gradual cooling; elevated temperatures and fast cooling rates resulted in little or no microbial activity. In situ rates of cooling are anticipated to be slow enough to allow subsequent microbial rebound (Richardson et al. 2002). Thorough characterization of the subsurface environment after thermal treatment of DNAPL source zones has yet to be reported, but it is likely that the treated zone immediately after steam or hot water injection will be aerobic, given that air may be injected during treatment for the purposes of contaminant oxidation (Leif et al. 1998) or DNAPL mobility control (Kaslusky and Udell 2002). In contrast, redox potentials measured at a site after electrical resistive heating were found to be consistent with those required for reductive dechlorination (Beyke et al. 2000;Smith et al. 2000). Therefore, additional research is required to determine the effectiveness of employing microbial reductive dechlorination after thermal treatment of DNAPL source zones.
Co-solvent flushing. Alcohols have been used as co-solvents to enhance recovery of NAPLs through either solubilization or mobilization (displacement) [ Figure 4; for description of the mechanisms and implementation of co-solvent flushing technologies, see Advanced Applied Technology Demonstration Facility (AATDF) (1997); Augustijin et al. (1997); Falta (1998)]. During solubilization, NAPL remains relatively immobile throughout recovery. In contrast, mobilization relies upon reduced capillary forces resulting from a decrease in interfacial tension to facilitate release and displacement of NAPL ganglia, which are recovered as an organic liquid or free product. Mobilization and solubilization are not mutually exclusive processes; co-solvent floods may be designed to favor either mechanism through a detailed understanding of system phase behavior (Brandes and Farley 1993;Falta 1998). Although selection of alcohols to promote partitioning leading to reductions in the density difference between phases (e.g., Lunn and Kueper 1999) can mitigate downward migration of DNAPL, field implementation of mobilization co-solvent floods have been limited to the treatment of light NAPL source zones (Falta et al. 1999). Other field tests employing the use of co-solvents focused on enhanced removal through solubilization (Jawitz et al. 2000;Rao et al. 1997). Use of high concentrations of alcohols (> 70%) in co-solvent flushing may result in gravity override (bypassing) and reduced source-zone bioactivity. Gravity override can be limited with careful design of injection systems to counter buoyancy forces (Jawitz et al. 2000). Although flushing with concentrated alcohol solutions may negatively affect microbial activity, long-term monitoring results (> 3 years) from a site where co-solvent flushing was employed suggest that general bioactivity may rebound as alcohol concentrations decrease (Annable 2003;Mravik et al. 2003). It is unclear, however, how the populations critical to reductive dechlorination respond to alcohol flushing. In general, if harmful impacts on the microbial community can be avoided or are shown to be less disruptive than currently perceived, the addition of short-chain alcohols such as ethanol may prove to be a feasible method for stimulating posttreatment reductive dechlorination.
Surfactant-enhanced aquifer remediation. SEAR refers to in situ flushing technologies that use surfactants to overcome many of the limitations experienced during pump-andtreat remediation of DNAPL source zones (Figure 4; for mechanistic and practicable descriptions of SEAR, see, e.g., AATDF (1997); Jafvert (1996); Pennell and Abriola 1997)]. Generally, surfactants are molecules that preferentially accumulate at surfaces or interfaces based upon their amphiphilic molecular structure. Both anionic and nonionic surfactants have demonstrated potential for use in NAPL-contaminated aquifer remediation (Baran et al. 1994;Dwarakanath et al. 1999;Pennell et al. 1993;Shiau et al. 1994). SEAR technologies are similar to co-solvent flushing in that the general mechanisms of Article | Mass removal and dechlorination for source-zone remediation Environmental Health Perspectives • VOLUME 113 | NUMBER 4 | April 2005 source-zone mass removal are solubilization and mobilization (Figure 4 inset). As is the case with most aggressive remediation approaches, SEAR leverages greater upfront capital expenditures than traditional pumpand-treat remediation for higher efficiency. More than 90% recovery of contaminant mass has been demonstrated within DNAPL source zones in short time periods at the field scale Londergan et al. 2001;Ramsburg et al. 2005). The efficiency of SEAR makes it an attractive alternative to pump-and-treat remediation where hydraulic control allows for near complete capture of injected surfactant. One drawback to the use of surfactant solutions designed for high contaminant solubilization is the possibility of downward migration of the relatively dense solubilized plume or mobilized free-product DNAPL before recovery. Plume plunging behavior, however, may be mitigated through the addition of alcohols to the surfactant solution (Kostarelos et al. 1998) and careful design of the hydraulic flow regime/control system . Concerns over downward migration of mobilized DNAPL may be alleviated by using SEAR technologies that reduce DNAPL density in situ before mobilization (Ramsburg et al. 2003;Ramsburg and Pennell 2002;Yan et al. 2003) Use of readily biodegradable, food-grade surfactants minimizes concerns over the fate of unrecovered surfactant, yet the effect of such surfactants on microbial populations responsible for reductive dechlorination within the swept zone is only now beginning to be explored. Although most anionic and nonionic surfactants considered for application are completely degradable under aerobic conditions (Swisher 1987), degradation of alkylphenol ethoxylates (e.g., Triton X-100) has been shown to generate products (e.g., alkylphenols) that are persistent, toxic, and estrogenic (e.g., Ahel et al. 1994aAhel et al. , 1994bStephanou and Geiger 1982;White et al. 1994). Residual levels of readily degradable, food-grade surfactants, however, will likely promote the establishment of anaerobiosis, potentially facilitating conditions conducive for reductive dechlorination.
Application of biodegradable anionic surfactants at field sites has typically been accompanied by high concentrations of 2-propanol (∼40 g/L) and sodium chloride (as high as 7 g/L) to increase contaminant solubilization capacities > 60 g/L (e.g., Brown et al. 1999). Thus, posttreatment conditions will likely have elevated concentrations of anionic surfactant, alcohol, and sodium chloride, which could inhibit or prevent microbial activity. Unfortunately, no long-term monitoring results have been reported, limiting the understanding of microbial activity after treatment with these formulations. In contrast, longterm monitoring results from a field test conducted using a biodegradable, food-grade, nonionic surfactant (without alcohol or salt addition) indicate that surfactant degradation stimulated microbial activity within the treated source zone (Ramsburg et al. 2004).
Implications for coupling physicalchemical treatment with microbial reductive dechlorination. Existing evidence suggests certain physical-chemical source-zone treatment technologies are more promising for the stimulation of microbial activity as a posttreatment polishing step. Although air sparging, chemical oxidation, and steam flooding may generate an aerobic environment suitable for subsequent metabolic or co-metabolic oxidation, SEAR and co-solvent flushing appear to be the most promising physical-chemical treatments for integration with the microbial reductive dechlorination process. Note that in this assessment, the possibility that DNAPL contaminant distributions resulting from aggressive treatment may be technology specific has not been considered because of the scarcity of data. Residual alcohol or surfactant solutions contribute to oxygen depletion and establishment of anaerobic conditions after aggressive treatment. Further, residual flushing solution may serve as a source of reducing equivalents and stimulate the reductive dechlorination process. Although other technologies may eventually be successfully integrated with posttreatment microbial reductive dechlorination, SEAR seems particularly applicable because of limited toxicity on the microbial community, the establishment of reducing conditions, and the release of reducing equivalents for stimulation of the reductive dechlorination process. Thus, the ultimate fate of the residual surfactant solution and its effect on the dechlorinating population must be considered.
Although microbial degradation of surfactants in aerobic environments is well documented (Swisher 1987), it is uncertain how surfactants typically selected for SEAR are degraded in anaerobic environments. Linear alcohol ethoxylates are degraded to fermentable substrates under anaerobic conditions (Huber et al. 2000), and the degradation of nonionic surfactant has been reported under methanogenic conditions (Yeh et al. 1999). It is therefore, likely that fermentation of unrecovered surfactant will serve as an indirect source of reducing equivalents by producing hydrogen and organic acids, whose slow anaerobic oxidation will generate additional hydrogen to support the chlororespiring populations.
The residual surfactant concentrations, however, may also alter the bioavailability of a contaminant (Colores et al. 2000;Pennell et al. 2001;Rouse et al. 1994). Yeh et al. (1999) investigated the bioavailability of hexachlorobenzene (HCB) in the presence of nonionic, ethoxylated sorbitan surfactants (i.e., Tween series) in a methanogenic mixed culture obtained from contaminated sediment. At low surfactant concentrations (< 10 mg/L) there was no apparent change in rate or extent of HCB dechlorination. At a surfactant concentrations above the critical micelle concentration (CMC), enhanced HCB dissolution occurred, and although dechlorination rates decreased, the dechlorination end point remained unchanged. Complete inhibition of reductive dechlorination was observed at a surfactant concentration of 1,000 mg/L. However, Yeh et al. (1999) hypothesized that the observed inhibition was likely due to toxic effects of high surfactant concentrations rather than micellar sequestration of HCB. These results are supported by a recent study using a PCE dechlorinating consortium and a matrix of anionic, nonionic, and cationic surfactants (McGuire and Hughes 2003). McGuire and Hughes (2003) observed that the nonionic surfactant Tween 80 [polyoxyethylene (20) sorbitan monooleate] exhibited the least impact on dechlorination (both rate and extent) and thus speculated that the number of ethylene oxide groups present on the surfactant molecule affects surfactant toxicity. In fact, Bury and Miller (1993) and Guha et al. (1998) demonstrated that contaminants (in these studies nonchlorinated hydrocarbons) sequestered in the micellar phase may remain bioavailable. The response of the dechlorinating microbial community to surfactants is poorly understood, and future research should explore possible stimulatory or inhibitory effects in a heterogeneous environment where local surfactant concentrations may be well above the CMC.
Mathematical Assessment
Although microbial reduction of PCE in DNAPL source zones may be feasible, the relatively low dissolution enhancement factors (3-to 6-fold) reported imply that source longevity would still be measured in multiple decades. Alternatively, if uncertainties in the source zone microbial environment after physical-chemical treatment can be overcome, multiple order-of-magnitude reductions in source-zone mass removal obtained via active physical-chemical treatment might be combined with posttreatment biopolishing to substantially reduce source longevity. Ultimately, it may be possible to devise a posttreatment source-zone strategy that minimizes operations and maintenance efforts while still meeting regulatory standards at down-gradient points of compliance.
The potential benefits of tailoring physical-chemical treatments to stimulate microbial reductive dechlorination may be illustrated through a straightforward mathematical modeling analysis that compares source longevity for four hypothetical DNAPL source-zone scenarios ( Figure 5) under three management strategies: a) natural gradient dissolution (natural dissolution), b) enhanced reductive dechlorination (source-zone bioremediation), and c) physical-chemical treatment followed by source-zone biopolishing (SEAR plus enhanced reductive dechlorination). The four hypothetical field scenarios were selected to span the range of behavior that may be expected in the field and are characterized by a ganglia-to-pool (GTP) ratio, which is a measure of the distribution of mass between low saturation ganglia regions and high saturation pool regions in the source zone. The formation properties, spill scenario, and SEAR characteristics were taken from a recent numerical modeling study that was based on a pilot-scale SEAR demonstration at the Bachman Road site in Oscoda, Michigan Lemke and Abriola 2003;Lemke et al. 2004). These properties are summarized in Table 2. Scenario 1 assumes NAPL is entrapped as residual globules and ganglia at a uniform saturation throughout the source zone ( Figure 5A). This scenario has an infinite GTP ratio (IGP) and would be characteristic of an ideal site that had perfectly uniform hydraulic properties and where DNAPL was released over a reasonably wide area. Cleanup of this site is modeled using a simplified hydraulic approach (Brusseau 1996), which is based on mass-balance calculations. Scenario 2 is perhaps more realistic. It is representative of a situation with the NAPL entrapped as residual ganglia ( Figure 5B), although some pooling has occurred because of permeability contrasts [high GTP ratio (HGP), GTP > 1.0]. This DNAPL saturation distribution was generated following the methods outlined by Lemke and Abriola (2003) and Lemke et al. (2004). Using this methodology, the release of NAPL into a nonuniform permeability field is simulated using an laboratory-validated numerical multiphase simulator (MVALOR; Dekker and Abriola 2000; Lemke et al. 2004;Rathfelder et al. 2001). Natural dissolution or SEAR is then simulated using a separate numerical simulator (MISER) that has been used to accurately simulate SEAR in laboratory experiments (Rathfelder et al. 2000(Rathfelder et al. , 2001 and was used in the design of a recent SEAR pilot-scale test . Scenario 3 was also generated using this same methodology ( Figure 5C). Here, however, formation properties were configured so that the resultant saturation distribution was dominated by pools [low GTP ratio (LGP), GTP < 1.0; for Article | Mass removal and dechlorination for source-zone remediation Environmental Health Perspectives • VOLUME 113 | NUMBER 4 | April 2005 471 details, see Lemke et al. (2004)]. Scenario 4 assumes all mass is immobilized in six idealized, rectangular, fully saturated (S n = 1) pools with no ganglia remaining ( Figure 5D). This scenario is an extreme case where the GTP ratio is equal to zero (ZGP). Cleanup in this scenario was modeled using an analytical solution to the two-dimensional advection-dispersion equation following the methods of Johnson and Pankow (1992). It should be noted that, in contrast to the HGP and LGP scenarios (1 and 4), which result from the use of numerical models that incorporate more of the physics of the problem (e.g., hysteretic DNAPL migration, nonuniform flow, ratelimited dissolution), the IGP and ZGP scenarios are nonphysical, idealized end-members intended to bracket behavior that may be observed in the field. Although the distribution of mass in the source zone is different in each of the four scenarios, the amounts of mass in the source zone, the source-zone (i.e., domain) volume, the aqueous-phase contaminant solubility during a given process (i.e., SEAR or natural gradient dissolution), and the average hydraulic flux through the source zone are identical. The source longevity in scenarios 1-4 using each of the three remediation strategies was arbitrarily defined as the time when 99.9% NAPL was removed from the source zone. The second and third management strategies, source-zone bioremediation and SEAR plus biopolishing, used a simplified bioenhancement factor taken from the literature to quantify the improvement in dissolution because of microbially mediated aqueous-phase degradation. Reductive dechlorination enhanced-dissolution factors ranging from 3-to 6-fold have been reported (Cope and Hughes 2001;Yang and McCarty 2002). For this simplified example, an enhancement factor of 5 was assumed. This enhancement factor was reported in column studies in which NAPL ganglia were uniformly distributed, chlororespirers were present and active, and there were no limitations on microbial growth (Cope and Hughes 2001;Yang and McCarty 2000). It is unlikely that these conditions could be obtained at real sites, and thus, the enhancement factor of 5 is likely optimistic. However, in an effort to determine the benefits of aggressive mass removal before source-zone biopolishing (management strategy 3) versus bioremediation alone, favorable source-zone bioremediation (management strategy 2) was assumed.
Calculated values of source longevity for each of the three management strategies for all four scenarios are reported in Table 3, and percent mass removal as a function of time is presented in Figure 6. As might be expected, source longevity for scenario 1 (IGP) and scenario 4 (ZGP) tends to bracket the cleanup behavior of the more complex scenarios (HGP and LGP). Application of physical-chemical source-zone treatment (a 10-day surfactant flush of 4% Tween 80) before biopolishing is shown to reduce the source longevity regardless of scenario conditions. The magnitude of this reduction, however, depends on the level of pooling in the NAPL source zone ( Figure 6A). If, for example, the LGP scenario is assumed to be representative of a typical small-scale site, the 10-day SEAR followed by biopolishing will result in a 53 and 91% decrease in source longevity, in comparison with results of sourcezone reductive dechlorination alone and natural dissolution conditions, respectively ( Figure 6B). In this scenario, conducting SEAR operations for an additional 15 days (25 days total) would result in removal of 98.5% of the DNAPL mass, thereby reducing source longevity to 4 years. Thus, results presented in Table 3 and Figure 6 suggest that physical-chemical treatment followed by enhanced microbial activity could greatly reduce source longevity and associated longterm risk.
Bachman and Sages
The co-solvent flood at the former Sages dry cleaning facility (Jacksonville, Florida) and the Bachman Road SEAR site (Oscoda, Michigan) serve as documented case studies where field evidence supports the conclusion that physical-chemical source-zone removal may be coupled with reductive dechlorination. A comparison between observations at the Sages and Bachman sites is shown in Table 4. It is important to recognize that these posttreatment monitoring data provide only a snapshot of conditions (at 1,280 days for Sages and 450 days for Bachman) in a transient environment. Although the evolutions of the conditions at the Sages and Bachman sites are described in more detail in Mravik et al. (2003) and Ramsburg et al. (2004), respectively, we provide a summary below to facilitate analysis of the observed stimulation of microbial reductive dechlorination after physical-chemical treatment.
At the Sages site, 34,000 L of a solution consisting of 95% (vol) ethanol and 5% (vol) water were flushed through a DNAPL source zone over a period of 3.5 days followed by a 4.5-day water flood used to recover injected fluids (Jawitz et al. 2000). This co-solvent flood was successful in removing 43 L of PCE-DNAPL from the subsurface, and extraction well data indicate 92% of the ethanol introduced during the flush was recovered (Jawitz et al. 2000). Posttreatment characterization conducted approximately 1 month after the cessation of flushing activities indicated that DNAPL remained after treatment (Sillan 1999) and that the average PCE and ethanol concentrations in the extraction wells were ∼ 120 µM and ~230 mM, respectively (Mravik et al. 2003). Results from longer-term sampling at the Sages site indicate that PCE concentrations within the source zone rebounded to pretreatment levels approximately 150 days after treatment and that ethanol concentrations remained in excess of 160 mM for approximately 350 days (Mravik et al. 2003). Although ethanol toxicity remains a concern, elevated concentrations of hydrogen and acetate in the treated zone suggest microbial activity (Mravik et al. 2003 positive when analyzed via nested polymerase chain reaction with Dehalococcoides-targeted primers (Mravik et al. 2003). Additionally, microcosm studies with aquifer material from the Sages site indicate that sulfate-reducing and methanogenic populations rebounded after exposure to elevated concentrations of ethanol (Ramakrishnan et al. 2005). Although the survival and activity of dechlorinating populations within the treated zone have not been demonstrated to date, observations of significant cis-DCE production (up to 242 µM) at monitoring points located within the treated zone are indicative of microbial reductive dechlorination. At the Bachman Road site, a pilot-scale field demonstration of SEAR was conducted to remove PCE-DNAPL from beneath a former dry cleaning facility. For this source-zone treatment, 68,400 L of an aqueous solution containing 6% (weight) Tween 80 were introduced over a period of 10 days, with 2 additional days of active water flooding Ramsburg et al. 2005). Approximately 95% of the injected surfactant was recovered along with > 19 L of PCE. Posttreatment site monitoring indicates that PCE concentrations were reduced by two orders of magnitude from pretreatment levels at many locations within the treated zone and, in contrast to the Sages site, did not rebound after 450 days (Ramsburg et al. 2004). Surfactant concentrations decreased steadily over time, and after 270 days, surfactant was not detectable at most sampling points within the treated zone (12 µM detection limit).
Before the SEAR treatment, substantial reductive dechlorination had not occurred in the source zone. However, significant concentrations of PCE degradation products were measured within the treated zone 270 days after treatment (Table 4). Acetate and formate, likely products of Tween 80 fermentation, were observed at levels as high as 4,600 µM and are indicative of anaerobic microbial degradation of the surfactant (Ramsburg et al. 2004). Organic acids are known to support reductively dechlorinating populations present in the Bachman aquifer (He et al. 2002(He et al. , 2003a(He et al. , 2003bSung et al. 2003), and PCE-to-cis-DCE transformation within the treated source zone is consistent with laboratory microcosm studies conducted with aquifer material from the Bachman Road site (He et al. 2002). VC, however, was detected at only 3 of 26 sampling locations within the source zone. The apparent accumulation of cis-DCE at most observation locations may indicate that PCE-to-cis-DCE degrading organisms are predominating within the treated zone.
These two examples from field sites suggest that physical-chemical source-zone treatments are capable of stimulating organisms responsible for degrading residual level contaminants.
At these sites, data support the conclusion that ethanol and Tween 80 were metabolized by active microbial communities, resulting in an increased production of hydrogen and acetate. The availability of these electron donors, in turn, promoted reductive dechlorination activity. Although such enhanced bioactivity within source zones may occur at sites contaminated on much larger scales (e.g., Hill Air Force Base; Londergan et al. 2001), it is important to recognize that sites such as Sages and Bachman are representative of numerous small-scale chloroethene source zones existing in communities across the United States (e.g., State Coalition for Remediation of Drycleaners 2004). These smaller sites not only are significant sources of dissolved phase contamination but are often more problematic because a) they typically occur in proximity to areas of higher population, increasing risk and limiting hydraulic isolation (i.e., containment) options, and b) the relatively low NAPL saturations and smaller treated volumes at these sites increase treatment costs as quantified by conventional metrics (dollars per cubic meter of treated soil or dollars per liter of NAPL recovered). Higher costs per volume (treated soil or NAPL) result from a threshold cost associated with establishing a treatment system regardless of site size. Many innovative source-zone technologies offer efficient mass removal at the expense of greater, upfront capital expenditures (Rao et Mravik et al. 2003 100-4,600 (100) µM Ramsburg et al. 2004 2002). Decreased source longevity resulting from aggressive treatment, however, results in lower operational and maintenance costs making many innovative approaches economically viable when compared against long-term pump-and-treat remediation (e.g., Ramsburg and Pennell 2001). A staged treatment approach that employs microbial reductive dechlorination after aggressive mass removal may thus provide a cost-effective option for reduction of both source longevity and risk. The need for integrating treatment technologies for groundwater cleanup has become more apparent (Jackson 2003;Rao et al. 2002) since first being advocated by the NRC's Committee on Ground Water Cleanup Alternatives (NRC 1994). Thorough site characterization is critical for design of any treatment train remedy (Jackson 2003). Sitespecific tailoring of physical-chemical treatment for stimulation of posttreatment bioactivity must be based upon an accurate understanding of the location and extent of DNAPL, as well as hydrogeology and pretreatment microbial parameters. Co-solvent and surfactant flushing are very promising approaches because they can be tailored to enhance posttreatment reductive dechlorination. It should be noted, however, that ISCO may provide another means of polishing of residual-level contamination subsequent to other source-zone remediation technologies. Additionally, ISCO may be an attractive follow-on treatment alternative at sites where characterization efforts demonstrate that dechlorinating populations cannot be readily stimulated or augmented.
Conclusions
Taken in total, literature data, example calculations, and case studies presented above support a position of cautious optimism regarding the potential of combined physical-chemical/ reductive dechlorination remedial methods for the effective treatment of chlorinated solvent source zones. The literature review, however, suggests a number of areas requiring further investigation before the performance of such methods can be fully assessed and optimized. Given the number of remediation sites at which natural attenuation of chlorinated solvents has been documented (Wiedemeier et al. 1999), and the knowledge that many of the flushing solutions themselves stimulate bioactivity in laboratory tests, one would anticipate that stimulation of indigenous microorganisms in a source zone after physical-chemical treatment would be common. Therefore, the lack of widespread evidence for bioremediation after physical-chemical treatment indicates either that microbial activity is occurring but lacks documentation (e.g., the indicators of bioremediation are not monitored) or that the posttreatment environment does not favor microbial activity. It is important that future field demonstrations of source-zone flushing technologies are designed to systematically investigate a) the source-zone (dechlorinating) microbial community, before, during, and after the treatment process, and b) contaminant and transformation product concentration distributions after treatment. Indeed, to date, most field observations of enhanced reductive dechlorination in treated source zones have been fortuitous, with little thought devoted to microbial processes in the initial design and implementation of the treatment monitoring scheme. Specific culture-dependent (e.g., microcosms) and culture-independent (nucleic acid-based) tools for assessment of the microbial community are now available for this characterization effort (He et al. 2003a(He et al. , 2003bHendrickson et al. 2002;Löffler et al. 2000;Morse et al. 1998).
Future field demonstrations may also be enhanced through exploitation of results obtained from microbial laboratory investigations. Laboratory-scale studies conducted under conditions representative of a sourcezone environment (i.e., in the presence of organic liquid) provide heuristic, as well as quantitative, guidance for implementation of posttreatment bioremediation. Substrate amendment strategies that favor chlororespiring populations by maintaining a low concentration of hydrogen may be adapted from the laboratory to the field. However, additional work will be required to explore the effect of unrecovered flushing solutions (e.g., alcohol or surfactant) typical of a posttreatment sourcezone environment on the metabolism of chlorinated NAPLs by chlororespiring organisms. The discovery of numerous dechlorinating populations capable of converting PCE to cis-DCE and recognition of the importance of Dehalococcoides populations in the transformation of chloroethenes to ethene will likely improve future bioaugmentation strategies and further enhance posttreatment biopolishing. Although enhanced NAPL dissolution by partially dechlorinating populations has been demonstrated, it remains to be seen if complete detoxification (e.g., ethene formation) in source zones is feasible. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2010-10-08T00:00:00.000 | 11789689 | {
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} | pes2o/s2orc | Optical Sensing Properties of Dithiocarbamate-Functionalized Microspheres, Using a Polyvinylpyridine-Polyvinylbenzyl Chloride Copolymer
In this study, a new modified optical chemical sensor based on swellable polymer microspheres is developed using a 5% copolymer of polyvinylpyridine-polyvinyl-benzyl chloride microspheres functionalized as the corresponding dithiocarbamate. This sensor demonstrated significant enhancements in sensitivity, dynamic range and response time. These improvements are related to the presence of pyridine in the polymer backbone, which is believed to increase the space between the groups, thus decreasing steric hindrance, and hence increasing substitution of the dithiocarbamate group. The hydrophilicity of pyridine also allows free movement of the solvent and analyte to and from the inside of the microspheres. These dithiocarbamate-derivatized polymer microspheres were embedded in a hydrogel matrix of polyvinylalcohol cross-linked with glutaraldehyde. This sensor responded selectively to Hg2+ solutions of different concentrations (1 × 10−5 M to 0.1 M). The observed turbidity measured as absorbance varied between 1.05 and 1.75 units at a wavelength of 700 nm. The response is based on the interaction between the metal cations with the negative charges of the deprotonated dithiocarbamate functional group, which led to neutratization of the charges and thus to polymer shrinking. As a result, an increase in the turbidity of the sensing element due to a change in the refractive index between the hydrogel and the polymer microspheres occured. The changes in the turbidity of the sensing element were measured as absorbance using a conventional spectrophotometer.
Introduction
Recently, interest has been directed towards developing optical chemical sensors based on polymer swelling and shrinking [1][2][3][4][5][6][7]. Holtz et al. prepared a sensing element from a crystalline colloidal array of polymer spheres and a hydrogel that swells and shrinks in the presence of certain analytes [8]. Meanwhile, Liu et al. used a hydrogel containing various molecular recognition receptors that are subject to polymer swelling for the detection of different chemical species [9]. Some of these sensors involved use a variety of functional groups attached chemically to the backbone of polyvinylbenzyl chloride polymer. Seitz et al. developed an optical sensing element from lightly cross-linked chemically derivatized polymer microspheres with dimensions of a few micrometers. These microspheres were suspended in a hydrogel membrane which changed its volume reversibly in response to changes in analyte concentrations. This chemical sensing is based on the changes in the optical properties of the membrane that accompany swelling and shrinking. On swelling the refractive index of the microspheres becomes closer to that of the hydrogel, resulting in a decrease in the membrane turbidity [4,6,10].
In previous work, we developed a sensor in which polyvinylbenzyl chloride microspheres were aminated with ethylenediamine, and then converted to the respective dithiocarbamate by reacting the product with carbon disulfide. The resulting dithiocarbamate functionality was selected due to its formation of stable chelates with heavy metal ions of environmental concern, and to its negligible affinity towards alkali and alkaline earth metal ions typically present in real samples [2,11]. The response to metal cations is due to the neutralization of the negative charges on the deprotonated dithiocarbamate that lead to shrinking of the microspheres. This results in a change in the refractive index between the microspheres and the hydrogel membrane which can be measured as turbidity. This sensor has some limitations, particularly, due to its low sensitivity and long response time. To overcome these limitations, the sensing element was modified by using a partially hydrophilic copolymer and a different aminating group. This modification led to more substitution during the functionalization of the microspheres with dithiocarbamate.
In this work, partially hydrophilic polyvinylpyridine-polyvinylbenzyl chloride copolymer microspheres were aminated with ethanolamine and then transformed into the corresponding dithiocarbamate copolymer. The resulting microspheres were entrapped in a polyvinylalcohol membrane cross-linked with glutaraldehyde to form an optical sensing element. The latter demonstrated significant improvement in both dynamic range sensitivity and response time.
Instruments
Absorption measurements related to turbidity were performed on a Perkin-Elmer Lambda 5 UV-visible spectrophotometer. The pH measurements were recorded on a Jenway pH meter (3,310) with a combination glass electrode and a tolerance of ±0.01 pH units. A Fourier transform infrared spectrophotometer (Nicolet Avatar 370DTGS) was used to obtain IR spectra.
Synthesis of the dithiocarbamate polymer
One gram of (polyvinylpyridine-polyvinylbenzyl chloride) copolymer 1 was soaked in few milliliters of dimethylformamide for several days and then immersed in 7.5 mL of ethanolamine (2) at room temperature and stirred for one week. The product 3 was washed several times with distilled water then the excess ethanol amine was removed under reduced pressure.
Next one gram of the aminated copolymer microspheres 3 was stirred with a mixture of 15 mL of 2-propanol, 5 mL CS 2 , and 20 mL DMF for one hour at room temperature. This was followed by the addition of 5 mL of 10% aqueous NaOH and the resulting solution was stirred for five days. The dithiocarbamate copolymer 4 was then filtered and washed several times with distilled water and dried under reduced pressure (Scheme 1).
Polymer capacity
The capacity of dithiocarbamate copolymer microspheres 4 towards metal ions was determined by initially soaking a 0.1 gram of the derivatized copolymer in 20 mL of aqueous 0.1 M HgCl 2 and stirring overnight. Then, the copolymer was filtered and washed extensively with distilled water. The unadsorbed metal ions on the copolymer were determined by inductively coupled plasma (ICP). Then the adsorbed metal ions on the copolymer were calculated.
Optical measurements
The sensing element prepared as described previously was stuck on the inner sidewall of a cuvette which was then secured in the cell holder of a conventional Perkin Elmer spectrophotometer [2,6], such that the membrane was positioned in the path of the light beam. The solution in the cuvette was changed using a disposable pipette, starting with the lower concentrations of analyte and proceeding to the higher ones, with an interval of 10 minutes between each spectrum run. The response of the sensor to pH was performed starting with the lower pH and proceeding to the higher ones (2.0-12.6). The reproducibility of the sensor response was evaluated by cycling between blank and 0.1 M Hg 2+ ions several times. Readings as turbidity absorbance at wavelength (700 nm) were taken after 10 minutes of introducing the solution in the cuvette. Between each reading, Hg 2+ ions were eluted by a saturated solution of EDTA and then washed extensively with distilled water until a blank reading was obtained. The response time of the sensing element towards 0.1 M of Hg 2+ ions was obtained by measuring the change in turbidity as absorbance with time, until a steady state was reached. After the sensor responded to metal cation, a saturated solution of EDTA was added, followed by basic buffer solution and finally washing successively with distilled water to regenerate the sensing element.
Characterization of the dithiocarbamate polymer
The introduction of the dithiocarbamate groups into the copolymer backbone was characterized by the disappearance of the C-Cl bond stretching at 800 cm −1 and 600 cm −1 and the appearance of peaks at ~1,500 cm −1 which are related to the C-N vibration of CS 2 -NR 2 bond and peaks between 1,200 cm −1 and 1,050 cm −1 which are related to the C=S vibration of the CSS bond (Figure 1).
Capacity studies showed that the amount of Hg 2+ ions absorbed was 13.5 mmol per gram of the copolymer as opposed to 1.1 mmol per gram of the dithiocarbamate functionalized polyvinylbenzyl chloride polymer. The use of the copolymer improved significantly the sensitivity and increased the dynamic range of the sensor due to the presence of the pyridine moiety in the polymer backbone. The latter acted to space out the polymer reducing steric hindrance and thus resulting in more substitution.
Sensor evaluation
When the sensing element was examined with different pH buffer solutions (2.0-12.6), insignificant changes in absorbance were observed. This probably results from the presence of both basic (amine groups) and acidic groups (CS 2 H) situated on the dithiocarbamate copolymer 4. The nitrogen (hard base) and the sulfur (soft base) were both protonated at low pH, where positive charges on nitrogen repel each other causing the polymer to be in a swollen state. As the pH is raised, the sulfur on the dithiocarbamate group becomes deprotonated and so the resulting negative charges repel each other causing the polymer to stay in a swollen state. This behavior does not lead to any significant and observable shrinking process, and thus no detectable changes in absorbance ( Figure 2). Surprisingly, the deprotonated dithiocarbamate copolymer showed no optical response towards tested heavy metal cations ( Ni 2+ , Cu 2+ , Cr 3+ ,Pb 2+ , Zn 2+ , Cd 2+ ), while it showed a very high affinity towards the soft metal cation Hg 2+ (Figures 3,4). The response of our sensing element towards Hg 2+ was tested by using different aqueous solutions with concentrations ranging from 1 × 10 −5 M to 0.1 M Hg 2+ . At this concentration range, the turbidity increased from 1.05 to 1.75 measured as absorbance ( Figures 5,6), as opposed to 0.74 to 0.86 in the previously tested dithiocarbamate polymer derived from polyvinylbenzyl chloride [2]. The increase in turbidity with Hg 2+ concentration is probably due to the formation of a complex between Hg 2+ and the deprotonated dithiocarbamate groups, thus causing the polymer microspheres to shrink due to neutralization of the negative charges on the sulfur atoms. This shrinkage increased the difference in the refractive index between the hydrogel and the microspheres, which resulted in an increase in the turbidity of the sensing; this was measured as absorbance.
Thus, the use of polyvinylpyridine-polyvinylbenzyl chloride copolymer instead of polyvinylbenzyl chloride polymer improved the sensitivity and increased the dynamic range. The presence of the pyridine group in the polymer backbone apparently increased the space between groups in the polymer backbone. This decreased the steric hindrance, and resulted in more substitution.
The response time of the sensing element toward Hg 2+ ions was obtained by recording the change in turbidity as absorbance of 0.1 M Hg 2+ at 700 nm vs. time. The absorbance increased with time until it reached a steady state. The response time was significantly shorter with the copolymer than that obtained with poly vinyl benzyl chloride. Thus, the copolymer took 30 seconds to reach 90% response while the polymer needed 10 minutes under the same conditions ( Figure 7) [2]. This fast response is probably due to the presence of the hydrophilic pyridine group in the copolymer which enhanced the movement of the analyte and solvent across the microspheres during the shrinking process. The reproducibility of the sensing element was examined by taking the change in turbidity as absorbance of a 0.1 M Hg 2+ aqueous solution at 700 nm. The absorbance for the blank (1.109) and 0.1 M Hg 2+ (1.648) stayed almost constant during the ten runs, and according to the standard deviation calculations (4.714 × 10 −4 ), this sensing element is highly reproducible. The stability of the sensor was examined by measuring the response to Hg 2+ during several weeks; one run was taken every week. This sensing element was found to be stable for several weeks during which it gave a positive response towards Hg 2+ ions (Figure 8). Both the reproducibility and stability of the sensor are related to the utilization of the very small particles (1-3 µm) of the copolymer which provided mechanical stability and flexibility during multiple swelling and shrinking cycles. This result is an indication of the high stability of the dithiocarbamate functional group under the conditions of the experiment.
As expected the dithiocarbamate copolymer showed no response towards alkali and alkaline earth metal cations (Figure 9). This is because the sulfur atoms of the dithiocarbamate group are soft ligands and do not interact with hard metal cations (alkali and alkaline earth metals). The presence of these metal cations will not affect the optical properties of the sensor, this is demonstrated by the significant similarity in response towards Hg 2+ ions in both tap and distilled water ( Figure 10). Thus, this sensor has the potential to be applied to real samples.
Conclusions
A new improved optical chemical sensor based on swellable polymer microspheres has been developed using a dithiocarbamate functional group covalently bonded to a backbone of polyvinyl-pyridine-polyvinylbenzyl chloride copolymer. This gave better performance compared to previously prepared sensors. It showed better sensitivity, reproducibility, stability and shorter response time towards Hg 2+ ions. In addition, no significant response to the heavy metal ions ( Ni 2+ , Cu 2+ , Cr 3+ ,Pb 2+ , Zn 2+ , Cd 2+ ) and the alkali and alkaline earth metal ions was detected. | v3-fos-license |
2018-04-03T05:14:48.585Z | 2017-02-14T00:00:00.000 | 14937303 | {
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} | pes2o/s2orc | Repair of a Bacterial Small β-Barrel Toxin Pore Depends on Channel Width
ABSTRACT Membrane repair emerges as an innate defense protecting target cells against bacterial pore-forming toxins. Here, we report the first paradigm of Ca2+-dependent repair following attack by a small β-pore-forming toxin, namely, plasmid-encoded phobalysin of Photobacterium damselae subsp. damselae. In striking contrast, Vibrio cholerae cytolysin, the closest ortholog of phobalysin, subverted repair. Mutational analysis uncovered a role of channel width in toxicity and repair. Thus, the replacement of serine at phobalysin´s presumed channel narrow point with the bulkier tryptophan, the corresponding residue in Vibrio cholerae cytolysin (W318), modulated Ca2+ influx, lysosomal exocytosis, and membrane repair. And yet, replacing tryptophan (W318) with serine in Vibrio cholerae cytolysin enhanced toxicity. The data reveal divergent strategies evolved by two related small β-pore-forming toxins to manipulate target cells: phobalysin leads to fulminant perturbation of ion concentrations, closely followed by Ca2+ influx-dependent membrane repair. In contrast, V. cholerae cytolysin causes insidious perturbations and escapes control by the cellular wounded membrane repair-like response.
IMPORTANCE Previous studies demonstrated that large transmembrane pores, such as those formed by perforin or bacterial toxins of the cholesterol-dependent cytolysin family, trigger rapid, Ca 2ϩ influx-dependent repair mechanisms. In contrast, recovery from attack by the small -pore-forming Staphylococcus aureus alpha-toxin or aerolysin is slow in comparison and does not depend on extracellular Ca 2ϩ . To further elucidate the scope of Ca 2ϩ influx-dependent repair and understand its limitations, we compared the cellular responses to phobalysin and V. cholerae cytolysin, two related small -pore-forming toxins which create membrane pores of slightly different sizes. The data indicate that the channel width of a small -pore-forming toxin is a critical determinant of both primary toxicity and susceptibility to Ca 2ϩdependent repair. P ore-forming proteins are widely used by bacteria to directly damage cells (1), promote intracellular growth (2,3), or introduce virulence factors into the cytosol (4,5). Intriguingly, nucleated cells are able to restore structural and functional plasma membrane (PM) integrity after damage by bacterial pore-forming toxins (PFTs) (6), permitting, for example, recovery from major Staphylococcus aureus alpha-toxindependent losses of cellular ATP (7). Restoration of PM integrity has been also documented for streptolysin O (SLO) (8), pneumolysin (9), aerolysin, and listeriolysin (LLO) (10); it occurs in various cell types in culture and has been shown in Caenorhabditis elegans to operate in vivo (11). Efficient repair of the PM after wounding or attack by proteins forming large pores, such as SLO, perforin, or complement, is thought to require Ca 2ϩ influx (8,(12)(13)(14)(15). Downstream mechanisms include endocytosis of lesions and replacement of the PM by lysosomal exocytosis (15)(16)(17)(18)(19)(20)(21)(22) and/or blebbing of the PM and ectocytosis (23,24). These pathways might act in a complementary manner (25).
Caveolin has been implicated in endocytosis of SLO pores (22). More recently, a requirement of the endosomal complex required for transport (ESCRT) for membrane repair after damage by laser light has been reported; this pathway could also be involved in the repair of membrane pores (26). Notably, the recuperation of cells from an attack by small -pore-forming S. aureus alpha-toxin or aerolysin is significantly slower than that of cells treated with SLO or LLO, and it proceeds in the absence of extracellular Ca 2ϩ (10,27,28). Furthermore, comparative studies showed that recovery following attack by S. aureus alpha-toxin or aerolysin, but not by SLO or LLO, involves p38 mitogen-activated protein kinase (p38 MAPK), autophagy, and phosphorylation of the ␣-subunit of eukaryotic initiation factor 2 (eIF2␣) (10,28,29). Evidently, the mode and efficacy of PM repair and cellular recovery depend on the type of PFT (reviewed in reference 5). In order to comprehend differential cellular tolerance for various PFTs and PFT-producing bacteria, it will be important to elucidate the scope and limitations of Ca 2ϩ influx-dependent repair. Here, we have investigated cellular responses to phobalysin P (PhlyP) and the orthologous Vibrio cholerae cytolysin (VCC) (30)(31)(32)(33)(34)(35), two related small -PFTs of Photobacterium damselae subsp. damselae and V. cholerae, respectively. V. cholerae is the notorious cause of a profuse, life-threatening diarrhea in humans. P. damselae subsp. damselae is a pathogen of marine animals that may infect wounds and lead to hyperaggressive necrotizing soft tissue infection or sepsis in humans. In addition to other proteins (36,37), VCC and PhlyP are considered to serve as virulence factors of these bacteria (35,38,39). We exploited the similar, yet distinct structures of these toxins to gain insight into the function or failure of Ca 2ϩ influx-dependent repair after attack by small -PFTs.
PhlyP and VCC perturb ion concentrations in epithelial cells with different kinetics.
PhlyP is a small -PFT that is related to VCC (39), but in contrast to VCC, it lacks a C-terminal -prism domain (Fig. 1A). Moreover, homology-based modeling of the PhlyP transmembrane pore using the known structure of VCC (40) as a scaffold predicted a wider narrow point of the channel (Fig. 1B) and fewer charged residues clustering in the channel-forming region of PhlyP (Fig. 1C). Therefore, in spite of their homology-50% identity on the amino acid level-it is conceivable that PhlyP and VCC exert different effects on target cells. This conjecture was confirmed by the finding that only PhlyP made epithelial cells (HaCaT cells) permeable to propidium iodide (PI) (39), prompting us to also compare changes of ion concentrations in epithelial cells. Loss of intracellular K ϩ is a hallmark of PM permeabilization by all PFTs investigated so far. Treatment of HaCaT cells with purified PhlyP caused dose-dependent loss of cellular K ϩ within 2 min; little further decrease was observed thereafter ( Fig. 2A). In contrast, the loss of K ϩ was progressive in samples treated with VCC (Fig. 2B). Given that PhlyP made cells permeable to PI (molecular weight [MW] of 668.4), we surmised that it would also permit influx of Ca 2ϩ ions. Cells permeabilized by PhlyP retained the exquisitely Ca 2ϩ -sensitive probe Fluo-8 AM (MW of~1,000) (see Fig. S1A in the supplemental material), which was exploited to detect whether the toxins caused changes of intracellular calcium ion concentrations [Ca 2ϩ ] i . PhlyP (400 ng/ml) led to a significant increase of fluorescence in Fluo-8 AM-loaded cells within 30 s after exposure (Fig. 2C); half-maximal effects were reached at 100 ng/ml and saturation at~200 ng/ml (data not shown). VCC at 400 ng/ml led to a final increase of fluorescence like that of PhlyP (Fig. 2D), although 100 ng/ml VCC remained ineffective (data not shown). Conspicuously, the VCC-dependent increase in fluorescence commenced significantly later than the PhlyP-dependent increase (~60 s versus~12 s; P ϭ 9.5 ϫ 10 Ϫ8 ). This raised the question of whether the two toxins increased [Ca 2ϩ ] i via different mechanisms. Purinoceptors have been implicated in cellular responses to PFT and in the regulation of Ca 2ϩ influx (41)(42)(43). Therefore, we tested the effect of suramin, an inhibitor of P2 receptors, on PFT-dependent changes of [Ca 2ϩ ] i . Suramin exerted a moderate inhibitory effect on the PhlyP-dependent rise of [Ca 2ϩ ] i ( Fig. 2C) but virtually blocked the VCC-dependent increase (Fig. 2D).
Epithelial cells replenish K ؉ after perforation by PhlyP. To investigate whether PhlyP-treated epithelial cells were able to recover, we measured cellular K ϩ levels immediately after a brief incubation with toxin or after incubation for various times in the absence of toxin. Following incubation of cells with PhlyP (100 ng/ml for 10 min at 37°C), the cellular K ϩ levels were reduced to~10%, but they returned to normal within 1 h after the removal of unbound toxin (Fig. 3A). A similar recovery was observed when cells were treated with 500 ng/ml PhlyP (see Fig. S1B in the supplemental material). In contrast, the loss of cellular K ϩ in response to VCC was sustained (Fig. 3A). Notably, the combination of both toxins behaved like VCC alone, indicating that the rescue process, apparently triggered by PhlyP, cannot save cells simultaneously intoxicated by VCC (Fig. 3B). Although incubation of cells with PhlyP for 8 min sufficed to cause significant influx of PI, membrane integrity was reconstituted after the washing out of PhlyP. Resealing was observed whether cells were treated with purified PhlyP (data not shown) or extracellular products (ECPs) from strain AR119, a P. damselae subsp. damselae strain expressing PhlyP ( Fig. 3C) (39,44).
PhlyP elicits a wounded membrane repair-like response, but VCC does not. Because EGTA prevented the restoration of K ϩ levels ( Fig. 4A), and because the depletion of K ϩ was irreversible in Ca 2ϩ -free medium (see Fig. S1C in the supplemental material), we investigated whether mechanisms proposed to act downstream from Ca 2ϩ influx-dependent repair of large membrane pores (15,18,(22)(23)(24) were also involved here. Therefore, we measured the release of -hexosaminidase, a marker of lysosomal exocytosis (45). Notably, PhlyP causes no leakage of lactate dehydrogenase (39), and EGTA blocked the release of -hexosaminidase (data not shown), demonstrating that -hexosaminidase release faithfully reported lysosomal exocytosis. PhlyP induced release of the enzyme from HaCaT cells (Fig. 4B), but VCC was ineffective ( Fig. 4B and C). In line with a role of lysosomal exocytosis for recovery from PhlyP attack, desipramine (DPA), an inhibitor of acid sphingomyelinase (ASM), which impairs the reversal of SLO-dependent membrane permeabilization (18), reduced the replenishment of cellular K ϩ without altering the initial toxin-dependent loss of this ion (see Fig. S1D). Consistent with this, inhibition of ASM did not aggravate the VCC-dependent loss of K ϩ (see Fig. S1E). PhlyP caused the formation of large, dynamic blebs in HaCaT cells, some of which appeared to detach to form large vesicles (see Movie S1), and blebbistatin stalled the formation of free vesicles (see Movie S2). However, neither alone nor in combination with DPA did blebbistatin alter the replenishment of cellular K ϩ after attack by PhlyP (see Fig. S1D), thus not supporting a major role of blebbing for cellular recovery from PhlyP. And yet, PhlyP led to increased endocytosis of fluorescently labeled bovine serum albumin (BSA), a cargo of caveolar uptake (see Fig. S2A), Small Pore-Forming Toxins May Trigger or Avoid Repair ® and SDS-stable oligomers were coisolated with caveolin and the exosomal marker protein flotillin in supernatants of target cells, suggesting sequential endocytosis and exocytosis of PhlyP (see Fig. S2B). The wounded membrane repair-like response following membrane perforation by SLO has been demonstrated by RNA interference (RNAi) to depend on caveolin (22), and a current model is depicted in Fig. S2D. Here, we exploited mouse embryonal fibroblasts (MEF) lacking caveolin expression (MEFcav Ϫ/Ϫ ) to investigate whether this protein is also important for cellular defense against the small -pore-forming toxin PhlyP. As in HaCaT cells, VCC and PhlyP both caused loss of K ϩ from wild-type MEF (MEFwt) or MEFcav Ϫ/Ϫ (Fig. 4D and E). Also, restoration of the intracellular K ϩ concentration was not observed after treatment with VCC (Fig. 4E), whereas replenishment of K ϩ was efficient in PhlyP-treated wild-type cells. Importantly, replenishment of K ϩ failed in MEFcav Ϫ/Ϫ exposed to PhlyP (Fig. 4D). That PhlyP triggered a wounded membrane repair-like response in MEF was further suggested by the facts that it increased the fraction of cells carrying ceramide ( Fig. 4F and G) and that it led to exposure of a luminal epitope of the lysosomal marker protein LAMP-1 at the cell surface (Fig. 4H) (17); only minor effects were discernible after treatment with VCC. The protective role of caveolin was confirmed when we analyzed PI influx by flow cytometry. Exposure to PhlyP for 5 min led to some influx of PI in wild-type and caveolin-deficient cells. However, wild-type cells soon excluded PI again (15 min), whereas the fraction of MEFcav Ϫ/Ϫ positive for PI had increased (Fig. 4I). By 45 min, two-thirds of MEFcav Ϫ/Ϫ but only about one-fourth of wild-type cells were heavily stained with PI. Recovery from PhlyP is not blocked by the p38 MAPK inhibitor SB203580. In contrast to PhlyP, other small -pore-forming toxins, i.e., aerolysin and S. aureus alpha-toxin, have been previously shown to trigger not Ca 2ϩ influx-dependent repair but slower, p38 MAPK-dependent recovery processes (28). Like VCC, PhlyP activates p38 MAPK (see Fig. S3A in the supplemental material). However, an inhibitor of activated p38 MAPK, SB203580, did not impede the replenishment of cellular K ϩ in PhlyP-treated cells (see Fig. S3B).
Channel narrow points impact fluxes of Ca 2؉ . The differential effects of PhlyP versus VCC on cellular homeostasis, influx of vital dyes, and repair could be due at least in part to differences in their transmembrane channels. Specifically, the bulky side chain of W318 in the VCC channel forms a heptad, reminiscent of the phenylalanine clamp in the anthrax protective antigen pore (4,40), and it could restrict the flux of ions or dyes. In contrast, serine 341, predicted to form the narrow point in PhlyP pores, is expected to be less obstructive. To investigate a potential impact of channel narrow points on toxin function, we generated single-amino-acid exchange mutants of the VCC and PhlyP protoxins pVCC and pPhlyP, in which tryptophan at position 318 (W318) of pVCC and serine at position 341 of pPhlyP were swapped, creating mutants pVCC(W318S) and pPhlyP(S341W) (Fig. 5A). HaCaT cells were loaded with Fluo-8 AM and treated with wild-type or mutant toxins, and fluorescence was recorded; the experiment was performed in the presence or absence of suramin. Intriguingly, pPhlyP(S341W)hereinafter termed pPhlyP S/W-caused a significantly lower suramin-insensitive increase of fluorescence than pPhlyP (Fig. 5B). For comparison of wild-type and mutant VCC (Fig. 5C), the mature toxins were generated with trypsin, because maturation by cellular proteases appeared comparably inefficient; VCC(W318S)-termed VCC W/S below-caused increases of fluorescence similar to those caused by wild-type VCC, but only in the case of VCC W/S was a significant portion of that signal insensitive to suramin (Fig. 5C).
Narrowing the PhlyP channel limits primary damage and repair; widening the VCC channel enhances toxicity. Next, we asked whether the above-described point mutations had an impact on membrane damage or repair. Cells were treated with wild-type or mutant protoxins, stained with PI and Hoechst stain, and examined by fluorescence microscopy. Only minimal influx of PI was noted upon a short incubation with either wild-type or mutant pVCC, but loss of membrane integrity progressed von Hoven et al.
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inexorably despite washing out of toxin ( Fig. 6A and B; see also Fig. S4 in the supplemental material). Notably, cells deteriorated more rapidly after exposure to pVCC W/S, as suggested by particularly strong influx of PI and nuclear condensation (see Fig. S4 and 6B and C, respectively). The swapping of serine and tryptophan in PhlyP led to more pronounced changes: only in half of the cells was influx of PI observed, and this only to a small extent, upon treatment with pPhlyP S/W, while the majority of cells treated with wild-type pPhlyP were brightly stained. Surprisingly, pPhlyP S/W-treated cells continued to permit low-level ingress of the dye, while membrane integrity was restored in pPhlyP-treated cells. Quantification of SDS-stable oligomers and analysis by electron microscopy did not indicate alterations in the ability to form oligomers (see Fig. S5). Individual PhlyP pores were of equal or slightly higher conductance than mutant pores for all salts tested, but the traces for the PhlyP S/W mutant pores showed increased levels of flickering and frequent breakdown of conductance, which lasted for seconds in some cases (see Fig. S6). We also found that PhlyP pores (mutant or wild type) showed higher conductance than VCC pores (about 300 versus 22 pS in KCl) and Fig. 1A. Colored letters in cytolysin domain indicate residues exchanged in mutants. (B and C) HaCaT cells were pretreated or not with 150 g/ml suramin, loaded with Fluo-8 AM in the presence or absence of inhibitor, and exposed to pPhlyP or pPhlyP S/W (B) or to VCC or VCC W/S (C) at 400 ng/ml. Fluorescence intensity was recorded at intervals of 5 s for a period of 3 min. Mean values Ϯ SE are shown (n Ն 5). P values were determined with Student's t test.
Small Pore-Forming Toxins May Trigger or Avoid Repair ® were rather cation selective, while VCC was moderately anion selective (46). Differential behavior of pPhlyP and pPhlyP S/W was also evident in MEF cells. First, the influx of PI was more pronounced in response to pPhlyP (see Fig. S7A). Second, pPhlyP caused a far stronger release of -hexosaminidase in HaCaT cells (Fig. 7A) or caveolin-deficient MEF (Fig. 7B) than did pPhlyP S/W. This release was completely blocked by EGTA (data not shown). The binding of pPhlyP to wild-type or knockout MEF was equal (Fig. S7B), but MEFcav Ϫ/Ϫ cells were more sensitive to pPhlyP (Fig. S7A). The difference between PhlyP and pPhlyP S/W was more pronounced in wild-type cells (Fig. S7A). Similarly, DPA sensitized MEFwt for both PhlyP and pPhlyP S/W (see Fig. S7C). Thus, late steps of the wounded membrane repair-like response (ASM-and caveolin-dependent steps; see Fig. S2D) appear to be involved in ongoing defense of MEF against PhlyP or pPhlyP S/W. The different capacities of PhlyP and pPhlyP S/W to trigger the release of -hexosaminidase surfaced when caveolin-dependent tolerance was disabled (Fig. 7B).
DISCUSSION
The present work reveals that different small -pore-forming toxins may either trigger or subvert calcium influx-dependent repair. Furthermore, the data suggest that the channel width of small -pores codetermines the kinetics and degree of primary damage, as well as susceptibility to repair. PhlyP, in contrast to the closely related VCC, caused fulminant breakdown of membrane integrity but permitted resealing by a process which until now has only been implicated in the repair of much larger membrane lesions, for instance, pores formed by cholesterol-dependent cytolysins. The modeling-based hypothesis that the narrow point in the PhlyP channel is wider than that in VCC is supported by conductance measurements.
Repair of PhlyP pores involves Ca 2ϩ influx, lysosomal exocytosis, and caveolin, but MAPK p38 is dispensable, supporting the idea that Ca 2ϩ influx-dependent repair supersedes the requirement for alternative salvage pathways. In striking contrast to PhlyP, VCC subverts Ca 2ϩ influx-dependent repair. Thus, even brief exposure of cells to low concentrations of VCC sufficed to initiate the progressive demise of human epithelial cells. That nanomolar concentrations of VCC are required to increase [Ca 2ϩ ] i , although picomolar concentrations are sufficient to kill cells (34), provides an explanation for VCC's propensity to subvert repair. However, even concentrations of VCC sufficient to increase [Ca 2ϩ ] i did not elicit a membrane repair response. Inappropriate topology, timing, or the degree of VCC-dependent increases of [Ca 2ϩ ] i may be responsible: that VCC-dependent increases of [Ca 2ϩ ] i were blocked by suramin indicated that they are mediated by P2 receptors, G-protein-coupled receptors, or other targets of the drug, which might cause Ca 2ϩ fluxes not to occur in sufficient proximity to VCC pores to allow repair. Second, compared to the PhlyP-dependent Ca 2ϩ influx, the VCCdependent Ca 2ϩ influx was delayed and comparatively slight. Therefore, we believe that the progressive damage by VCC is due to inadequate Ca 2ϩ influx through the small and anion-selective pore (34,40), in the face of otherwise severe perturbations of cellular physiology (e.g., loss of K ϩ ). Sure enough, VCC proved to be an inefficient trigger of lysosomal exocytosis. Notably, the PhlyP-dependent responses were unable to compensate for VCC's inability to trigger repair in mixing experiments. This could happen if VCC inhibits a step of the repair program downstream from lysosomal exocytosis, for instance, caveolar endocytosis. Because the abilities of VCC and PhlyP to trigger or subvert Ca 2ϩ influx-dependent repair appeared to correlate with their different channel narrow points, mutational analysis was a sensible approach. Single residues presumed to form channel narrow points of PhlyP and VCC were swapped to reveal their contributions to functional phenotypes. Changes in the Ca 2ϩ influx resulting from mutations of channel narrow points might impact both the cytotoxic power Small Pore-Forming Toxins May Trigger or Avoid Repair ® and ability to trigger repair responses; it was not predictable which effect would prevail. And yet, the interpretation of the data obtained with these constructs was straightforward: W318 restricts the influx of calcium ions through VCC pores; the obstacle falls away in the W318S mutant. Conversely, the replacement of S341 in pPhlyP with tryptophan reduces the influx of calcium ions. A principle finding made with the mutant protoxins was that the effect of channel width on cellular responses depends on the molecular context. The wider narrow point of the PhlyP channel promotes lysosomal exocytosis and recovery. However, it fails to do so if transplanted to VCC; in fact, it enhances toxicity in that context. The reason could be that the moderately increased influx of Ca 2ϩ through mutant VCC pores is sufficient to enhance toxicity but too low to trigger Ca 2ϩ influx-dependent repair. As a matter of fact, pVCC W/S caused only slightly greater increases of Ca 2ϩ influx than did wild-type pVCC, and the increase in the -hexosaminidase release was statistically insignificant. That pVCC W/S did not elicit a stronger suramin-insensitive increase of [Ca 2ϩ ] i could be due to additional restraints in VCC pores. To sum up, VCC and PhlyP, two related small -PFTs, pose quite different challenges to cell autonomous defense, which may, at least in part, be attributed to different channel widths (Fig. 8): whereas PhlyP acts fast and thus could overrun host responses if present in sufficient quantities, VCC causes insidious damage and subverts membrane repair. The results highlight the function or failure of Ca 2ϩ influx-dependent repair as a defense against small -PFTs; this may help to better understand the pathogenesis of diseases caused by bacteria producing these widespread toxins.
MATERIALS AND METHODS
Toxins. The preparation of PhlyP and VCC was as described previously (39). In brief, PhlyP was purified by preparative isoelectric focusing and ion exchange chromatography from extracellular products of P. damselae subsp. damselae. Recombinant protoxins pVCC, pVCC W/S, pPhlyP, and pPhlyP S/W were expressed in Escherichia coli as N-terminally His 6 -tagged fusion proteins and purified by affinity chromatography; VCC was generated from pVCC with trypsin (33). Single-amino-acid-exchange mutants of pPhlyP and pVCC were generated with the aid of the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). For primer sequences and technical details, see Text S1 in the supplemental material.
In silico modeling of the PhlyP pore. The sequence of PhlyP was modeled on the X-ray structure of VCC (PDB identifier [ID] 3O44 [40]). Alignment and structural modeling were performed by using MODELLER version 9.13 (47). See Text S1 in the supplemental material for details.
Flame photometry for measurement of K ؉ . The loss and replenishment of cellular K ϩ levels after an initial loss is a valuable proxy of perturbation and reconstitution of membrane integrity after attack by small -PFTs and other PFTs (10, 28). Cellular K ϩ was quantified by flame photometry as described PhlyP, a small -barrel pore-forming toxin which forms comparatively wide pores, triggers rapid Ca 2ϩ influx, lysosomal exocytosis, and repair similarly to the large pore-forming streptolysin O (SLO). In contrast, small -barrel pore-forming toxins like Vibrio cholerae cytolysin (VCC) form narrower channels and subvert this response. CDC, cholesterol-dependent cytolysins.
von Hoven et al. previously (10). In brief, cells were washed three times with ice-cold K ϩ -free choline buffer. Cells were subsequently lysed by incubation for 30 min in choline buffer-0.5% Triton X-100 at room temperature on a shaker. Lysates were analyzed for K ϩ with an M401 flame photometer (Sherwood, United Kingdom) using propane gas. Fluo-8 AM-based Ca 2؉ assay. PFT-induced changes of [Ca 2ϩ ] i in HaCaT cells were monitored by using Fluo-8 AM from Santa Cruz Biotechnology, Inc., in a TriStar LB 941 instrument from Berthold Technologies, as detailed in Text S1 in the supplemental material.
Fluorescence microscopy. Immunofluorescence analysis of ceramide and the lysosomal marker protein LAMP-1 was performed with MEF because the available antibodies yielded unspecific staining in HaCaT cells. The staining protocols for LAMP-1 and ceramide were as described in the supplemental material.
PI influx. The PI influx assay was performed as described previously (39). See Text S1 in the supplemental material for details.
Statistics. The data shown are from Ն3 independent experiments if not otherwise stated. Error bars represent plus-or-minus standard errors of the means. The statistical significance of differences between mean values was assessed with the two-sided Student's t test or with one-way analysis of variance (ANOVA) for multiple comparison; significance was assumed when the P value was Յ0.05. | v3-fos-license |
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} | pes2o/s2orc | Carcinoembryonic Cell Adhesion-Related Molecule 2 Regulates Insulin Secretion and Energy Balance
The Carcinoembryonic Antigen-Related Cell Adhesion Molecule (CEACAM) family of proteins plays a significant role in regulating peripheral insulin action by participating in the regulation of insulin metabolism and energy balance. In light of their differential expression, CEACAM1 regulates chiefly insulin extraction, whereas CEACAM2 appears to play a more important role in regulating insulin secretion and overall energy balance, including food intake, energy expenditure and spontaneous physical activity. We will focus this review on the role of CEACAM2 in regulating insulin metabolism and energy balance with an overarching goal to emphasize the importance of the coordinated regulatory effect of these related plasma membrane glycoproteins on insulin metabolism and action.
General Introduction
Since the discovery of carcinoembryonic antigen (CEA) in 1965 as tumor-specific antigen in human colonic carcinoma, research on this family of proteins has mounted, in particular focusing on one of its members, the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) plasma membrane glycoprotein [1]. Consistent with its ubiquitous expression and its regulation by metabolic [2,3] as well as immunological factors [4], CEACAM1 exerts several pleiotropic functions that have been well characterized. From the metabolic standpoint, CEACAM1 promotes insulin clearance and mediates a downregulatory effect on fatty acid synthesis by acute insulin pulses [5,6]. It also regulates inflammatory response [7]. CEACAM2 (previously known as Biliary Glycoprotein 2 (Bgp2)) is a close relative plasma membrane glycoprotein of CEACAM1. Consistent with its differential tissue and cell-specific expression [8], CEACAM2 exerts distinct as well as some overlapping functions with CEACAM1. CEACAM2 is involved in spermatid maturation [9,10], platelet activation and adhesion [11] and blood pressure regulation [12]. CEACAM2 is also involved in regulating insulin secretion [13,14] and energy expenditure [15,16]. The role of CEACAM2 in metabolism is the subject of this review.
Gene Structure of CEACAM2
In contrast to Ceacam1 gene that is detected in both pre-and postnatal developmental stages [8], Ceacam2 gene is not expressed during the embryonic stages in mice but starts to appear at three weeks postnatally and its expression continues to increase linearly to adulthood [9].
Mouse Ceacam2 and Ceacam1 loci are located on murine chromosome 7 about 62 kb apart [17]. Their genomic sequences share~79.6% homology [8]. Both genes have nine exons, the seventh undergoes alternative splicing to give rise to an early stop codon resulting in two different transcripts that are distinguished by a long (-L) or a short (-S) intracellular tail, containing or lacking conserved phosphorylation sites, respectively [18,19]. This pair of transcripts contains four IgG-like loops in its extracellular domain (CEACAM-4L/4S).
In addition to exon 7, both exons 3 and 4 undergo alternative splicing to produce two isoforms with two instead of four IgG loops (CEACAM-2L/2S). The incidence of this additional alternative splicing is high in Ceacam2 and yields a coding domain sequence of 1020 bp encoding CEACAM2-2L and another of 816 bp encoding CEACAM2-2S. Both transcripts share the same N-terminal tail with a signaling peptide and two extracellular loops with one being of V-type IgG and the other of C2-type IgG.
CEACAM2-2L undergoes glycosylation at the extracellular domain to elevate the apparent molecular mass from 37 kDa of apoprotein to 52 kDa. CEACAM2 loses glycosylation as well as the ability to form cis-or trans-polymers after deletion of the first V type IgG domain [10]. The amino acid sequence of the intracellular domains of CEACAM1 and CEACAM2 share~93% homology, in particular at the putative tyrosine phosphorylation sites at Y 488 and Y 515 in CEACAM1-4L and Y 307 , Y 334 in CEACAM2-2L.
Ceacam2 transcripts are detected in spleen, testis and prostate [8,17,19,20] and in pooled sorted non-β pancreatic cells [13]. At the protein level, CEACAM2 is expressed in kidneys, uterus, crypt cells and the villi lining the intestinal segment beginning with the distal jejunum, neuroendocrine cells of the ileum and platelets [8,9,11,13,15,17,20]. CEACAM2 protein is also expressed in several neuronal populations in the brain, including the ventromedial hypothalamus (VMH) and other centers involved in feeding behavior and rewards, such as hippocampus, striatum, olfactory bulb, and the globus and ventral pallidus [15]. In contrast, CEACAM2 is virtually absent in tissues that constitute the main sites of insulin action in the periphery (liver, white adipose tissue and skeletal muscle), as opposed to CEACAM1 that is predominantly expressed in the liver and its transcripts are detected in adipose tissue at a minimal level but not in skeletal muscle, under physiologic conditions.
Role of CEACAM2 in Insulin Secretion
Fluorescence-activated cell sorting of isolated islets revealed a relatively higher level of CEACAM1 expression in pancreatic β-cells [21] as opposed to CEACAM2 that is predominantly expressed in non-β pancreatic cells [13]. Despite its expression in β-cells, global null deletion of Ceacam1 does not alter glucose-stimulated insulin secretion or β-cell area [21]. In contrast, global deletion of Ceacam2 causes an increase in β-cell secretory function [13]. This occurs without affecting basal plasma levels of hormones (insulin, glucagon and somatostatin) or without significantly changing the areas of pancreatic cells (α-, βand δ-cells), as shown by immunohistochemical analysis [13]. Moreover, pooled islets isolated from global Ceacam2 knockout (Cc2 −/− ) mice release normal levels of insulin as compared to islets from wild-type mice in response to both glucose and potassium chloride. Together, this suggests that CEACAM2 regulates insulin secretion via an extra-pancreatic rather than a cell-autonomous regulatory mechanism that directly involves pancreatic cells.
In light of its expression in VMH [15], a glucose-sensing center in the brain [31], it is also possible that CEACAM2 regulates insulin secretion primarily via a neuronal-mediated mechanism [32,33]. This possible mechanism remains to be tested.
Role of CEACAM2 in Insulin Clearance
Insulin metabolism is regulated by insulin secretion from pancreatic β-cells and by its clearance, which occurs mainly in hepatocytes and to a lower extent in renal proximal tubule cells [34]. Upon its pulsatile secretion [35], insulin is rapidly transported via the portal vein into hepatocytes through the fenestrae in the liver sinusoidal endothelium to undergo degradation [36]. In this manner, the liver clears up to 80% of secreted insulin during its first pass [37]. In contrast, insulin transport in extrahepatic insulin target tissues is tightly regulated by endothelial cells [38][39][40], demonstrating a role for these cells in the regulation of peripheral insulin extraction.
Insulin clearance is mediated by receptor-mediated insulin uptake into the cell followed by its degradation in lysosomes as well as in endosomes [41]. Upon its phosphorylation by the insulin receptor tyrosine kinase, CEACAM1 forms a complex with the insulin receptor to increase the rate of insulin uptake and its target to the degradation process in hepatocytes [6] as well as in proximal tubular cells [42]. Several genetically modified loss-and gain-of-function mouse models targeting CEACAM1 in the liver demonstrated a key role for the upregulatory effect of CEACAM1 on receptor-mediated insulin uptake in maintaining insulin sensitivity and limiting de novo lipogenesis in liver in the face of higher insulin levels in the portal than systemic circulation. The role of CEACAM1 in regulating insulin clearance and its underlying mechanism has recently been reviewed [6].
In contrast to CEACAM1, CEACAM2 is not detected to a significant extent in hepatocytes but rather in murine kidney, an important site for insulin extraction. Whether CEACAM2 promotes receptor-mediated insulin uptake in renal proximal tubule cells is currently under investigation. Consistent with the dependence of this function on the phosphorylation of the conserved tyrosine residue in the highly homologous intracellular domain of these related CEACAM membrane glycoproteins [43,44], CEACAM2 is expected to mediate insulin clearance in renal proximal tubular cells. Intact insulin clearance in male Cc2 −/− null mice does not rule out a potential role for CEACAM2 in insulin extraction since it likely results from their intact CEACAM1 expression [15] and its dependent hepatic and renal insulin uptake. However, a potential role for CEACAM2 in extracting endogenously released insulin may not be as critical as that of CEACAM1 given the failure of insulin to regulate its transcription as it does to Ceacam1 promoter transcriptional activity [2,3,13,45]. In light of the suppressive effect of glucose on Ceacam2 mRNA levels [13], the role of CEACAM2 in glucose-stimulated insulin secretion is predictably more physiologically significant than its potential role in insulin clearance.
Role of CEACAM2 in Food Intake: Effect on Insulin Action
Food intake and energy balance are regulated by leptin-dependent neuronal signals in the arcuate nucleus (ARC) as well as in the dorsomedial (DMH) and ventromedial hypothalamus (VMH) [46]. Using immunohistochemical analysis, we detected CEACAM2 in neuronal hypothalamic populations like VMH, hippocampus, striatum, olfactory bulb and the globus and ventral pallidus [15] that are implicated in the regulation of feeding behavior [15,47,48]. Consistently, both male and female global Cc2 −/− null mice display hyperphagia without changes in plasma leptin levels at its onset. This suggests that hyperphagia in Cc2 −/− mice is not primarily caused by changes in leptin sensitivity [49]. Given that hypothalamic Ceacam2 mRNA is induced by fasting and reduced upon refeeding in response to glucose release [13,15], it is possible that hyperphagia in Cc2 −/− null mice develops at least in part, from altered glucose sensing activity [31] of the VMH as a consequence of the loss of neuronal CEACAM2.
Hyperinsulinemic-euglycemic clamp analysis demonstrated insulin resistance in skeletal muscle but not in the liver or adipose tissue of Cc2 −/− females [15], resulting from increased fatty acids uptake followed by incomplete fatty acid β-oxidation and consequently, lipotoxicity [50,51]. Given that CEACAM2 is not expressed in skeletal muscle [8], this points to central dysregulation of insulin action in these mice. Since VMH is a key site of leptin regulation of glucose uptake in skeletal muscle but not white adipose tissue [52], it is conceivable that cellular leptin resistance in Cc2 −/− females links CEACAM2 to leptin-dependent signaling pathways regulating glucose uptake and energy dissipation [53][54][55]. Thus, it is possible that peripheral insulin resistance in Cc2 −/− females is caused, at least in part, by altered leptin-dependent signaling pathways in VMH regulating glucose disposal and energy dissipation [54,56].
Young Cc2 −/− males exhibit increased fatty acid uptake in skeletal muscle and in the mitochondria to undergo complete fatty acid β-oxidation. This led to insulin sensitivity and lower total fat mass. With age, fat mass and visceral adiposity increase, while the metabolically active lean mass decreases in parallel to reduced glucose uptake in skeletal muscle that constitutes a main site of energy expenditure [57]. Since CEACAM2 is not expressed in skeletal muscle [8], the progressive age-related decline in energy dissipation in Cc2 −/− males likely stems from central dysregulation of peripheral glucose disposal, as is the case for their female counterparts [15]. Given that hypothalamic Ceacam2 mRNA level remains intact with age, unlike that of Ceacam1 that declines progressively until it reaches a loss by >70% at nine months of age to contribute to hyperphagia and disturb energy balance [58], it is likely that reduced Ceacam1 mRNA amplifies the adverse effect of Ceacam2 deletion on the hypothalamic control of glucose disposal and energy expenditure in older Cc2 −/− males.
Moreover, at this older age, male mutants develop insulin resistance in liver in addition to skeletal muscle [14]. The hepatic insulin resistance likely results from impaired CEACAM1-dependent hepatic insulin clearance pathways and resultant chronic hyperinsulinemia [14]. The progressive decrease in hepatic Ceacam1 mRNA stems from a compromised ability of insulin to induce Ceacam1 transcription under conditions of hyperphagia-driven insulin resistance [2,3] and from PPARα activation by lipolysis-derived fatty acids [3]. Reduced hepatic CEACAM1 levels provide a positive feedback mechanism on fatty acid β-oxidation [3] to prevent hepatic steatosis in aged Cc2 −/− males and to produce acetyl-CoA with the overarching goal to prevent glycolysis and reroute pyruvate to gluconeogenesis and glucose-6-phosphate to the glycogen synthetic pathways [3,14,59]. This is consistent with a role for reduced hepatic CEACAM1 levels in limiting fasting hyperglycemia [60] that could result from excessive increase in insulin secretion in aged Cc2 −/− males.
Pair-feeding experiments show that hyperphagia causes insulin resistance in female and male Cc2 −/− mutants at two and nine months of age, respectively [14,15]. Subsequently, Cc2 −/− mutants develop compromised energy expenditure and reduced locomotor activity [14,15]. The resulting energy imbalance leads to increase in body weight and visceral obesity at about six months of age in females [15] and at about nine months of age in males [14]. In contrast to female mice, young Cc2 −/− males exhibit increased sympathetic tone to white adipose tissue, consistent with induced brown adipogenesis in this depot and higher energy dissipation [16,61]. With age, the surrogate markers of brown adipogenesis (Ucp1 and Dio2) [62] and activated sympathetic tone (Ucp1, Adβ2r and Adβ3r) [63] are progressively reduced in white adipose tissue, consistent with reduced energy expenditure and increased visceral obesity in older Cc2 −/− males [14]. This age-related disturbance in energy dissipation could result, at least partially, from a loss of CEACAM2 at the VMH that contributes significantly to the central regulation of energy balance [64,65]. Thus, the hypermetabolic state (manifested by complete β-oxidation in skeletal muscle, increased brown adipogenesis in brown and white adipose depots, and increased sympathetic tone to adipose tissue), appears to offset the negative effect of hyperphagia in young Cc2 −/− males and maintain them insulin-sensitive until 8-9 months of age when they become hypometabolic exhibiting lower spontaneous physical activity than their wild-type counterparts and developing systemic insulin resistance [14,16].
Hyperphagia can also result from chronic hyperinsulinemia and insulin resistance [66][67][68][69], which develops in Cc2 −/− females at~2 months of age arising chiefly from increased insulin secretion [13,15]. In males, the persistent increase in insulin secretion, in part mediated by the higher plasma GLP-1 secretion [13], is offset by a parallel increase in CEACAM1-mediated insulin clearance, resulting in normoinsulinemia in the young until~9 months of age when chronic hyperinsulinemia develops largely from impaired hepatic insulin clearance that fails to counter the sustained elevation in insulin secretion [14]. Impaired insulin extraction in older males, results from the age-related progressive decline in hepatic CEACAM1 levels [14,58]. Nonetheless, hyperinsulinemia induces the transcriptional activity of SREBP-1c to stimulate the expression of lipogenic genes [70], such as fatty acid synthase (FASN), followed by their activation. Since the rise of hypothalamic FASN activity mediates hyperphagia independently of leptin [71][72][73], it is likely that hyperphagia is sustained by hyperinsulinemia-driven increase in hypothalamic FASN activity in Cc2 −/− mutants [14,15]. Additionally, elevated hypothalamic FASN activity could contribute to dysregulated central control of peripheral glucose disposal and reduced fatty acid β-oxidation in skeletal muscle of Cc2 −/− females and Cc2 −/− males at ≥9 months of age [14,15,72].
Hyperinsulinemia can also induce FASN activity in the liver. With the progressive decrease of hepatic CEACAM1 expression as Cc2 −/− males age, the counterregulatory CEACAM1-dependent negative effect of insulin on hepatic FASN activity [74] is abolished, giving rise to excessive lipid formation and re-esterification in the liver, followed by its redistribution to the white adipose depot for storage and subsequently, visceral obesity [58]. The resultant increase in lipolysis [58] as well as the pro-inflammatory state [75] contribute to systemic insulin resistance that develops in Cc2 −/− males at ≥9 months of age [14,15].
Conclusions
Based on the phenotype of Cc2 −/− mice, we propose that at fed state, when glucose is released, CEACAM2 expression rapidly declines in the entero-endocrine cells (as well as the neuroendocrine cells of the hypothalamus) [15] to stimulate insulin secretion via GLP-1-dependent mechanisms (Graphical Abstract). This in turn, induces CEACAM1-dependent hepatic insulin clearance [2,21,44] to maintain normoinsulinemia and insulin sensitivity. Given that GLP-1 prompts transition into the fasting state [76], this may initiate a negative feedback mechanism to recover hypothalamic CEACAM2 expression and subsequently, limit food intake and insulin secretion (Graphical Abstract). Further studies are needed to decipher the mechanisms underlying the role of CEACAM2 in controlling food intake but our data show that both leptin-dependent and leptin-independent hypothalamic pathways are implicated. Nonetheless, involvement of CEACAM2 in the central regulation of feeding behavior in addition to energy dissipation in skeletal muscle and insulin secretion is consistent with its expression in VMH, which contributes to the central regulation of energy balance and glucose disposal via sympathetic relay to peripheral tissues [65,77,78]. Moreover, the observed sexual dimorphism in obesity in Cc2 −/− null mutants further links CEACAM2 to the regulation of obesity and insulin resistance by VMH since lesions in this neuronal population cause obesity more commonly in female than male rodents [65]. The phenotype of Cc2 −/− mice provides an in vivo demonstration that CEACAM2 in the neuroendocrine cells of ileum and hypothalamus downregulates insulin secretion by suppressing GLP-1 release in male and female mice (Figure 1). Becasuse insulin upregulates hepatic CEACAM1 expression [2,3], the decrease in insulin secretion by CEACAM2 lowers hepatic CEACAM1 expression to limit insulin clearance and maintain normoinsulinemia in the face of restricted insulin secretion. CEACAM2 also limits food intake in both males and females but its deletion causes a reduction in sympathetic nervous activity in females only. This sexual dimorphism in terms of energy expenditure causes sex-dependent regulation of insulin action with Cc2 −/− females developing insulin resistance and Cc2 −/− males developing insulin sensitivity until about 8-9 months of age. The progression of insulin resistance in age-dependent manner in Cc2 -/-males [14] appears to involve the differential reduction of CEACAM1 in the hypothalamus [58] as well as in the liver [14]. The former contributes to leptin resistance and reduced spontaneous physical activity [79] and the latter to hyperinsulinemia-driven energy imbalance and systemic insulin resistance, at least partly by blunting hepatic insulin action [58,80]. This links insulin clearance to insulin secretion in the overall systemic regulation of physiologic insulin levels and provides further evidence for the impact of the coordinated regulatory effect of CEACAM proteins in insulin metabolism and action. The progression of insulin resistance in age-dependent manner in Cc2 −/− males [14] appears to involve the differential reduction of CEACAM1 in the hypothalamus [58] as well as in the liver [14]. The former contributes to leptin resistance and reduced spontaneous physical activity [79] and the latter to hyperinsulinemia-driven energy imbalance and systemic insulin resistance, at least partly by blunting hepatic insulin action [58,80]. This links insulin clearance to insulin secretion in the overall systemic regulation of physiologic insulin levels and provides further evidence for the impact of the coordinated regulatory effect of CEACAM proteins in insulin metabolism and action. | v3-fos-license |
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} | pes2o/s2orc | Chemical Profiling of Jatropha Tissues under Different Torrefaction Conditions: Application to Biomass Waste Recovery
Gradual depletion of the world petroleum reserves and the impact of environmental pollution highlight the importance of developing alternative energy resources such as plant biomass. To address these issues, intensive research has focused on the plant Jatropha curcas, which serves as a rich source of biodiesel because of its high seed oil content. However, producing biodiesel from Jatropha generates large amounts of biomass waste that are difficult to use. Therefore, the objective of our research was to analyze the effects of different conditions of torrefaction on Jatropha biomass. Six different types of Jatropha tissues (seed coat, kernel, stem, xylem, bark, and leaf) were torrefied at four different temperature conditions (200°C, 250°C, 300°C, and 350°C), and changes in the metabolite composition of the torrefied products were determined by Fourier transform-infrared spectroscopy and nuclear magnetic resonance analyses. Cellulose was gradually converted to oligosaccharides in the temperature range of 200°C–300°C and completely degraded at 350°C. Hemicellulose residues showed different degradation patterns depending on the tissue, whereas glucuronoxylan efficiently decomposed between 300°C and 350°C. Heat-induced depolymerization of starch to maltodextrin started between 200°C and 250°C, and oligomer sugar structure degradation occurred at higher temperatures. Lignin degraded at each temperature, e.g., syringyl (S) degraded at lower temperatures than guaiacyl (G). Finally, the toxic compound phorbol ester degraded gradually starting at 235°C and efficiently just below 300°C. These results suggest that torrefaction is a feasible treatment for further processing of residual biomass to biorefinery stock or fertilizer.
Introduction
The gradual depletion of world petroleum reserves together with the impact of environmental pollution by increasing exhaust emission has created an urgent need to develop alternative energy sources [1,2,3]. To address these issues, Jatropha curcas has been the focus of intensive research, because its seeds contain high levels of oil that is a valuable source of biodiesel. Unfortunately, large amounts of waste are generated by biodiesel production, and these waste include Jatropha tissues obtained after harvesting and pruning as well as the seed cake generated by oil extraction. These represent potential valuable resources of carbon for industrial and agricultural use. For example, Gunaseelan reported that the production of J. curcus plantation on rain fed dry land at a density of 4444 plants/ha yielded 1.42 ton/ha oil extraction, whereas residual biomass from de-oiled cake, pruned leaves, and fruit hulls yielded 4.83 ton/ha [4]. Furthermore, it was reported that the energy gain from biodiesel exhibited 53 GJ, whereas the energy gain from the anaerobic fermentation of residual biomass exhibited 36 GJ. Therefore, large amounts of residual biomass of Jatropha plantation can be used to recycle industrial energy through anaerobic fermentation [5,6].
Plant lignocellulosic biomass possesses undesirable properties such as high oxygen content, low calorific value, hydrophilicity, and high moisture content [4,7]. In addition, the chemical composition of plant lignocellulosic biomass is heterogenous, making the design and operation of biorefinery stock production more complicated. Therefore, a key challenge is to develop efficient and cost-effective conversion technologies for maximizing the utilization of lignocellulosic biomass. We have recently reported how ionic liquids can break down strong intermolecular hydrogen bonds in crystalline cellulose [8,9]. However, at present, this pretreatment technology is rather highly expensive and not environmentally friendly. Furthermore, we have elucidated that anaerobic fermentation sludge can break down crystalline cellulose, and these anaerobic microbiota can immediately produce biogas such as methane [5,6,10]. However, these microbiota can immediately metabolize glucose and oligosaccharides; therefore, it is quite difficult to extract biorefinery stock from cellulosic material.
On the other hand, torrefaction pretreatment is a thermal method for the conversion of biomass by heating it from 200uC to 300uC under atmospheric conditions in the absence of oxygen [11,12,13,14]. This process improves biomass properties and was therefore proposed as a potential solution to the problems described above [6,7,8,9]. Torrefied products such as gas, char, and tar (oil) can be used as chemical and energy resources and also as a fertilizer in the form of biochar [15].
Torrefaction has been applied to diverse biomass sources [16,17,18,19]. However, the physicochemical conversion of the plant biomass during torrefaction is largely uncharacterized. Detailed characterization of its chemical composition is required to determine how biomass can be most effectively applied. Therefore, we attempted to determine the physicochemical properties of torrefied Jatropha biomass.
Nuclear magnetic resonance (NMR) is widely used to analyze lignocellulose. The (2D)-NMR heteronuclear single quantum coherence (HSQC) method is useful for characterizing solubilized biomass such plant cell wall and particle organic matter [20,21,22,23,24,25,26]. The objective of the present study was to analyze the products of Jatropha biomass conversion using different torrefaction conditions. Torrefied products from different Jatropha tissues were analyzed using attenuated total reflectance Fourier transform infrared (ATR-FTIR) and NMR using polar and semipolar low and high molecular weight molecules (LMWMs and HMWMs, respectively. HMWMs refer to the DMSOsolubilized polymer fraction after water and methanol extractions, whereas LMWMs refer to the directly extracted metabolite fractions using water or methanol). Based on these analyses, we aimed to provide information that will guide more efficient application of biomass, such as biorefinery stock and fertilizer ( Figure 1).
Materials and Methods
Jatropha stems, seeds, and leaf tissues were harvested as described previously [20]. The stems were analyzed either as a whole or divided into xylem and bark; the latter included the phloem. The seed was divided into coat and kernel; the latter comprised the endosperm and embryo. The samples were freezedried and ground using an auto-mill machine (Tokken Co. Ltd, Japan). Powdered samples from the seed coats and kernels were subjected to oil extraction treatment by adding 1 ml of hexane per 100 mg of the sample. Note that the residual oil should be negligible, given that neither FT-IR nor NMR detected corresponding oil signals. The samples were incubated for 5 min at 50uC with shaking, centrifuged at 14000 rpm for 5 min, and then the supernatants discarded. This procedure was repeated five times.
LMWMs were extracted with 100% methanol and then with distilled water. One milliliter of methanol was added to 100 mg of the sample, incubated for 5 min at 50uC with shaking, centrifuged at 15000 rpm for 5 min, and then the supernatant was discarded. This procedure was repeated four times, and the samples were then dried and extracted with Milli-Q water using the same procedure as methanol extraction but without using methanol.
Torrefaction was conducted using an EXSTAR TG/DTA (Thermo Gravimetric/Differential Thermal Analysis) 6300 (SII Nanotechnology Inc., Tokyo, Japan) under a nitrogen atmosphere. The samples were heat-treated at 5uC/min starting at 24uC up to either 200uC, 250uC, 300uC, or 350uC. When the maximum temperature was reached, it was maintained for 10 min.
The torrefied and non-torrefied samples were analyzed in triplicate by ATR-FTIR using a Nicolet 6700 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a KBr Figure 1. Schematic conceptual figure of the analysis of heat treated Jatropha biomass and its future industrial and agricultural application. Torrefaction was applied and compounds present in the biomass were analyzed using FTIR and NMR techniques. FTIR characterized the total residue generated by the treatments, and NMR characterized samples prepared according to their solubilities and molecular weights. These analyses indicate that torrefaction can be more efficiently applied to utilize biomass (A). Scheme of Jatropha curcas biodiesel production from farming, pruning, harvesting, and oil extraction. These processes generate biomass from plant tissues and the seed cake (B). doi:10.1371/journal.pone.0106893.g001 disk. The ATR Smart iTR accessory with a high-pressure clamp (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. Spectra (4500-650 cm 21 ) were obtained using triangular apodization with a resolution of 4 cm 21 and an interval of 1 cm 21 . Background and sample spectra were normalized from 32 scans. The baseline and ATR corrections for penetration depth and frequency variations were conducted using OMNIC software supplied with the equipment.
After FTIR analysis, LMWMs were extracted with 100% methanol and distilled water as described previously [20]; however, the supernatant was collected in the present study. The remaining pellets were freeze-dried and ball-milled for 12 h using a P-5 Ball-mill machine (Fritch, Co. Ltd, Germany) programmed to grind for 10 min at 10 min intervals. The samples were suspended in dimethylsulfoxide (DMSO)-d 6 /pyridine-d 5 (4:1) using 60 mg of the sample and 600 mL of solvent. The mixture was incubated at 50uC for 30 min with shaking, centrifuged at 15000 rpm for 5 min, the supernatant collected as HMWMs, and then analyzed using the 1 H-13 C-HSQC NMR method. It was performed at 318 K with 32 scans as described previously [27,28,29]. The central DMSO solvent cross-peak was used as the internal reference (dC 39.5, dH 2.49 ppm).
LMWM samples collected from 108 mg of torrefied products were dried and dissolved in 600 mL of methanol-d 4 (CD 3 OD) and deuterium oxide (D 2 O), both containing 1 mM sodium 2,2dimethyl-2-silapentane-5-sulfonate (DSS). The mixtures were analyzed using the 1 H-NMR method at 298 K with 64 scans. The DSS signal was used as an internal reference (0.0 ppm). All NMR measurements were obtained using a AvanceII-700 spectrometer (Bruker, MA, USA) equipped with an inverse triple resonance CryoProbe with a Z-axis gradient for 5-mm sample diameters operating at 700. 153 MHz 1 H frequency. The assignment of the signals in the NMR spectra was performed using SpinAssign (http://prime.psc.riken.jp/) according to previous reports [30,31,32,33,34,35,36].
For the phorbol ester degradation assay, torrefaction of phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate (Sigma-Aldrich, St. Louis, MO, USA) as a standard was conducted using an EXSTAR TG/DTA 6300 as described above. The torrefied samples treated at different temperatures were analyzed using 1 H-NMR at 298 K with 64 scans.
ATR-FTIR analysis
The treatment temperature was defined through a preliminary thermogravimetric analysis where all the samples were vaporized at a heating rate of 5uC/min from 24uC to 500uC. This analysis showed differential TG peaks at approximately 200uC, 250uC, 300uC, and 350uC ( Figure S1).
To determine the change in chemical composition of Jatropha biomass during torrefaction, six different Jatropha tissues were treated at four different temperatures (200uC, 250uC, 300uC, and 350uC) and analyzed using ATR-FTIR. Table 1 show the spectra and assignment of functional groups. The peaks were assigned according to the previous reports [20,23,37,38,39]. Figure 2 shows heat-map like bird's eye viewing of FTIR spectra. The horizontal axis shows wave number and the vertical axis shows different samples, and the signal intensity (arbitrary unit) are shown according to the color key. Top nine samples in the vertical axis are standard compounds such as lignin, sugars, peptide, and fatty acid. The corresponding peaks from low molecular metabolites such as glucose, xylose, and linoleic acid were not present in both non-treated and torrefied biomass spectra. The comparison of three kinds of Jatropha varieties of kernel and seed coat did not show any remarkable changes, whereas non-treated stem biomasses from different species (Poplar, Wheat, and Jatropha) also did not exhibited remarkable changes. Likewise, mechanical treatment for both Poplar and Kernel samples also did not show remarkable changes. However, heat treatment to Jatropha tissues exhibited dramatic changes in FTIR spectra. The intensity of peak number 1 gradually decreased with increased temperature. Some of the observed difference between the 200uC-treated and nontreated samples may represent dehydration of compounds [39], whereas the decrease at higher temperatures may represent the cleavage of intramolecular hydrogen bonds [37].
We observed that biomass degradation differed according to tissue type ( Figure S2). For the seed coat, xylem, stem, and leaf, the intensity of peak 12 (assigned as C-O stretching in cellulose and hemicellulose) and 14 (C-H deformation of amorphous cellulose) were unchanged at 200uC and 250uC, and a decrease was observed at 300uC and 350uC. For the kernel and bark, in contrast, the decrease in the intensity of peaks 12 and 14 started at temperatures lower than 200uC and 250uC, respectively. The decrease in these peaks is a hallmark of the reduction of cellulose and hemicellulose content by volatilization and condensation during char formation [40]. Thus, cellulose and hemicellulose degradation may start at 200uC in the kernel, 250uC in the bark, and 300uC in the seed coat, xylem, stem and leaf.
The tissue-specific degradation patterns were also observed for lignin and aromatics compounds (peaks 5, 6, 7, 8, 9, 10, and 11). For the seed coat and xylem, a rise in the temperature caused a gradual decrease in their peak intensities, although the peaks were present at 350uC. Therefore, it is possible that lignin and aromatic compounds gradually decomposed over large temperature ranges. However, for the bark, leaf, and stem, the same peaks increased in intensity with an increase in the temperature, particularly peak 5, which was assigned as an aromatic skeletal vibration, and peak 9, which was assigned as C-H cellulose +C-O of syringyl ring derivatives. Chen et al. reported that the presence of peaks 5 and 9 and peaks around 750 cm 21 represent calcium oxalate monohydrate [41]. This mineral widely occurs in plants, including the bark tissues and in the stone cells of phloem and leaf [42]. Calcium oxalate is known as thermal degradation over 500 degree C due to removal of carbon monoxide. It is possible to verify the heat resistance of calcium oxalate in the bark, leaf, and stem because the peak persists even at high temperatures, which is in agreement with the results of Sen et al. [40].
For the kernels, the pattern of the peaks assigned to aromatic compounds and lignins was different. Peaks 5-11 decreased as the temperature rose. It is possible that overlapping signals were derived from amino acids, as the kernel is rich in amino acids and poor in lignin [20]. Thus, it is possible that the degradation of amino acids started at 250uC and continued at higher temperatures.
NMR analysis of HMWMs
The 1 H-13 C HSQC NMR spectra of the seed coats treated at 200uC (lighter) or 300uC (darker) were divided into four different regions as follows: lignin side-chain (red), polysaccharide (blue), polysaccharide anomeric (purple), lignin aromatic (brown), and other aliphatics (green) ( Figure 3A). By observing both spectra, it is possible to verify the difference in signal intensities and numbers of signals. The degradation of HMWMs was analyzed in detail using the area of assigned signals from 1 H-13 C HSQC NMR to generate a heat map ( Figure 3B, S3A).
We performed correlation analysis of the signals to determine the origin of certain polysaccharide residues. There was a high correlation among the signals assigned as (1-4) Figure S5). Other residues may have been derived from different polysaccharides, but there was not a high level of correlation.
Properties of cellulose. Figure 3B shows a heat map generated using the value of the area under the peak of each of the assigned polysaccharide residues and is scaled from 0 to 1. The heat map reveals an increase in the area values from the nontreated to 300uC-treated samples, but the signals were either undetectable or very low for all the samples heated to 350uC. The increase in the peak area may be explained by the cleavage of the glycoside linkages in the cellulose polymer that produced oligomers, which are more soluble in DMSO-pyridine and consequently increase the value. In all the samples except leaves, the highest intensity was generated by heating to 300uC, indicating that cellulose decomposition possibly started at low temperatures, increased significantly at 300uC, and was largely completed at 350uC. The values for leaves were the highest when heated to 250uC, decreased at 300uC, and were undetectable at 350uC ( Figure S3B).
Pyrolysis of cellulose present in different types of biomass causes a primary fragmentation reaction associated with the cleavage of glycosidic bonds, which reduces the degree of polymerization and yields a tarry pyrolyzate containing levoglucosan, other anhydrosugars, oligosaccharides, and glucose decomposition products. It is followed by a secondary cracking reaction that produces volatiles. Fragmentation occurs at temperatures ranging from 200uC to approximately 300uC, and degradation occurs at higher temperatures [43,44]. Our present FTIR and NMR results show that in most samples, cellulose was mainly fragmented to oligomers at temperatures up to 300uC, and degraded at higher temperatures ( Figure 3B).
Properties of hemicelluloses. The products of thermal degradation of glucuronoxylan residues in the xylem, seed coat, bark, and stem showed a very similar pattern. Both end groups (a-D-Xylp-R and b-D-Xylp-R) were no longer detected in (1-4)-b-D-Xylp and acetylated xylopyranoside residues after treatment at lower temperatures ( Figure 3B, S3B). The signal intensity of acetylated xylopyranoside decreased with increasing temperatures, and low levels were detected at 300uC. In contrast, the signal intensity of (1-4)-b-D-Xylp residues increased at 250uC, followed by an abrupt decrease at 300uC. At 350uC the signals were no longer detectable. Thus, it is possible that glucuronoxylan degradation started at its end groups.
In leaves, a-D-Xylp-R was not detected in the samples that were not heated. For the seed coats and xylem, the intensities of the peaks of acetylated xylopyranoside residues were low at 300uC and 250uC, and the intensities of peaks of b-D-Xylp-R were undetectable at 350uC and 300uC ( Figure S3B). Signals representing 2-O-Ac-Xylp, 3-O-Ac-Xylp, and a-D-Xylp were not detected in the kernel samples; therefore, it is possible that glucuronoxylan is not present or was present at low levels, and that the (1-4)-b-D-Xylp residues may have been derived from a different polysaccharide ( Figure S3B).
The degradation patterns of the other residues heated to 200uC or 300uC were not well defined compared with cellulose (Figure 3, S3). This difference can be explained by the heterogeneous composition of hemicellulose, in contrast to cellulose, that can vary greatly within a given biomass species [20]. In general, hemicellulose degrades between 150uC-350uC [18,45], in agreement with our results showing that different residues of hemicellulose decomposed mainly between 200uC-300uC.
Properties A heat map of the lignin units and substructure signals is shown in Figure 3C. Amino acids signals are known to overlap the signals from lignin, and thus, it was not possible to verify the decomposition pattern in the kernel, leaf, and bark, all of which contain a relatively high content of amino acids. The analysis of the heat map revealed that different lignin units degraded at discrete temperatures, depending on the sample. In the seed coats, H was no longer detected at 200uC; cinnamyl alcohol XI at 250uC; S and S9 at 300uC; and G and G9 at 350uC. In stems, G9 was no longer detected at 250uC; H, S, and S9 at 300uC; and G at 350. In xylem, H was no longer detected at 250uC; G9 and S at 300uC; and G and S9 at 350uC ( Figure 3C).
Lignin decomposes over a broad temperature range, because its various oxygen functional groups have different thermal stabilities, and therefore their scission occurs at different temperatures. Further, the composition and structure of the lignin complex varies according to biomass type, reaction temperature, heating rate, and the degradation pattern [46,47]. These properties can explain the difference in the decomposition pattern observed here.
Compared with the units, the substructures degraded at lower temperatures. In the seed coat samples, the substructures A-H/G (threo) and A-H/G (erythro) degraded at 300uC, G at 350uC, A-S at 200uC, and S at 250uC ( Figure 3C). Similar results were observed for the other samples. Therefore, the thermal decomposition of lignin started with cleavage of the linkage followed by the degradation of the units. The degradation of G and S, S and A-S occurred at lower temperatures compared with G and A-G/H ( Figure 3C). The ''A'' between syringyl units is easier to cleave than that between guaiacyl units; therefore, S degradation occurred at an earlier stage of heating than G degradation [47]. Melkior et al. also demonstrated that during thermal decomposition, S moieties are decomposed to G by demethoxylation [48].
NMR analysis of LMWMs
Water soluble (polar) LMWMs. From the spectra of products generated by heated stems, it was possible to determine the predominant presence of maltodextrin signals at 200uC and particularly at 250uC. Cellobiose and succinate were present at relatively low levels. The intensity of maltodextrin signals increased at 250uC compared with 200uC, and signals were no longer detected at 300uC and 350uC ( Figure S4A). The signals corresponding to maltodextrin in seeds were higher at 200uC, lower at 250uC, and undetectable at 300uC and 350uC ( Figure S4B). From the score and loading plots of principal component analysis (PCA), all the samples treated at 200uC and stems treated at 250uC showed high intensity maltodextrin signals, because maltodextrin contributes to PC1 in the negative direction, and these plots are grouped at the negative side of PC1 in the score plot ( Figure 4A, B). The plots for 300uC and 350uC are grouped along the positive side of PC1, indicating the absence of maltodextrin signals Thus, signals from maltodextrin were detected at 200uC and 250uC and no longer detected at 300uC and 350uC.
Maltodextrin (C 6 H 10 O 5 )nH 2 O n is a polymer of saccharides that consists of glucose units primarily linked by a-1,4 glucosidic bonds and is derived from the hydrolysis of a-1,4 glucosidic bonds in starch. Thus, the presence of maltodextrin at 200uC and 250uC may indicate the decomposition of starch to low molecular weight molecules by the cleavage of the a-1,4 glucosidic bond. Previous high-resolution magic angle spinning NMR studies of intact Jatropha tissues indicated detection of sucrose signals [49]. However, this technique is not so adequate for the detection of immobile macromolecules, such as starch. Because the Jatropha stem possesses photosynthetic tissues on its green bark, the origin of starch may be attributed to these photosynthetic products. The starch in stems mainly decomposed to maltodextrin at 250uC, and maltodextrin and starch degraded at 300uC and 350uC, respectively. In the other samples, decomposition of starch mainly occured at 200uC, and starch and maltodextrin degraded at 300uC and 350uC. This result is in agreement with that of Liu et al. (2008), showing that starch is mostly degraded at 300uC when heated at 5uC/min, which is the same rate used in our study [50].
Methanol soluble (semipolar) LMWMs. We assigned the signals from metabolites generated by the products of fatty acid metabolism as heptadecane, tetracosanoic acid, hexadecanoic acid, and linoleic acid; sugars as b-D-fructose 6-phosphate; amino acids and compounds derived from amino acid metabolism as Lvaline, N-acetyl-L-aspartate, and 5-oxoproline ( Figure 4C). From PCA we identified three groups with different degradation patterns as follows: 1) stem, bark, and xylem; 2) seed coat, and 3) kernel and leaf. The 1 H-NMR spectra and PCA for the bark, xylem, and stem were similar even at different temperatures. In these cases, the signals detected corresponded predominantly to fatty acids, which are common pyrolysis products that are generated by different biomass pyrolysis processes [51] (Figure 4C, D, S4C, D). The signal intensities of seed coats were compared with the other samples and therefore clustered separately (data not shown).
The high level of amino acids present in the kernels and leaves may have contributed to the different pattern of degradation, because L-valine, N-acetyl-L-aspartate, and 5-oxoproline contribute to the negative direction of PC1 and PC2 ( Figure 4A). The spectra of the kernels ( Figure S4D) and leaves (data not shown) show higher signal intensities for amino acids that were derived from the kernels at 250uC and from the leaves at 200uC. Therefore, peptides and amino acids may be degraded to LMWMs at 200uC and 250uC; with a higher decomposition rate at 250uC for the kernels and 200uC for leaves. LMWMs degraded at 300uC and 350uC. These results are in agreement with those of other studies that analyzed the thermal decomposition of amino acid using TG methods. In these studies, different types of amino acids were responsible for the highest weight-loss rate at Figure 5. Analysis of the thermolysis process of the phorbol ester. Thermogravimetric-Differential Thermal Analysis of phorbol 12-myristate 13-acetate (A) and phorbol 12,13-dibutyrate (B). Stacked plots of 1 H-NMR spectra heated phorbol 12-myristate 13-acetate at 200uC, 250uC, 300uC, 350uC, and 500uC (C), and changes in corresponding signal intensities against torrefication temperature (D). The coloring highlighted arrows on each signals in (C) are corresponded to same color in (D). Colors were classified as follows; aromatic (magenta), methyl (yellow), acetyl (red), lipid chain (cyan) and methyl derived from lipid chain (green). doi:10.1371/journal.pone.0106893.g005 Chemical Profiling of Jatropha Torrefied Biomass PLOS ONE | www.plosone.org approximately 300uC, which is the same temperature reported to decrease their signal intensities [52,53,54].
N-acetyl-L-aspartate is not present in plant tissue and is abundantly present in animal brain tissue. Therefore, it has been the focus of numerous studies on its roles in the nervous system. We identified this molecule using SpinAssign, and the four signals that were detected had the following chemical shifts: dC 39 It is therefore possible that this compound was generated by pyrolysis, but this must be verified using specific analyses.
Decomposition of phorbol ester
The optimum temperature for torrefaction of phorbol 12myristate 13-acetate and its di-butyrate form was first determined using thermogravimetric analysis at a heating rate of 5uC/min from 24uC to 500uC. Differential TG peaks were first detected in the samples heated at 250uC (5% weight decrease), peaked at 296uC (57% weight decrease), and decomposed at 350uC (94% weight decrease) ( Figure 5A). We noted that a similar toxic compound, phorbol 12-myristate di-butyrate, exhibited a very similar thermal degradation property ( Figure 5B). These torrefied samples (heated to 200uC, 250uC, 300uC, 350uC, and 500uC) were analyzed using 1 H-NMR ( Figure 5C), and the changes in relative signal intensities are shown in Figure 5D. Some aromatics and ester signals decreased from 250uC, suggesting that major moieties decomposed at lower temperature than side-chain aliphatics. Thus, this phorbol ester degraded at temperatures just below 300uC ( Figure S6), indicating the suitability of these temperatures to detoxify biorefinary-stock and fertilizers.
FTIR and NMR results. The NMR and FTIR results suggested that the decrease in the intensity of peaks 12 to 14 between 300uC and 350uC was caused by the degradation of cellulose. The decrease between 250uC and 300uC was caused by the degradation of glucuronoxylan and between 200uC and 300uC by starch degradation. Because starch is composed of glucose residues as is cellulose, its degradation may have contributed to peak 12.
The decrease in peak 13 between 250uC and 300uC was more accentuated in the bark and kernel samples, which contain relatively lower levels of hemicellulose and cellulose [15]. Although the contribution of starch to peak 13 may be higher in these samples, cellulose residues degraded at 350uC, and xylopyranosebased residues (2-O-Ac-Xylp, 3-O-Ac-Xylp, a-D-Xylp-R, b-D-Xylp-R, (1-4)-b-D-Xylp) degraded at temperatures higher than 250uC, which may characterize the degradation of starch.
Conclusions
Torrefaction of Jatropha tissues decomposed cellulose to oligosaccharides at increasing rates from 200uC to 300uC, and at 350uC cellulose and oligosaccharides derived from cellulose degraded. Each tissue yielded similar results. However, the decomposition of hemicellulose differed among the samples and was likely caused by the differences in the structures and compositions of hemicellulose samples. The analysis of LMWMs showed that starch decomposed to maltodextrin at 200uC and 250uC and degraded at 300uC and 350uC. Fatty acids that are commonly found in tar generated by torrefied biomass were present in all the samples heated to each temperature. Lignin decomposes over a wide temperature range. Lignin degrades in the order H, S, G. The substructures A-S, AH/G largely degraded at 250uC and 300uC, respectively, and their subunits degraded at 350uC. Amino acids and peptides (LMWMs) decomposed at 200uC and 250uC and degraded at higher temperatures. Phorbol ester degraded at temperatures just below 300uC. In most cases, heating biomass at 200uC had little effect, heating at 250uC produced smaller molecules, and complete degradation occurred at 300uC and 350uC. Figure S1 Thermogravimetric-Differential Thermal Analysis of Jatropha tissues. The temperature was risen from 45uC to 500uC with a heating rate of 5uC/min. (TIF) Figure S2 The FTIR spectra of Jatropha tissues. Assignment of peaks in the FTIR spectrum of non-treated stem (A). The | v3-fos-license |
2016-06-02T01:01:25.250Z | 2013-03-01T00:00:00.000 | 2176576 | {
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} | pes2o/s2orc | Ultrasound Exposure Improves the Targeted Therapy Effects of Galactosylated Docetaxel Nanoparticles on Hepatocellular Carcinoma Xenografts
Purpose The distribution of targeted nanoparticles in tumor tissue is affected by a combination of various factors such as the physicochemical properties of the nanoparticles, tumor hemoperfusion and tumor vascular permeability. In this study, the impact of the biological effects of ultrasound on nanoparticle targeting to liver carcinoma was explored. Methods The copolymer MePEG-PLGA was used to prepare the galactosylated docetaxel nanoparticles (GDN), and the physical and chemical properties as well as the acute toxicity were then assayed. The impact of ultrasound exposure (UE) on tumor hemoperfusion was observed by contrast-enhanced ultrasonography (CEUS), and the distribution of docetaxel in tumors and liver were detected by high performance liquid chromatography (HPLC). In the GDN combined with UE treatment group, the mice were injected intravenously with GDN, followed by ultrasound exposure on the human hepatocellular carcinoma xenografts. Twenty-eight days post-administration, the tumor growth inhibition rate was calculated, and the expression of Survivin and Ki67 in tumor tissues were determined by immunohistochemistry assay and quantitative real-time PCR. Results The mean size of prepared liver-targeting nanoparticles GDN was 209.3 nm, and the encapsulation efficiency was 72.28%. The median lethal dose of GDN was detected as 219.5 mg/kg which was about four times higher than that of docetaxel. After ultrasound exposure, the tumor peak - base intensity difference value, examined by CEUS, increased significantly. The drug content in the tumor was 1.96 times higher than in the GDN treated control. In vivo, GDN intravenous injection combined with ultrasound exposure therapy achieved the best anti-tumor effect with a tumor growth inhibition rate of 74.2%, and the expression of Survivin and Ki67 were significantly decreased as well. Conclusion Ultrasound exposure can improve targeting nanoparticles accumulation in the tumor, and achieve a synergism antitumor effect on the hepatocellular carcinoma xenografts.
Introduction
Hepatocellular carcinoma is one of the most common malignant tumors in the world, with a global incidence rate exceeds 626,000 per year, ranking it third among the most lethal malignant tumors [1]. At present, the standard treatment for hepatocellular carcinoma includes surgical resection and minimally invasive treatment. However, due to multiple nidi and rapid deterioration from the disease, only about 30% of patients can take advantage of surgical resection or liver transplantation [2]. For most inoperable patients, transcatheter arterial chemoembolization is the preferred treatment, yet the 5-year survival rate is merely 26% [3]. In recent years, tumor targeting therapy has advanced due to its high efficacy and fewer side effects [4][5][6]. In theory, conjugating ligands or antibodies to nanoparticles can selectively transport the chemotherapeutics to the targeted tumor. Yet, the biodistribution of targeted nanoparticles after intravenous injection is influenced by various factors, such as reticuloendothelial system elimination, the physicochemical properties of the nanoparticles, the size of vascular endothelial pores, and the tumor hemoperfusion. Xu prepared galactosylated docetaxel nanoparticles and studied their distribution, finding that the amount of drug accumulating in hepatocellular carcinoma xenografts was much lower than in the liver, or even in the kidney [7]. Hence, improving the accumulation of targeted drug in the tumor is the goal of hepatocellular carcinoma targeted therapy.
The transmission of ultrasonic waves in biological tissues produces thermal effects, acoustic cavitation, mechanical effects, and other modifications, which were found to help improve the anticancer effects [8][9][10]. With acoustic energy increases, the biological effects including the raised tissue temperature and improved vascular and cell permeability help the conventional drugs transport through the vascular and cell membrane. However, the drug transportation has bidirectional property; that is the drugs can either transport to the tumor cells or return to the blood circulation. In this study liver targeted nanoparticles were prepared and ultrasound exposure was used to help increase the vascular permeability and improve targeted nanoparticles transport to the liver cancer cells. Nanoparticles can then bind to and enter the cells by endocytosis, further improving the tumor targeting therapy effects.
Preparation and Characterization of GDN
The galactosylated cholesterol, cholesten-5-yloxy-N-(4-((1-imino-c-b-D-thiogalactosylethyl) amino) butyl) formamide (Gal-C4-Chol) was synthesized by the method described previously [11,12], which targets to the asialoglysoprotein receptor on the hepatoma cell membrane. GDN were prepared using the method of modified emulsification-solvent evaporation. MePEG-PLGA 100 mg, docetaxel 5 mg and Gal-C4-Chol 15 mg were dissolved in 1 ml dichloromethane, then injected into 5 ml of 3% polyvinyl alcohol aqueous solution, and sonicated at 50W for 30sec, repeating five times. Then 10 ml of distilled water was added and the mixture magnetically stirred to remove the dichloromethane. The mixture was centrifugated at 15 0006g for 10 min, and then we collected the precipitation as GDN, that was vacuum freeze-dried and sterilized by Co 60 irradiation. Docetaxel nanoparticles (DN) were prepared without Gal-C4-Chol.
The particle size of GDN was measured by a ZETASI-ZER3000HS laser particle size analyzer (Malvem Instruments Ltd, UK). The superficial morphology was observed using scanning electron microscopy. The encapsulation efficiency was determined by HPLC at 30uC with a mobile phase of methanol/ H 2 O (75/25, v/v) and a flow rate of 1.0 ml/min.
In vitro Release of GDN
Five mg of GDN in 50 ml phosphate buffer solution (pH 7.2, including 0.02% sodium azide and 0.1% Tween 80) was placed in an orbital shaker and maintained at 37uC and 80 rpm for measurement of in vitro release. At 3, 6, 12, 24, 48 and 72 hrs, samples were centrifuged using a centrifugal filter device at 50006g for 10 min, then the filtrates were collected to detect the docetaxel content by HPLC.
Ethics Statement
This study was carried out in strict accordance with the Guidelines of the Animal Care and Use of Fujian Medical University. The protocol was approved by the Committee on the Ethics of Fujian Medical University. All efforts were made to minimize suffering.
Acute Toxicity Test
Fifty male and 50 female Kunming mice weighing 18-22 g were randomly divided into two groups, the docetaxel and GDN groups. Each group was then divided into five dosage treatment groups of 10 mice. The drugs were injected via the tail vein followed by observations for eight weeks. Toxic reactions and the number of deaths were recorded, and the median lethal dose (LD 50 ) was calculated according to the modified Karber's method [13].
Influence of Ultrasound Exposure on Tumor Temperature
BALB/c nu-nu male nude mice, weighing 16-18 g, were provided by Shanghai SLAC Laboratory Animal Center. The mice were kept in a pathogen free environment, and allowed free access to chow and water.
The human hepatoma cell line HepG2 (Shanghai Cell Bank, Chinese Academy of Sciences) was grown in RPMI-1640 medium containing 10% fetal bovine serum at 37uC under 5% CO 2 and saturated humidity. Cells were collected and adjusted to a density of 1610 7/ ml, and 0.2 ml injected into the right armpit of nude mice to create the model of hepatocellular carcinoma.
Twelve hepatoma-bearing nude mice with a tumor diameter of 1.7-2.0 cm were used to detect the influence of ultrasound exposure (frequency 840 kHz, acoustic intensity 0.75w/cm 2 ) on tumor temperature. The ultrasonic probe covered the whole tumor, the exposure time was set at 1, 2, 3 and 4 min and the tumor temperature was detected using an electronic thermometer, and used to create the time-temperature curve. Tumor Contrast-enhanced Ultrasonography Twelve hepatoma-bearing mice with a tumor diameter of 1.7-2.0 cm were divided into two groups, control group and UE group (n = 6 each). In the control group, acoustic contrast agent SonoVue (5 ml/kg), a sulfur hexafluoride-filled microbubble, was injected by bolus via the tail vein, and then the tumor was treated by CEUS (SEQUOIA 512, Siemens, center probe frequency 10 MHz, mechanical index 0.25). In the UE group, the tumors were subjected to ultrasound exposure for 2 min, and followed by CEUS examination. The contract-enhanced index, arriving time (AT), time to peak (TTP), and peak-base intensity difference (PBD) were determined by the quantitative analysis software, Syngo US Workplace.
Biodistribution
Twenty hepatoma-bearing nude mice with a tumor diameter of 1.2-1.5 cm were randomly assigned to four groups (n = 5), DN, GDN, DN combined with UE and GDN combined with UE. In all groups, the mice were injected intravenously with a docetaxel dose of 10 mg/kg. The mice in groups combined with UE achieved additional ultrasound exposure for 2 min every two hours, repeating three times, right after DN or GDN injection. Two hours after the last treatment, the mice were sacrificed by exsanguination, and the tumor and liver were collected. The tissues were homogenized with 50% methyl cyanides, centrifuged at 12, 0006g for 10 minutes, and the supernatant sampled to determine the docetaxel content using an HPLC assay.
In vivo Antitumor Effects
Twenty-five mice with a tumor diameter of 1.2-1.5 cm were randomly divided into five groups (n = 5). The mice were anaesthetized using ether and received treatment every week, the doses of DN and GDN were calculated according to the encapsulated docetaxel dose of 10 mg/kg.
Immunohistochemistry Examination
The tumors were dissected to perform immunohistochemistry. The expression of Survivin and Ki67 in tumor cells were detected by the SP method. The primary antibodies, mouse anti-human polyclonal antibodies were used to detect the expression of Survivin and Ki67. Negative-control sections were incubated in blocking buffer alone without primary antibody. Each section underwent 3, 39-diaminobenzidene staining, and haematoxylin restaining. With a 200 times magnification, in 10 microscopic fields, the ratio of positive stained tumor cells to the total tumor cells was calculated for analyses.
Expression of Survivin and Ki67 by Quantitative Real-time PCR
According to the manufacturer's instructions, tumor total RNA was extracted and reverse transcribed to cDNA. The SmRNA expression of Survivin and Ki67 were determined by the real-time fluorescence quantitative PCR method on an ABI 7300 PCR instrument. Each reaction was run in triplicate and contained 2 ml of template, along with 1.6 ml primers in a final reaction volume of 25 ml. The sequences of the primer pair for Survivin were 59-CAG ATT TGA ATC GCG GGA CCC -39 (sense) and 59-CCA AGT CTG GCT CGT TCT CAG -39 (antisense) (expected size: 208 bp). The sequence of the primer pair for Ki67 were 59-AAC ACC TAC AAA ATG ACT TCT-39 (sense) and 59-CTT CAC TCT TAC TTT CCA CAG-39 (antisense) (expected size: 146 bp). The sequence of the primer pair for GAPDH were 59-GCA CCG TCA AGG CTG AGA AC -39 (sense) and 59-ATG GTG GTG AAG ACG CCA GT-39 (antisense) (expected size: 143 bp). Cycling parameters were 95uC for 2 min, following by 40 cycles of 95uC for 30s, 60uC for 20s, and 72uC for 20s. Data were obtained as average CT values and normalized against control as gCT. Expression changes in genes transcript between model and treatment group are shown as 2 gg CT.
Statistical Analysis
All data are presented as mean 6 standard deviation using SPSS 16.0 statistical software package. A Mann-Whitney U test and single-factor analysis of variance were carried out, and P,0.05 is used to indicate a significant difference and P,0.01 a very significant difference.
Characteristics of GDN
The prepared GDN were spheroid shaped (Figure1), with a mean size of 209.3 nm and polydispersivity index of 0.40. The drug encapsulation efficiency was detected as 72.28%.
In vitro Release of GDN
The in vitro release of GDN was steady and gradual with an accumulated release of 78.5% in 72 hours. There was no initial burst of drug release (Figure 2).
Acute Toxicity Test
The LD 50 of docetaxel was determined to be 47.3 mg/kg. The LD 50 of the GDN was significantly higher, 219.5 mg/kg. Table 3
Influence of Ultrasound Exposure on Tumor Temperature
As showed in Figure 3, as the time of ultrasound exposure was extended, the tumor temperature increased steadily. After exposure for 4 min, the tumor temperature reached 39.3uC, which was 5.1uC higher than the pretreatment temperature.
Biodistribution
The content of docetaxel accumulating in the hepatoma xenograft in the GDN group was detected as 1.4660.33 mg/g protein, which was higher than in the DN group 0.8960.12 mg/g protein (P,0.05). After ultrasound exposure, the drug content in DN combined with UE increased to 1.3560.29 mg/g protein, which was about 1.52 times higher than that in DN group (P,0.05). In GDN combined with UE group, the drug content increased significantly, reaching 2.8660.52 mg/g protein, which was 1.96 times higher than that in GDN group (P,0.05) (Figure 4).
Contrast-enhanced Ultrasonography
After intravenous injection of SonoVue, the tumors in both control and UE groups showed rapid and homogeneous enhancement, lasting about 90 sec. Compared to the control group, the values of AT and TTP in the UE group were slightly lower, while the PBD was much higher, and the difference was significant (P,0.05) ( Table 1).
In vivo Antitumor Effects
Hepatoma cell tumors grew continually following subcutaneous transplantation in nude mice. Compared to the model group, the Table 3 tumor volumes were smaller in all the docetaxel treated groups, especially in the GDN+UE group ( Figure 5). Table 2 shows that all treatments inhibited tumor growth, in particular, the GDN+UE group displayed the most powerful antitumor effect, with a significant difference compared with all other groups (P,0.05 or P,0.01).
Immunohistochemistry
In the GDN injection combined with ultrasound exposure treatment group, the expression of Survivin, an apoptosis inhibitor marker, and Ki67, a proliferation marker, in the tumor decreased. The percentage of positively stained tumor cells was lower than in the other groups, and the difference was significant compared with all other groups (P,0.05 or P,0.01) (Table 3, Figure 6 and 7).
Determination of Survivin and Ki67 SmRNA Expression
The SmRNA expression of Survivin and Ki67 were significantly down-regulated in all docetaxel treated groups compared with control group, especially the GDN+UE treated group (P,0.01). Compared with the GDN+UE group, the difference was highly statistically significant for the DN group (P,0.01), and there was also a significant difference for the GDN and DN+UE groups (P,0.05) ( Table 4).
Discussion
As an efficient chemotherapeutics with a wide antitumor spectrum, docetaxel can inhibit cell microtubule depolymerization, the synthesis of DNA, RNA or protein, leading to necrocytosis, and has been widely applied in treatment of breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer and others tumors [14][15][16][17]. However, docetaxel has a very poor water-soluble performance. Currently, Tween 80 is often used in clinical preparation to assist with water solubility, which tends to induce allergic reactions [18]. In this study, the preparation of targeted nanoparticles with the carrier of MePEG-PLGA resolved the water solubility problem and thus avoided allergic reactions induced by the solvent. The prepared targeting nanoparticles released the drug with the biodegradation of the MePEG-PLGA, and showed certain sustained release function, which may be the main contributor of significant reduction of the lethal dose 50 in mice.
Generally, nanoparticle with small size, good hydrophilicity and low zeta potential can effectively avoid identification and elimination by the reticuloendothelial system, and extend their plasma half-life [19]. In this study, the amphiphilic MePEG-PLGA was used to prepare the targeted nanoparticles. PEG is located in the outer shell and improves its hydrophilic performance; thereby effectively avoiding clearance by the reticuloendothelial system. The vascular pore in normal tissue is less than 100 nm, yet due to the incomplete vascular structure, the pore is increased to about 700 nm in malignant tissue [20]. The mean size of the galactosylated docetaxel nanoparticles was about 209.3 nm, which was the range between the normal tissue and the malignant tumor vascular pore. After intravenous injection, more nanoparticles can aggregate in malignant tissues and obtain a better passive drug targeting effect.
The asialoglysoprotein receptor (ASGPR) was initially discovered and characterized in mammalian liver by Ashwell [21]. The receptor specifically binds glycoproteins containing terminal galactosyl groups on their oligosaccharide chain [22][23][24]. Nanoparticles conjugated with a galactosyl group can positively target liver or hepatocellular carcinoma, and enter the cells by endocytosis mediated by ASGPR [25,26]. The prepared galactosylated docetaxel nanoparticles displayed a good liver targeting effect, after intravenous injection, the content of GDN accumulating in hepatocellular carcinoma was 1.64 times higher than that of docetaxel nanoparticles without galactosylation.
In vivo, targeted nanoparticles should first permeate the vascular wall and aggregate in the tumor tissue, then bind to the receptors and enter the cells. The result of biodistribution of galactosylated docetaxel nanoparticles showed that the amount of drug accumulating in the liver was much higher than that in the hepatoma xenograft; the tissue hemoperfusion and vascular permeability may be the main reason.
A large number of studies have indicated that ultrasound exposure can increase tissue temperature in an acoustic intensity and time dependent manner. In this study, the acoustic intensity was set as 0.75W/cm 2 . After ultrasound exposure for 2 minutes, the tumor temperature was increased by 2.9uC. When the exposure time rose to 4 minutes, the temperature increase reached 5.1uC, which would injure the skin of the mouse. So an ideal ultrasound parameter combination to improve tumor targeting therapy effects would be an acoustic intensity of 0.75W/cm 2 with an exposure time of 2 minutes.
After ultrasound exposure, the peak-base intensity difference value, examined by CEUS, increased significantly, indicating that ultrasound exposure can improve the tumor blood supply. And the drug content in tumor tissue increased remarkably, further demonstrating that ultrasound exposure can enhance the tumor hemoperfusion, increase the vascular permeability, and thus improve the targeted nanoparticles accumulation in the tumor.
Ultrasound exposure significantly improved the antitumor effect of galactosylated docetaxel nanoparticles on the hepatoma xenograft, which showed a tumor growth inhibition rate of 74.2%. It also inhibited the expressions of Survivin and Ki67 in hepatoma cells. Survivin is the strongest apoptosis inhibitor in the currently known family of apoptosis inhibitor protein, and is highly expressed in liver cancer tissues [27][28][29]. Meanwhile, Ki67 is a marker with the highest sensitivity in the appraisal of cell proliferation and can satisfactorily reflect the differentiation of liver cancer cells [30][31][32]. The over-expressed Survivin and Ki67 is closely related with the biological characteristics of hepatocellular carcinoma; such as peplos invasion, tumor metastasis, and patient prognosis.
In conclusion, ultrasound exposure can produce bioeffects, and raise the tumor hemoperfusion, increase vascular permeability and the drug content in tumor tissues, hence improve the antitumor effects of galactosylated docetaxel nanoparticles on hepatoma xenografts. | v3-fos-license |
2020-08-06T09:03:59.210Z | 2020-08-04T00:00:00.000 | 222245486 | {
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} | pes2o/s2orc | White Bread Fortified with Calcium from Eggshell Powder
This paper presents the preparation of white bread with different hen eggshell powder additions. The aim of this paper is to resolve at least two important aspects: the waste recovery from food industry and fortification of white bread with minerals (calcium). The addition of eggshell powder to the white bread preparation was up to 2% and an increase in bread quality was on elasticity and humidity starting with 0.5% eggshell powder addition. Bread aging occurred 10 to 12 h after its baking. After 24 h, all physico-chemical properties of bread crust and crumb in the case of fortified bread with calcium had a positive effect.
Due to a high consumption of hen eggs, a large amount of egg waste is discarded, some studies being focused on eggshell-derived components as a renewable resource. This massive amount of waste could potentially be used in different applications by offering great material for industrial and structural applications. Eggshell is a natural bio-ceramic composite, with a combination of both inorganic and organic components that have exceptional characteristics. The unique chemical composition and substantial availability make eggshells a potential source for bio-based calcium carbonate [8].
The egg (Figure 1) consists of a protective layer, i.e., eggshell (ES), two membranes associated with ES (ESM), an egg white and yolk. Egg is very important in human nutrition due to protein and nutrients present in liquid state as egg white and yolk. The solid protective layer (ES) and its associated membranes (ESM) are considered waste [9]. The protective layer consists of the outer layer (cuticle), below the cuticle is the layer of calcium carbonate (testa) and finally the inner layer (mammillary layer) [9]. Main components of the protective layer are carbonates, phosphates and sulphates of calcium and magnesium and organic matter, the percentage of calcium carbonate being of about 90% [9]. In Traces of Na, K, Mn, Fe, Cu, and Sr metals are also present in the protective layer. ESM have cca. 60% proteins, i.e., collagen (35%), glucosamine (10%), chondroitin (9%) and hyaluronic acid (5%), and small amounts of inorganic components e.g. Ca, Mg, Si, Zn [9]. Study of ES structure has been a fascinating area of research in the recent years [10].
The nutritional value of wheat flour, excepting the initial quality of wheat, is determined by the degree of extraction that substantially alters the content of vitamins and mineral salts. Nowadays, due to the grinding process and the environmental conditions, at industrial level, flour or white bread enrichment has been made with various minerals, vitamins, enzymes, etc. The amounts of vitamins and mineral salts added to the flour for enrichment vary from country to country, starting from 0-1110 mg Ca/453 g flour (Ca as Ca3(PO4)2, CaCO3, CaHPO4 × 2H2O, CaSO4) [11]. From the nutritional point of view, 1000 mg of Ca for adults and 1200 mg for pregnant and young people are daily recommended, while for women aged over 45 years, a daily intake of 1500 mg of Ca is recommended. The amount of Ca can be supplemented in the case of people suffering from hypertension, cardiovascular disease, osteoporosis and dyslipidaemia [12].
According to the data reported in the literature, the content of Ca in ES is 70-95%. Higher values are in the crust of the raw egg and low values in the shell subjected to heat treatment. In a mass of 0.457 g of ES is found about 0.320 g of Ca [11]. ES is probably the best natural source of Ca and it is easily digested and absorbed.
Growing interest is now focused to ES powder because it can be used as a mineral source for fortification of different food products, e.g., yoghurt [13], biscuits [14], chocolate cakes [15], bread [16][17][18]. Based on this information, this study contributes to the valorisation of food waste (ES) to obtain a new product: white bread enriched in Ca as a natural source. The white bread was chosen as application from two points of view: it is a basic food of population all over the world and its minerals content is lower than that of bread obtained from integral wheat flour.
By using the natural materials, is wanted to achieve the following aspects: the shelf life of a product to be as long as possible and also the negative effects of additives to be eliminated in the same time. Using ES as additive, the foods can be fortified with mineral substances. Also is known that soils are poor in minerals and grains default. Through the large amounts of Ca and Mg in ES and then in bread, a fortification with minerals of white bread is aimed.
The novelty and the originality of this paper is the using of hen ES powder at bread fabrication in order to obtain bread with higher nutritive properties than those of bread currently available on the market.
Raw materials
The raw materials used for fortification of white bread with Ca from ES powder were: white wheat flour 650 type, yeast, water, salt and ES from the waste of our household.
To obtain ES powder, it was necessary to wash the ESs for many times. The membranes were separated and finally the ESs were dried into an oven at 50°C for 10 min to avoid any further contamination. The dried ESs were grounded, followed by pulverization process to obtain particles size of about 125 µm (similar with flour particles size). Drying temperature may lead to a decreasing in the amount of Ca from ES powder according to the experiments published in the literature [18,19].
Bread obtaining
The bread was prepared by using a bread machine having the first kneading time of 10 min (first fermentation 10 min), the second kneading time of 10 min (second fermentation 35 min) and baking https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (7) process of 65 minutes. The recipe is presented in Table 1. Table 2.
Results and discussions
The percentage of ES powder added to the bread preparation was up to 2% and an increase in bread quality was observed especially on elasticity and humidity starting with 0.5% ES powder addition. Bread aging, which involved essential changes in bread quality, occurred after 10 to 12 h and increased with the duration of storage. After 24 h, all physico-chemical properties of bread crust and crumb in the case of fortified bread with Ca had a positive effect (Table 3, Figures 2-4). This shows that the ES powder plays a preservative role through the freshness of the bread obtained. These ESs can therefore be called natural additives used to increase the preserving and Ca supplements for bread fortification as a nutritional supplement. The obtained bread was also sensory analyzed (Table 4) with a scoring scale of maximum 20 points for the evaluation of organoleptic characteristics [19]. The maximum total score was 19/20. The addition of ES powder improves the aging and flavor qualities, but the bread has a chew inconvenience, a screech.
The differences in volume and crumb structure for various percentages of ES powder are shown in Figure 5.
The bread has toasted brown crust -golden, slightly crispy. Bread with added ES powder is well cooked, presents elastic crumb, has uniform colour and the knife blade stays clean. The crumb bread consists of pores, with walls formed during the baking process from compact coagulated gluten mass, partially swollen and gelatinized starch granules being found inside the pores' walls. In fresh bread, the gelatinized starch granules are in contact with the mass of coagulated proteins throughout their surface, with no visible delimitation between them. In aged bread, partially gelled starch granules are visibly separated from each other, due to the fact that a thin layer of air forms around their surfaces, as the volume of the starch granules is reduced and the proteins on the pore walls does not undergo any change [19]. ESs having adsorption properties absorb this air and thus can be explained the effect of increasing bread consistency in which ESs are added, respectively, the breaking of the bread is prevented.
Is is considered the process of demolding starch as a cause of bread aging [19]. During the baking process, the starch absorbs the water released by the coagulating, swelling, partially gelled proteins, passing from the initial crystalline state in an amorphous state [19]. In our study, it is possible to explain the increase in bread consistency, through the water released during the baking process, that will be absorbed by the ES powder too, and after the baking process, this amount of water can be gradually released.
During bread aging time, water diffuses from gluten to starch, contributing to the reorganization of macromolecules of amylose and amylopectin as well as their transition from amorphous state to crystalline state. In fresh crumb free water represents 75%, in which a number of substances are solubilized forming the aqueous phase between starch and gluten [19].
In our study, we could explain the aging process by the addition of ES powder. The ES powder is interposed by the water adsorbed between starch and gluten either in the baking process or after the bread baking. In the aged crumb of bread without ES, the water drops to half, causing the concentrating of aqueous phase and decreasing of elasticity. In our research an increasing in elasticity can be observed, so we could say that the aging process with the characteristic consequences is delayed.
Conclusions
In food industry from Romania, the bakery industry occupies an important place in the production of consumer goods, bread being a staple food in the regular daily diet because the body provides some important substances that are necessary for vital activity. The using of ES in bakery industry brings several advantages: waste recycling in food industry, nutritionally enriching with Ca, increasing of bread preserving. The nutritional value of bread is an important element to the daily ration of food and the subject of wide research in the field of nutrition. This value is conferred not only by energy intake (calories), based on their increased groove-sugars (carbohydrates), proteins and lipids (fats), but also by the contribution of all components in those products, that are easily assimilated by the human body.
The final product obtained has pleasant taste and smell, but also better developed and the interaction of Ca from ES powder determines the freshness of bread. | v3-fos-license |
2021-10-22T15:39:21.974Z | 2021-09-01T00:00:00.000 | 239462742 | {
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} | pes2o/s2orc | Amylase Production by Aspergillus niger and Penicillium Species by Solid-State and Submerged Cultivation Using Two Food Industrial Wastes
Amylase enzymes are starch degrading enzymes and have received a great deal of attention due to their perceived technology importance and economic benefit. Amylase enzymes are considered important enzymes used in starch processing industries for the hydrolysis of polysaccharides like starch into simple sugar constituents. This enzyme is also involved in the commercial production of glucose. Solid-state cultivation and submerged cultivation have tremendous potentials for enzyme amylase production by using different solid substrates like rice bran, wheat bran, coconut oil cake, and groundnut oil cake which are rich in starch. These agro-industrial wastes are considered cheap raw materials for the production of amylase. Wastewater from the industry like brewery can also be used as a liquid substrate for submerged cultivation. It may have the possibility of depurination of wastewater. In the present study, Aspergillus niger and Penicillium species were isolated and their amylase activity was determined by the starch hydrolysis method. Enzyme production was done by using coconut oil cake as a substrate for solid-state fermentation and brewery wastewater as a substrate for submerged fermentation. The enzyme produced by the organisms was extracted and enzyme assay was done by the Dinitrisalicilic method (DNS method). The protein estimation was done by Lowry Folin’s method. The qualitative assay was carried out by performing Gas Chromatography-Mass Spectroscopy (GC-MS).
INTRODUCTION
Enzymes are proteins and consist of long chains of amino acids that fold to produce a three-dimensional structure. Each amino acid sequence produces a specific structure, which has different properties. Enzymes are also responsible for many important biochemical reactions in microorganisms, plants, animals, and human beings. Enzymes differ in their function so that they have the unique ability to facilitate biochemical reactions without undergoing any change themselves. This catalytic ability makes enzymes unique.
Enzymes are biological catalysts with high selectivities. They have been used in the food industry for hundreds of years and play a vital role in many other industries (Detergents, textile manufacturing, pharmaceuticals, pulp, and paper). Recently, enzymes are becoming increasingly important in sustainable technology and green chemistry. They are also produced by various microorganisms including bacterial and fungal species (Esfahanibolandbalaie et al. 2008). Generally, enzymes are active at mild temperatures. Above specific temperature, the enzyme is denaturated. It has characteristic pH at which their activity is maximum. Extreme pH values result in electrostatic interactions within the enzyme, leading to the inactivation of enzymes (Prasanna 2005). Other important factors that affect the enzymatic effect are the enzyme concentration, treatment time, additives such as surfactants and chelators, and mechanical stress. The enzyme can break down a particular compound. The molecule that an enzyme acts upon is known as its substrate, which is then converted into a product or products. Some of the most common include amylase which converts starch into simple sugars, proteases that break down proteins, cellulases that break down cellulose, and lipases that split fats into glycerol and fatty acids (Alva et al. 2007). For each type of reaction in a cell, different enzymes are involved and they are broadly classified into six categories such as hydrolytic, oxidizing and reducing, synthesizing, lytic, transferring, and isomerizing. The important characteristic of enzymes is their catalytic function.
Microorganisms particularly have been considered as a treasure of useful enzymes. In recent years, using microorganisms as biotechnological sources of enzymes that are industrially important has stimulated great interest in the exploration of extracellular enzymatic activity in many microorganisms (Pandey et al. 2000). The first industrially produced enzyme was amylase from a fungal source in 1894, which was used for the digestive disorder treatment. Amylases are a class of enzymes that acts as a catalyst in the hydrolysis of starch into simple sugars such as glucose and maltose (Farzana et al. 2016). Amylase is commonly present in human saliva, Vol. 20, No. 3, 2021 • Nature Environment and Pollution Technology where it begins the chemical process of digestion. Starch degrading enzymes such as amylase have been receiving a great deal of attention and interest due to their perceived technological significance and economic benefits. This enzyme is also used for the industrial production of glucose (Saranraj & Stella 2013).
Historically, around 1857 the application of enzymes in textile industrial processes began when malt extract was used to remove size from fabrics before the printing process. Starch is widely used as a sizing agent, because it is readily available, relatively cheap, and based on natural, also sustainable raw materials (Adinarayana et al. 2005). About 75% of the sizing agents used worldwide are starch and its derivatives. In medicinal and clinical areas, there are several processes that involve the application of amylases (Kundu A.K et al. 1970). Because of the increasing demand for these enzymes in various industries, there is more interest in developing enzymes with better and desirable properties such as raw starch degrading amylases that is suitable for industrial applications and their cost-effective production methods (Rodriguez, S.C. and Sanroman, A.M. 2006). They can be obtained from several sources, such as plants, animals, and microorganisms (Saleem et al. 2014).
Although many microorganisms are able to produce this enzyme, some of the most widely used for their industrial applications are Bacillus licheniformis, Bacillus amyloliquefaciens, Aspergillus niger, Penicillium chrysogenum (Saranraj & Stella 2013). When compared to other microbial sources, the fungal amylases are preferred because of their more acceptable GRAS (Generally Recognized As Safe) status, the conditions such as hyphal mode of growth and good tolerance to low water activity (aw) and high osmotic pressure makes fungal species most efficient for bioconversion of solid substrates and thus attracting more interest as source of amylolytic enzymes suitable for industrial applications as given below (Singh et al. 2014 Solid-state cultivation is more simple, also requires lower capital, has superior productivity, reduce energy needs, requires simple fermentation media and absence of vigorous control of fermentation parameters, uses less water and produces lower wastewater, has easier control of bacterial contamination, and require a lower cost of downstream processing (Sivaramakrishnan et al. 2007).
Submerged fermentation is advanced and industrially important enzymes are commonly produced by using this method. Brewery industries produce large quantities of wastewater. The utilization of this wastewater as a substrate for submerged fermentation may reduce environmental pollution. Usage of solid substrates like coconut oil cake acts as a low-cost substrate for solid-state fermentation (Mabel et al. 2006).
Isolation of Aspergillus niger and Penicillium Species
Sabouraud Dextrose Agar (SDA) plates were prepared and a settle plate technique was performed. The SDA plates were kept open for 10 mins and incubated at room temperature for 4 days. After incubation, fungal species were observed on SDA medium.
Two different fungal cultures were selected based on their colony morphology and subcultured on SDA slants. These two fungal cultures were subjected to lactophenol cotton blue staining for observing the morphology.
Slide Culture Technique
A rectangular slab of SDA was prepared and was placed on a clean glass slide. The culture isolate was then inoculated and another glass slide was placed over the top of it to form a sandwich. This slide was kept inside a petri dish along with moist cotton and incubated for about 3 days at room temperature. After incubation, the coverslip was placed on a drop of lactophenol cotton blue stain and viewed under the microscope (45x). The morphology of A.niger and P.species were observed and photographed.
Determination of Amylase Activity
A.niger and P.species isolates were tested for amylase production by starch hydrolysis. Starch agar medium was prepared and inoculated with the isolated organisms and then incubated at room temperature for about 2-3 days. After incubation, the plates were flooded with the iodine solution, and the zone of clearance was observed around the microbial growth, which indicates the production of amylase. Based on the zone of clearance, the fungal isolates were used for further studies on the production of the enzyme amylase.
Enzyme Production
Enzyme production was done by two methods namely, Submerged fermentation (SmF) Solid-state fermentation (SSF)
Solid-State Fermentation (SSF)
Substrates like coconut oil cake were used as a solid substrate for solid-state fermentation. Ten g of coconut oil cake was weighed and hydrated with 10 mL of basal salt solution and adjusted with moisture content from 43-81% (Ramachandran et al. 2004). The substrate was sterilized by autoclaving at 12°C for 15 mins. 1% of inoculum was inoculated after sterilization and then incubated at room temperature for 6 days (Suganthi et al. 2011).
Enzyme Extraction for Submerged Culture
After incubation, the culture sample was filtered by using Whatman filter paper No.1. The paper-filtered media was used to perform analytical determinations (Mabel et al. 2006).
For Solid-state Culture
After incubation, 0.1 M phosphate buffer saline was prepared and pH was adjusted to pH 7.0. 22 mL of freshly prepared phosphate buffer saline was added to the substrate beds and shaken vigorously in a rotary shaker for 15-20 mins at 120 rpm.
The mixture was then filtered through a cheese cloth and the filtrate was subjected to centrifugation at 8000 rpm at 4℃ for 15 mins. The supernatant was collected in a clean fresh tube and it was filtered through a cheese cloth, the filtrate was used as the crude enzyme preparation (Suganthi et al. 2011).
Dinitro Salicylic Acid Method (DNS)
For standard preparation, different concentration of maltose (1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm) was prepared and DNS assay was performed. 6 tubes were arranged in a row of test tube rack and labeled as S1, S2, S3, S4, S5, S6, T1 (A.niger BW), T2 (Penicillium sps BW), T3 (A. niger CW), T4 (Penicillium sps CW). 1 mL of the respective stocks and samples were transferred to the labeled tubes. 1mL of DNS reagent was added to all the tubes, then all the tubes were heated at 100° for 5 mins. After heating, the tubes were allowed to cool. All the tubes were made into 10 mL with distilled water. Then it was transferred to cuvettes and the absorbance value was noted at 540 nm using a calorimeter. The graph was plotted with a concentration of maltose at the x-axis and absorbance value (OD value) at the y-axis.
Estimation of Protein Lowry-Folin's Method
A series of tubes were taken and labeled as Blank, S1, S2, S3, S4, S5, S6, T1, T2, T3, T4 respectively. 0.5 mL, 1mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of Bovine serum albumin (standard) was added to the S1, S2, S3, S4, S5, S6 respectively. 1 mL of samples were added to T1, T2, T3, T4. All the tubes were made up to 3mL with distilled water. Then 4.5 mL of Alkaline copper reagent was added to all the test tubes. 0.5 mL of Folin's reagent was added to all the test Vol. 20, No. 3, 2021 • Nature Environment and Pollution Technology tubes and incubated at room temperature for 10 mins. After incubation absorbance value was noted at 560 nm using a calorimeter. The graph was plotted with a concentration of protein at the x-axis and absorbance value (OD value) at the y-axis.
Gas Chromatography-Mass Spectroscopy (GC-MS)
The enzyme samples were qualitatively assayed by performing Gas Chromatography-Mass Spectroscopy (GC-MS). GCMS was performed by using equipment 7000 Series Triple Quad GC/MS for the enzyme samples to identify the amount and type of chemicals present in the sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. This equipment is a standalone capillary GC detector for use with the Agilent 7890A Series gas chromatograph. Mass spectroscopy works by the principle of ionizing chemical compounds to generate charged molecules and measuring their mass-to-charge ratio.
In GCMS, the sample is ionized. Molecules of the samples break into a charged fragment during the ionization process. Ions are separated based on their mass-to-charge ratio (m/z). Ions are detected by using a mechanism that has the ability to detect charged particles (e.g. electron multiplier). Results are finally displayed as spectra of the relative abundance as a function of the m/z ratio. Identification is carried out by correlating known masses to the identified masses or by a characteristic fragmentation pattern.
Isolation of Organisms
Fungal cultures like P.species and A.niger were isolated by the settle plate technique. The colony morphology on the SDA plate was observed as follows, · The colonies cottony in texture, initially white and later became gray-green or olive-gray (Fig. 1a). · Salt and pepper appearance and reverse turning pale yellow (Fig. 1b).
Slide Culture Technique
The morphology was microscopically observed by performing slide culture technique and the structure was identified by staining with lactophenol cotton blue stain as follows, · Dense, brush-like, spore-bearing structures. Simple or branched conidiophores and terminated by flask-shaped phialides. It confirms P.species (Fig. 1c).
· Dark brown in color, globose vesicle with primary and secondary sterigmata, and conidiospores cover the entire surface of the conidial head. It confirms A.niger (Fig. 1d).
Determination of Amylase Activity
A starch hydrolysis test was performed. Zone of clearance was observed around the colonies on each plate containing A.niger and P.species (Fig.1e). It indicates the ability of the isolated organisms to produce the enzyme amylase.
Submerged Fermentation
The brewery wastewater supplemented with nutrients was used as a liquid medium. A.niger and P.species were inoculated in two different conical flasks containing liquid medium and incubated (Fig. 2a, 2b).
Solid-State Fermentation
Coconut oil cake was used as a solid substrate. The substrate was inoculated with A.niger and P.species in two different conical flasks and incubated. After incubation, the mat growth was observed (Fig. 3a, 3b). It was then centrifuged and the supernatant (Fig. 3c, 3d) was filtered using a cheese cloth and used as a crude enzyme (Fig. 3e, 3f).
Assay of Amylase Activity Dinitrosalicyclic Acid Method
Dinitrosalicylic method was performed. The absorbance value was noted at 540 nm using a calorimeter for enzyme samples and standards (Fig. 4b). The graph was plotted with a concentration of maltose against absorbance (OD value) (Fig. 4a). From the graph, the concentration of amylase in the enzyme samples was determined (Fig. 4c).
Estimation of Protein by Lowry Folin's Method
The protein content of the samples was estimated using Lowry Folin's method. The absorbance value for the standards and test were noted at 560 nm using a calorimeter (Fig. 5b). The graph was plotted with concentration along the x-axis against absorbance value along the y-axis (Fig. 5a). The concentration of protein of crude enzyme extracts was estimated (Fig. 5c). (Tables 1, 2, 3 and 4).
DISCUSSION
Amylase is a vital enzyme involved not only in modern biotechnology but also employed in various industries like starch processing industries for the hydrolysis of polysaccharide and paper industries. Enzyme amylase obtained from fungal species has large applications in the food and pharmaceutical industries and also widely used for the preparation of oriental foods.
The present study states that the enzyme amylase can be produced by using food wastes like brewery wastewater and coconut oil cake that are cost-effective and easily available when compared to the conventional substrates. Using brewery wastewater as a substrate may also reduce environmental pollution. A.niger and P.species were isolated by using the settle-plate technique. The slide culture technique was carried out to observe the morphology of the organisms.
Starch hydrolysis test was performed, zone of clearance around the colonies indicates their ability to produce the enzyme amylase. Enzyme production was done by submerged cultivation using brewery wastewater as a liquid substrate, and solid-state fermentation using coconut oil cake as a solid substrate. Crude enzymes were obtained by using the performed. Qualitative analysis of enzyme extract was done by performing Gas Chromatography-Mass Spectroscopy (GC-MS).
CONCLUSION
Amylase enzyme was produced by using food industrial wastes such as coconut oil cake as a solid substrate for solid-state fermentation and brewery wastewater as a liquid substrate for submerged fermentation. It indicates that the utilization of these wastes as a substrate may decrease environmental pollution and also reduces the production cost of the enzyme amylase. | v3-fos-license |
2020-03-19T13:07:57.276Z | 2020-03-01T00:00:00.000 | 212750515 | {
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} | pes2o/s2orc | Cytotoxic Evaluation and Anti-Angiogenic Effects of Two Furano-Sesquiterpenoids from Commiphora myrrh Resin
Commiphora myrrh resin (Myrrh) has been used in traditional Arabic medicine to treat various inflammatory diseases. Two furano-sesquiterpenoids, 2-methoxyfuranodiene (CM1) and 2-acetoxyfuranodiene (CM2), were isolated from the chloroform fraction of the ethanolic extract of Arabic Commiphora myrrh resin. The cytotoxicity of the compounds was evaluated using human liver carcinoma, breast cancer cells (HepG2 and MCF-7, respectively) and normal human umbilical vein endothelial cells (HUVECs) cell lines. The development toxicity and anti-angiogenic activity of both compounds were also evaluated using zebrafish embryos. Cell survival assays demonstrated that both compounds were highly cytotoxic in HepG2 and MCF7 cells, with IC50 values of 3.6 and 4.4 µM, respectively. Both compounds induced apoptosis and caused cell cycle arrest in treated HepG2 cells, which was observed using flow cytometric analysis. The development toxicity in zebrafish embryos showed the chronic toxicity of both compounds. The toxicity was only seen when the embryos remained exposed to the compounds for more than three days. The compound CM2 showed a significant level of anti-angiogenic activity in transgenic zebrafish embryos at sublethal doses. Thus, we demonstrated the cytotoxic properties of both compounds, suggesting that the molecular mechanism of these compounds should be further assessed.
Introduction
Cancer is the second leading cause of death worldwide. In 2018, 18.1 million new cases and 9.6 million deaths were reported globally [1]. Medicinal plants, including plant resins, are known to contain compounds that may serve as new cytotoxic compounds to combat cancer [2]. Commiphora myrrh (family: Burseraceae) is a medicinal plant, which is known to produce a type of aromatic oleo-gum resin known as myrrh, that grows in Yemen and the southern regions of Saudi Arabia [3]. myrrh (family: Burseraceae) is a medicinal plant, which is known to produce a type of aromatic oleogum resin known as myrrh, that grows in Yemen and the southern regions of Saudi Arabia [3]. Over the years, it has been claimed that myrrh is very safe for human and animal use, and the medicinal properties of the biologically active constituents have been described in various in vitro and in vivo studies [4][5][6][7][8]. The anti-proliferative activities of different Commiphora species have also been reported [9]. However, the information regarding the compounds responsible for myrrh toxicity remains lacking. Recently, the adverse effects of high doses of the essential oils of myrrh in mice have been reported [10]. Meanwhile, a clinical study has also reported miscarriage in women who used a large amount of myrrh during pregnancy [11].
An investigation into the cytotoxicity of natural products can lead to the discovery of new anticancer agents. Hence, it is of the utmost importance to study the developmental toxicity of myrrh in appropriate in vitro and in vivo model systems, to investigate the cytotoxic compounds of this valuable traditional medicine. Many animal model systems are being used to assess the toxicity of newly designed compounds, natural extracts, and medicines. Recently, zebrafish have emerged as an excellent in vivo model system, not only for studying human diseases but also for the testing and target validation of a large number of compound libraries [12][13][14]. Herein, we describe the isolation of two furano-sesquiterpenoid compounds and report the cytotoxic activity against different cancer cell lines and zebrafish embryos. To the best of our knowledge, to date, there have been no reports concerning the cytotoxic activity of 2-methoxyfuranodiene and 2-acetoxyfuranodiene and this is the first cytotoxic evaluation of both compounds using in vitro cell lines and an in vivo zebrafish model.
Identification of Compounds
By employing 1D and 2D-NMR, mass analysis, and a comparison with literature data, the two isolated compounds were identified ( Figure 1).
Compound 1: 2-methoxyfuranodiene: white crystalline: C16H22O2. The structure was established with the help of 1D-NMR, 2D-NMR, COSY, NOSY, EIMS, and IR, the data for which has been compared with the published data [15].
Compound 2: 2-acetoxyfuranodiene: white crystalline: C17H22O3. The structure was established with the help of 1D-NMR, 2D-NMR, COSY, NOSY, EIMS, and IR, the data for which has been compared with the published data [15].
Cytotoxic Activity
To assess the cytotoxicity of compounds CM1 and CM2, an MTT assay was performed. As shown in Figure 2, both compounds presented high cytotoxic activity, thereby significantly decreasing the number of viable cells in a concentration-dependent manner. The dose-dependent inhibition rates of cell viability in HepG2, MCF7, and HUVEC cells are depicted in Figure 2. The
Cytotoxic Activity
To assess the cytotoxicity of compounds CM1 and CM2, an MTT assay was performed. As shown in Figure 2, both compounds presented high cytotoxic activity, thereby significantly decreasing the number of viable cells in a concentration-dependent manner. The dose-dependent inhibition rates of cell viability in HepG2, MCF7, and HUVEC cells are depicted in Figure 2. The median inhibitory concentration (IC 50 ) values of both compounds against the tested cells are presented in Table 1. The HepG2 cell line was highly sensitive to the cytotoxic effect, therefore it was selected for further assays in this study. The data are presented as the mean ± S.D. (*P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant compared to the control) for the three independent experiments carried out in triplicate.
Cell Cycle Analysis
To determine whether the decreasing number of live cells produced by the two compounds was due to cell growth inhibition, the cell cycle distributions of the control and treated cells were examined by flow cytometry after 48 h of incubation. As shown in figure 3, the percentage of cells in the S phase significantly decreased from 20.8 ± 0.04% in the control cells to 9.6 ± 0.4% and 7.1 ± 1.3% in the HepG2 cells treated with CM1 and CM2, respectively. Consequentially, the percentage of cells in the G2/M phase increased from 23.5 ± 0.74% to 31.9 ± 1.7% and 38 ± 2%, respectively. Our results indicate that both compounds inhibit cells proliferation in the S phase and caused a significant G2/M arrest. Collectively, these results suggest that the anti-proliferative effects of these compounds are associated with arrest in the cell cycle.
Cell Cycle Analysis
To determine whether the decreasing number of live cells produced by the two compounds was due to cell growth inhibition, the cell cycle distributions of the control and treated cells were examined by flow cytometry after 48 h of incubation. As shown in Figure 3, the percentage of cells in the S phase significantly decreased from 20.8 ± 0.04% in the control cells to 9.6 ± 0.4% and 7.1 ± 1.3% in the HepG2 cells treated with CM1 and CM2, respectively. Consequentially, the percentage of cells in the G2/M phase increased from 23.5 ± 0.74% to 31.9 ± 1.7% and 38 ± 2%, respectively. Our results indicate that both compounds inhibit cells proliferation in the S phase and caused a significant G2/M arrest. Collectively, these results suggest that the anti-proliferative effects of these compounds are associated with arrest in the cell cycle.
Apoptosis/Necrosis Assessment Using Flow Cytometry
Next, we examined the mode of cell death induced by CM1 and CM2, to clarify the mechanisms behind the anti-proliferative effects of both compounds using flow cytometry with Annexin V-FITC/PI staining. HepG2 exposure (48 h) to both compounds resulted in a significant change in the early and late apoptotic cell populations. We found that the treatments led to a significant increase in the early and late apoptotic + necrotic cells, from 0.7 ± 0.14% and 2.62 ± 0.19% in the control cells to 3. 91 ± 0.42% and 30.74 ± 1% in the CM1-treated cells and 7.13 ± 0.55% and 62.56 ± 5.4% in the CM2treated cells, respectively ( Figure 4). As a result, both compounds were proven to possess strong apoptotic induction abilities against HepG2 human liver cancer cells.
Apoptosis/Necrosis Assessment Using Flow Cytometry
Next, we examined the mode of cell death induced by CM1 and CM2, to clarify the mechanisms behind the anti-proliferative effects of both compounds using flow cytometry with Annexin V-FITC/PI staining. HepG2 exposure (48 h) to both compounds resulted in a significant change in the early and late apoptotic cell populations. We found that the treatments led to a significant increase in the early and late apoptotic + necrotic cells, from 0.7 ± 0.14% and 2.62 ± 0.19% in the control cells to 3. 91 ± 0.42% and 30.74 ± 1% in the CM1-treated cells and 7.13 ± 0.55% and 62.56 ± 5.4% in the CM2-treated cells, respectively ( Figure 4). As a result, both compounds were proven to possess strong apoptotic induction abilities against HepG2 human liver cancer cells.
Developmental Toxicity of 2-Methoxyfuranodiene and 2-Acetoxyfuranodiene in Zebrafish Embryos
The zebrafish embryos were treated with serial dilutions (0.1 to 30 µM) of both compounds individually. Treating the zebrafish embryos with different concentrations resulted in early and late lethality in zebrafish embryos. At concentrations higher than 50 µM, CM1 and CM2 treatments induced 100% embryonic lethality within 12 h of exposure. Generally, CM2 had a higher toxicity to zebrafish embryos compared to that of CM1. The LC50 values for CM2 and CM1 were 15 and 40 µM, respectively.
2-Methoxyfuranodiene and 2-Acetoxyfuranodiene Inhibited the Formation of Angiogenic Blood Vessels during Zebrafish Embryonic Development
The Tg(fli1:EGFP) zebrafish embryos were exposed to equal concentrations of either compound to obtain the comparative anti-angiogenic activity. The anti-angiogenic activities of CM1 and CM2 are presented in Figures 5 and 6, respectively. The control embryos developed normally (bright field image, Figure 5A). The inter-somatic blood vessels (white arrows in Figure 5B), which are angiogenic blood vessels, developed and grew normally in the control embryos. The zebrafish embryos that were treated with CM1 (15 µM) also developed normally up to 48 hpf ( Figure 5C, bright field image). CM1 did not interfere with the inter-somatic blood vessel formation process in the treated zebrafish embryos, and so a normal development and growth of inter-somatic blood vessels was observed
Developmental Toxicity of 2-Methoxyfuranodiene and 2-Acetoxyfuranodiene in Zebrafish Embryos
The zebrafish embryos were treated with serial dilutions (0.1 to 30 µM) of both compounds individually. Treating the zebrafish embryos with different concentrations resulted in early and late lethality in zebrafish embryos. At concentrations higher than 50 µM, CM1 and CM2 treatments induced 100% embryonic lethality within 12 h of exposure. Generally, CM2 had a higher toxicity to zebrafish embryos compared to that of CM1. The LC 50 values for CM2 and CM1 were 15 and 40 µM, respectively.
2-Methoxyfuranodiene and 2-Acetoxyfuranodiene Inhibited the Formation of Angiogenic Blood Vessels during Zebrafish Embryonic Development
The Tg(fli1:EGFP) zebrafish embryos were exposed to equal concentrations of either compound to obtain the comparative anti-angiogenic activity. The anti-angiogenic activities of CM1 and CM2 are presented in Figures 5 and 6, respectively. The control embryos developed normally (bright field image, Figure 5A). The inter-somatic blood vessels (white arrows in Figure 5B), which are angiogenic blood vessels, developed and grew normally in the control embryos. The zebrafish embryos that were treated with CM1 (15 µM) also developed normally up to 48 hpf ( Figure 5C, bright field image). CM1 did not interfere with the inter-somatic blood vessel formation process in the treated zebrafish embryos, and so a normal development and growth of inter-somatic blood vessels was observed (white arrows in Figure 5D). However, the development of secondary angiogenic blood vessels was severely affected by CM1. The presence of a sub-intestinal vein is quite clear in the control embryos (white arrow in Figure 5C), but the sub-intestinal vein failed to develop in CM1-treated embryos ( Figure 5D asterisk).
On the other hand, compound CM2 showed more efficient anti-angiogenic activity compared to that of CM1. As shown in Figure 6, CM2 affected the normal development of zebrafish embryos at a concentration of 15 µM. The embryos were much smaller in size, the body was curved, and there was no pigmentation (bright field, Figure 6C). CM2 also disrupted the inter-somatic blood vessel formation in the treated zebrafish embryos. At 48 hpf, the formation and development of the inter-somatic blood vessels in the control embryos were not affected ( Figure 6B). However, the formation of inter-somatic blood vessels in zebrafish embryos that were exposed to CM2 (15 µM) were severely affected. As is evident in Figure 6D, the majority of inter-somatic blood vessels did not emerge from the dorsal aorta ( Figure 6D, asterisk), some blood vessels formed but they did not grow and failed to connect to the dorsal longitudinal anastomotic vessel. The formation and growth of the sub-intestinal vein was normal in the control embryos ( Figure 6B). However, the sub-intestinal vein did not form in 100% (n = 30) of the CM2-treated zebrafish embryos.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 14 (white arrows in Figure 5D). However, the development of secondary angiogenic blood vessels was severely affected by CM1. The presence of a sub-intestinal vein is quite clear in the control embryos (white arrow in Figure 5C), but the sub-intestinal vein failed to develop in CM1-treated embryos ( Figure 5D asterisk).
On the other hand, compound CM2 showed more efficient anti-angiogenic activity compared to that of CM1. As shown in Figure 6, CM2 affected the normal development of zebrafish embryos at a concentration of 15 µM. The embryos were much smaller in size, the body was curved, and there was no pigmentation (bright field, Figure 6C). CM2 also disrupted the inter-somatic blood vessel formation in the treated zebrafish embryos. At 48 hpf, the formation and development of the intersomatic blood vessels in the control embryos were not affected ( Figure 6B). However, the formation of inter-somatic blood vessels in zebrafish embryos that were exposed to CM2 (15 µM) were severely affected. As is evident in Figure 6D, the majority of inter-somatic blood vessels did not emerge from the dorsal aorta ( Figure 6D, asterisk), some blood vessels formed but they did not grow and failed to connect to the dorsal longitudinal anastomotic vessel. The formation and growth of the sub-intestinal vein was normal in the control embryos ( Figure 6B). However, the sub-intestinal vein did not form in 100% (n = 30) of the CM2-treated zebrafish embryos. The CM2 treatment disrupted the inter-somatic blood vessel formation in the treated zebrafish embryos, the majority of the inter-somatic blood vessels did not form (white asterisks), and some blood vessels formed but they did not grow and failed to connect to dorsal longitudinal anastomotic vessel. Similarly, the sub-intestinal vein did not form in the transgenic zebrafish embryos that were treated with CM2.
Discussion
In recent years, the global increase in the incidence of cancer has had serious impacts on human health. In this regard, new therapeutic compounds derived from natural products could be a good option for avoiding adverse effects. Historically, C. myrrh has been widely used in traditional medicine for the treatment of several illnesses. Its therapeutic properties have been supported by practice and evidence-based research on terpenoids (especially furano-sesquiterpenes), which are the active compounds present in myrrh essential oil [16]. Moreover, phytochemical investigations concerning C. myrrh resin have shown it to contain heerabolene, acadinene, elemol, eugenol, cuminaldehyde, and numerous furano-sesquiterpenes, including furanodiene, furanodienone, curzerenone, and lindestrene [17]. Substantial furano-sesquiterpenoid compounds are present in the exudates of myrrh, and approximately 20 different types have been isolated and identified [18][19][20]. In this study, two pure compounds from the chloroform fraction of 80% v/v ethanol extract of C. myrrh resin were isolated using column chromatography. These two active compounds, identified as 2methoxyfuranodiene and 2-acetoxyfuranodiene, which are members of the furano-sesquiterpenoid family, have been previously isolated from C. myrrh gum [21]. However, their cytotoxic activity on human cancer cells is yet to be elucidated. Therefore, the present study aimed to investigate and explore the anti-proliferative potential of these two compounds on different cancer cells, with a focus on the different types of cell death induced. Based on the IC50 values, the results showed that CM2 was more active than CM1 in the tested cell lines, with HepG2 liver cancer cells displaying the most sensitivity (Table 1). The embryos were much smaller in size, the body was curved, and there was no pigmentation. (D) The CM2 treatment disrupted the inter-somatic blood vessel formation in the treated zebrafish embryos, the majority of the inter-somatic blood vessels did not form (white asterisks), and some blood vessels formed but they did not grow and failed to connect to dorsal longitudinal anastomotic vessel. Similarly, the sub-intestinal vein did not form in the transgenic zebrafish embryos that were treated with CM2.
Discussion
In recent years, the global increase in the incidence of cancer has had serious impacts on human health. In this regard, new therapeutic compounds derived from natural products could be a good option for avoiding adverse effects. Historically, C. myrrh has been widely used in traditional medicine for the treatment of several illnesses. Its therapeutic properties have been supported by practice and evidence-based research on terpenoids (especially furano-sesquiterpenes), which are the active compounds present in myrrh essential oil [16]. Moreover, phytochemical investigations concerning C. myrrh resin have shown it to contain heerabolene, acadinene, elemol, eugenol, cuminaldehyde, and numerous furano-sesquiterpenes, including furanodiene, furanodienone, curzerenone, and lindestrene [17]. Substantial furano-sesquiterpenoid compounds are present in the exudates of myrrh, and approximately 20 different types have been isolated and identified [18][19][20]. In this study, two pure compounds from the chloroform fraction of 80% v/v ethanol extract of C. myrrh resin were isolated using column chromatography. These two active compounds, identified as 2-methoxyfuranodiene and 2-acetoxyfuranodiene, which are members of the furano-sesquiterpenoid family, have been previously isolated from C. myrrh gum [21]. However, their cytotoxic activity on human cancer cells is yet to be elucidated. Therefore, the present study aimed to investigate and explore the anti-proliferative potential of these two compounds on different cancer cells, with a focus on the different types of cell death induced. Based on the IC 50 values, the results showed that CM2 was more active than CM1 in the tested cell lines, with HepG2 liver cancer cells displaying the most sensitivity (Table 1).
There are different modes of cell death: apoptosis, a genetically controlled or programmed process, and necrosis, a non-programmed or accidental process [22]. One feature of cancer cells is apoptosis Molecules 2020, 25, 1318 8 of 14 evasion and targeting apoptosis can terminate the uncontrolled growth of cancer cells and, hence, is considered the most successful non-surgical treatment. Many known anti-cancer drugs target different apoptotic pathways [23], and several natural compounds derived from plants affect apoptotic pathways that are blocked by cancer cells via various mechanisms [24]. Flow cytometry was utilized in this study to detect apoptosis/necrosis, which can be determined by the exposure of phosphatidylserine (PS) on the surface of apoptotic cells, another hallmark of apoptosis induction [25]. The loss of control over the normal cell cycle is another hallmark of cancer cells. Additionally, the cell cycle is a mechanism that is gaining increasing interest as an anti-cancer target [26]. In our study, an evaluation of the cell cycle phase distribution using flow cytometry was employed to determine whether the compounds altered the cell cycle of HepG2 cells. The results indicated that the percentages of treated HepG2 in the S phase decreased, whereas those in the G2/M phase increased.
In fact, several studies have shown that myrrh components induce apoptosis and arrest the proliferation of cancer cells [27]. In addition, apoptosis induction and the arrest of cell cycle progression were observed to be exerted by the compounds isolated from C. myrrh [28].
The furano-sesquiterpene class of compounds, which are known to possess interesting biological activities, contains a wide range of chemical compounds derived from natural products [29,30]. When the anti-cancer activities of furano-sesquiterpenes and the derivatives isolated from soft coral were tested against different types of cancer cells (leukemia, prostate, lung, breast, and cervix), some of these compounds were found to demonstrate promising activity against leukemia and prostate cancer cell lines [31]. Additionally, another furano-sesquiterpene isolated from soft coral was investigated to verify its apoptotic effects. Arepalli et al. found that the isolated compound inhibited the proliferation of several human cancer cell lines, mediated apoptosis, and induced cell cycle arrest in human leukemia cells (THP-1) [32].
The newly designed drugs that will be used to treat various human ailments, must first be tested in suitable animal models to assess the effective dose ranges and safety of the drugs. Various animal models are currently in use to evaluate the toxicity of chemically synthesized or natural products. The zebrafish has emerged as a very powerful tool for investigating the in vivo developmental toxicity of compounds on a large scale [33][34][35]. The small size, ease of genetic manipulations, and relatively economical cost has paved the way for zebrafish to be the best organism for human disease models [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53]. The zebrafish developmental toxicity study revealed the chronic toxicity of CM1 and CM2. Continuous exposure, for more than three days, resulted in the total lethality of exposed embryos. However, the general trend indicated that CM2 was more toxic compared to CM1 in zebrafish embryos. The in vivo toxicity correlated with the in vitro toxicity of these compounds in human cancer and normal cell lines. Studies related to the assessment of the toxicity profile of C. myrrh in any animal model are very limited, and there are no reports prior to this study that investigate the developmental toxicity of C. myrrh (or any compounds isolated from it) in zebrafish embryos. Prolonged use of C. myrrh has already been attributed to recurrent miscarriage and abdominal pain in one clinical case [11]. Zebrafish embryonic death, caused by exposure to furano-sesquiterpenoids isolated from C. myrrh, warrants that a careful dose monitoring system should be tested to avoid any adverse effects on fetus development. Various studies have reported the anti-angiogenic profile of myrrh within in vitro or in vivo systems [54][55][56][57]. During zebrafish embryonic development, the inter-somatic blood vessels and sub-intestinal vein are considered angiogenic blood vessels (which emerge from pre-existing blood vessels) and hence provide a quantitative tool to measure the angiogenic activity of natural products or synthetic compounds in live zebrafish embryos [58][59][60][61]. CM1 impeded the development of the sub-intestinal vein in the treated embryos. CM2 exhibited potent anti-angiogenic activity, as the angiogenic blood vessels, inter-segmental vessels, and sub-intestinal vein did not form in CM2-treated embryos.
In the present study, 2-methoxyfuranodiene and 2-acetoxyfuranodiene were found to be associated with anti-cancer effects, apoptosis, and cell cycle arrest in human liver cancer cells. These results validate previous studies where furano-sesquiterpene compounds exhibited obvious inhibitory effects on cancer cell proliferation. The molecular mechanisms associated with the anti-cancer activity of these compounds needs further investigation. However, the present findings suggest that the anti-proliferative activity of the compounds may be associated with their potential to induce apoptosis and inhibit angiogenesis.
Plant Material Collection and Compound Isolation
C. myrrh oleo-gum-resin was purchased from (Yasin Spices, Sana'a, Yemen) The plant identity was verified by Prof. Ramzi Mothana. A voucher specimen No. 12486 was deposited in the College of Pharmacy, Herbarium of Pharmacognosy Department, King Saud University, Riyadh, Saudi Arabia.
Preparation of the Crude Extracts
Air-dried oleo-gum resin of C. myrrh (1.5 kg) was extracted several times using a Soxhlet apparatus with 3 L of ethanol for 7 h. The acquired crude ethanolic extract was filtered, concentrated under reduced pressure using rotatory evaporator to give 54 g, the total alcoholic extract was suspended in H 2 O and successively partitioned with n-hexane, chloroform (CHCl3), and n-butanol (BtOH) to yield 5 g of n-hexane, 15 g of chloroform, and 10 g of butanol fractions.
Compound Isolation and Identification
The chloroform fraction (5 g) was applied onto the top of silica gel packed column (72 g, 80 × 3 cm). Elution began with 3% ethyl acetate: n-hexane, and the polarity was increased with ethyl acetate in gradient mode of elution analysis giving 18 fractions (20 mL each). Based on TLC behavior, the collected similar fractions were pooled together to yield eight main fractions. Fraction C (70 mg), eluted with 5% ethyl acetate:n-hexane, was re-chromatographed in a silica gel column (7.2 g, 60 × 1 cm) using chloroform: n-hexane solvent system. Elution mode from the column was gradient. According to their TLC behavior, similar fractions were collected to give seven sub-fractions. Sub-fraction 4, eluted with 20% CHCl3: n-hexane, yielded compounds CM-1 (25 mg) and CM-2 (28 mg). Several spectroscopic techniques were performed for structure elucidation including 1D and 2D-NMR.
Cytotoxic Activity Test for the Isolated Compounds (MTT Assay)
Several previous studies have been shown that the MCF-7 and Hepg2 cell lines showed sensitivity to the myrrh constituents [9]. To determine the cytotoxic activity of compounds CM1 and CM2, an MTT assay was employed. Briefly, 1 × 10 5 of HepG2 (liver) and MCF-7 (breast) cancer cells and normal HUVEC (human umbilical vein endothelial cells) cells were plated in tissue culture plates (24-well). After 24 h of incubation at 37 • C in 5% CO 2 , the cells were treated with various concentrations of both compounds (1-40 µM) for 48 h. To each well, 0.1 mL µL MTT (5 mg/mL) was incubated with cells for 2-4 h at 37 • C in 5% CO 2 . After incubation, acidified isopropanol (1 mL, 0.01 N HCL) was added to each well (forming soluble formazan), before the solution was placed on a shaker for 15 min. A microplate reader (Bio-Tek, Elx-800, Winooski, VT, USA) was used to record the optical density absorbance of the converted MTT at 570 nm. Untreated cells were considered as controls, while vinblastine was used as a positive control. For each compound tested, the IC 50 (concentration that decreases the growth of viable cells to half) was generated from the dose-response curves. Cell Viability (%) = (O.D. of treated sample)/(O.D. of untreated sample) × 100%.
Apoptosis Detection by Flow Cytometry (Annexin V-FITC/PI Staining)
HepG2 cells were seeded in a 6-well plate at a density of 1 × 10 6 cells/well. After 24 h, the cells were treated with CM1 and CM2 at the IC 50 concentration. Following exposure (48 h), both adherent and floating cells were collected by trypsinization and washed with ice-cold PBS. The pellet was re-suspended in ice cold 1× Annexin binding buffer (1 × 10 6 cells/mL) and incubated with 5 µL of Annexin V-FITC solution (Molecular Probes, CA, USA) and 5 µL of propidium iodide (PI, 50 µg/mL). The samples were mixed gently and incubated for 15 min in the dark. Thereafter, a binding buffer (400 µL) was added to each tube, and the results analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter, Brea, CA, USA). Data collection and analysis were performed using CXP software V. 3.0.
Cell Cycle Phase Analysis
After HepG2 cells were treated with the IC 50
Toxicity Screening of Zebrafish Embryos
Embryos from wild type or transgenic zebrafish were obtained by natural pair wise breeding, as described previously [63]. Wild type zebrafish embryos were treated with serial dilutions of 0.1, 0.3, 0.9, 1, 3, 9, and 30 µM, to assess the quantitative toxicity and LC 50 values of the compounds on developing zebrafish embryos. The transgenic Tg (fli1:EGFP) zebrafish embryos were exposed to sub LC 50 values, to check the activity of compounds on angiogenesis (development of blood vessels).
Angiogenesis
To assess the anti-angiogenic potential of the isolated compounds, the transgenic zebrafish line Tg (fli1: EGFP) was used. The endothelial cells (which make blood vessels) continuously express a green fluorescent protein under the fli1 promoter [64]. At six hours post-fertilization (hpf), the embryos were exposed to serial dilutions of compounds CM1 or CM2. The effects on the formation of blood vessels were checked in live zebrafish embryos, by observing the embryos using a Zeiss Axio Observer D1 Inverted fluorescent microscope paired with an FITC filter. Images were taken using ZEN software. The number of blood vessels in the control group were counted and used as a reference to quantify the number of missing blood vessels in the treated embryos.
Microscopy and Imaging
A Zeiss Axio Observer D1 Inverted fluorescent microscope was used to capture live images. ZEN software, provided by Zeiss, was used to process the images.
Statistical Analysis
All experiments were conducted independently and in triplicate. The results are presented as the mean ± standard deviation (SD). Statistical comparisons and charts were constructed using origin Lab software (version 8, Massachusetts, USA). Significant differences (p < 0.05) were determined using a Student's t-test. LC 50 values were calculated using probit analysis, as described by Finney [65].
Conclusions
When new bioactive compounds are investigated to elucidate their potential to treat cancer, their ability to inhibit cell proliferation and induce apoptosis are two of the major characteristics examined. Our findings indicate that the two tested compounds exerted potent cytotoxic effects and induced apoptosis. In addition, the compounds possess an ability to inhibit angiogenesis, suggesting the anti-cancer potential of these compounds, which should be further examined. | v3-fos-license |
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} | pes2o/s2orc | Theoretical Insights Into the Depolymerization Mechanism of Lignin to Methyl p-hydroxycinnamate by [Bmim][FeCl4] Ionic Liquid
Depolymerization of lignin into valuable aromatic compounds is an important starting point for its valorization strategies, which requires the cleavage of C-O and C-C bonds between lignin monomer units. The catalytic cleavage of these bonds is still difficult and challenging. Our previous experimental investigation (Green Chem., 2018, 20: 3743) has shown that methyl p-hydroxycinnamate (MPC) can be produced from molecular tailoring of H unit in lignin by the cleavage of the γ-O ester bond. In this study, the mechanism of [Bmim][FeCl4]-catalyzed depolymerization of lignin was investigated by using the density functional theory (DFT) method. The results reveal that [FeCl4]− anion of the catalyst plays a decisive role in the whole catalytic process, where two possible activation modes including three different potential reaction pathways can realize the depolymerization of lignin model compound. The calculated overall barriers of the catalytic conversion along these potential routes show that the third potential pathway, i.e., methanol firstly activated by [Bmim][FeCl4], has the most probability with the lowest energy barrier, while the second pathway is excluded because the energy barrier is too high. Also, the results illustrate that the solvent effect is beneficial to the reduction of the relative energy for the reaction to form the transition states. Hence, the obtained molecular level information can identify the favorable conversion process catalyzed by metallic ionic liquids to a certain extent, and it is desirable to enhance the utilization of biomass as a ubiquitous feedstock.
INTRODUCTION
Methyl p-hydroxycinnamate (MPC) is a promising platform chemical for a variety of fine chemicals, which can be used as valuable medicinal intermediates (compounds 1-3 in Scheme 1; Lin et al., 2013;Yang et al., 2015) and polymeric precursor monomers (compounds 4, 5 in Scheme 1; Tilman et al., 2009;Ji et al., 2012). It has been reported that MPC possesses a wide variety of biological properties such as antitumor, anti-inflammatory, anti-adipogenic, and neuroprotective activities (compounds 6-8 in Scheme 1; Lee et al., 2013;Vo et al., 2014). The current MPC production mostly relies on high-cost petrochemical feedstock, and corrosive acid or noble metal catalysts with complicated separation processes (Clark et al., 2000;Percec et al., 2006;Guzman et al., 2014). In this regard, finding a more environmentally friendly and lower-cost synthesis method for MPC production from renewable resources, such as lignocellulosic biomass, is very attractive with an urgent and challenging necessity and it can better meet the requirements of sustainable development as well.
Lignocellulosic biomass mainly comprises cellulose, hemicellulose, and lignin. Among these three constituents, lignin is the least used resource due to its complexity and recalcitrance to chemical process. However, lignin contains a large number of ring structures, including three monolignol precursors, i.e., p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S), with multiple C-O-C (ester, β-O-4 ′ , α-O-4 ′ , 4-O-5 ′ ), and C-C (β-β ′ , β-5 ′ , β-1 ′ , 5-5 ′ ) linkages. This advantage makes lignin highly potential to be a renewable source for various aromatic compounds. That is to say, it is critical to make full use of lignin by converting it into value-added chemicals. Catalytic depolymerizations, including catalytic oxidation, alcoholysis, and hydrogenolysis, are the most efficient ways to do so. These techniques always focus on the cleavage of C-O-C and C-C bonds to obtain low molecular weight products. However, the products are composed of a wide range of chemical compounds. To avoid this disadvantage and make the product more suitable for direct use as either industrial feedstock or fuel, more recently Li et al. (2018) proposed a remarkable method, that is different from previous C-O or C-C cleavage approaches. In particular, the selective catalytic tailoring the H unit (pCA) in herbaceous lignin for MPC production can be realized over metal-based ionic liquids (MBILs), as MPC has a similar structure to that of p-coumaric acid ester, a typical H structural unit in herbaceous lignin. In this case, the isolated yield of MPC upgraded to 71.1 mg g −1 under optimized reaction conditions with one of the MBILs, [Bmim] [FeCl 4 ]. While the calculation results of the narrow HOMO-LUMO energy gap between ester lignin and [FeCl 4 ] − anion preliminarily proved its superiority, the detailed reaction mechanism was not further provided.
It is well known that, ionic liquids (ILs), as a new kind of catalyst, have received significant attention in the field of biomass conversion owing to their high potential for the replacement of conventional acidic catalysts (Yang et al., 2017;Zhang et al., 2017). Hence, such a catalytic conversion from herbaceous lignin to MPC by the MBILs and similar strategy using ILs as the catalyst can be regarded as ground-breaking technologies, deserving more attention. In particular, it is noted that theoretical studies on lignin catalytic conversion with ILs are relatively rare, while considerable attention has been mainly focused on experimental studies. Our previous work illustrated that the geometric and interactions between 16 ILs and lignin model compound GG, and then identified sulfonic ILs having the strongest interaction with GG . Further, we investigated the reaction mechanism of the cleavage of β-O-4 bond in GG to guaiacol by -SO 3 H functionalized ILs . Given the versatility of IL catalysts Wang et al., 2015;Meng et al., 2017;Qian et al., 2018;Zhang et al., 2018) and our keen interest in the research of biomass (Cai et al., 2015;Suresh et al., 2017), an elegant approach for catalytic transform of lignin to structurally defined aromatic esters is put forward here. According to Otera (1993) and Hoydonckx et al. (2004), the lignin model compound, phenyl p-hydroxycinnamate (PPC), was used to produce MPC by cleavage of the γ-O ester linkage using MBIL [Bmim][FeCl 4 ] as catalyst (Scheme 2). The reaction mechanism was also raised by DFT calculations in details. By understanding all elementary steps involved in the reaction network mentioned above, we constructed a potential energy surface for all reaction routes and proposed a dominant reaction pathway, which will provide a guide for other researchers who are interested in developing efficient ways of depolymerizing lignin.
COMPUTATIONAL DETAILS
Scheme 2 shows the model system studied in this work and describes the conversion process of lignin model compound PPC to MPC catalyzed by [Bmim][FeCl 4 ] MBIL catalyst. All calculations were carried out under the framework of density functional theory at the B3LYP-D3 level (Stephens et al., 1994;Grimme et al., 2010) in Gaussian 09 D.01 program package (Frisch et al., 2013), including the optimization of the structures of all the reactants, products, intermediates, and transition states. The basis set SDD (Andrae et al., 1990;Igel-Mann et al., 2006) was used for Fe atom, and the 6-31+G(d,p) basis set (Harihara and Pople, 1973;Frisch et al., 1984) was applied for all the other atoms, which is referred to as B3LYP-D3/BSI hereafter, vibrational frequency analysis based on the optimized structures was carried out to verify the natures of all the stationary points (local minimum or first-order saddle point). Transition states were validated by performing intrinsic reaction coordinate (IRC) (Fukui, 1981;Barone and Cossi, 1998) calculations to ensure that each of them actually connected the desired reactant and product. Structures at the two ends of IRC paths were optimized to minima, which represent the stable geometrics of reactants and products. The single-point energies of all structures were then improved at M06-D3 /6-311++G(d,p) level (Grimme et al., 2010;Qu et al., 2014) for all atoms except Fe atom (SDD), which is referred to as M06-D3/BSII hereafter. To mimic the solvent effect on the reactions, the solvation model based on density (SMD) (Marenich et al., 2009;Bernales et al., 2012) in methanol solvent was applied for all gas phase structures. The final Gibbs free energies (G g ) in the gas phase are the summation SCHEME 1 | Schematic diagram of chemicals with MPC as platform compound (Li et al., 2018). SCHEME 2 | Conversion of lignin model compound to MPC according to Otera (1993) and Hoydonckx et al. (2004).
of thermal corrections to gas phase Gibbs free energies ( G c ) at the B3LYP-D3/BSI level and the single-point energies from the M06-D3/BSII level (E g ), referred to Equation 1. The solvation Gibbs free energies (G s ) are the sum of G c and the singlepoint energies from the M06-D3(SMD)/BSII level (E s ), referred to Equation 2. In the following content, the G s and G g were used to draw the relative free energy profiles. (1) When multi-component changes are involved in a reaction, the above-mentioned thermal corrections based on ideal gas phase model will overestimate the contributions of entropy to free energies for reactions with solvent, because the suppressing effect of solvent on the rotational and transitional freedoms of substrates is neglected, which also have been proved by experimental studies (Liang et al., 2008;Huang et al., 2011). In the present study, we adopt the approximate approach proposed by Martin et al. (1998) to correct all solute free energies. A correction of 4.3 kcal/mol applies to per component change for a reaction at 298.15 K and 1 atm, i.e., a reaction from m to n components has an additional correction of (n-m) × 4.3 kcal/mol. Many previous works (Qu et al., 2014(Qu et al., , 2015 have also proved the reliability of this protocol. So, we discuss the mechanism in terms of the corrected free energies in the following sections. The Cartesian coordinates and total energies (include in the gas phase and solvent corrected free energies) of all optimized structures are given in the Supporting Information.
RESULTS AND DISCUSSION
In this work, the depolymerization from lignin to MPC by cleavage of γ-O ester linkage, as catalyzed by MBILs, was performed under mild conditions (420 K) and this process is very different from typical lignin depolymerization by cleavage of the β-O-4 ether linkage. Therefore, we considered a different mechanism from a general reactivity profile (Sabot et al., 2007;Weng et al., 2011). Inspired by the alcoholysis mechanism (Otera, 1993;Hoydonckx et al., 2004), this work has put forward three potential pathways to explore the possible process of lignin depolymerization catalyzed by MBILs, as a complement to a series of researches on lignin (Cai et al., 2015;Long et al., 2016;Suresh et al., 2017). Herein, to visualize the three transformation paths for an in-depth understanding, the pathways are respectively indicated as a, b, and c. In pathway a, [FeCl 4 ] − anion of catalyst attacks PPC, and MPC is produced through acyl chlorination. Organic compounds containing ester link or acyl chloride link are ubiquitous, and strongly nucleophilic acyl chloride group compared to hydroxyl groups (Kuroki et al., 1998), can easily be transformed into the complex functionalized with the ester group (Kaynak et al., 2018). The conversion rate will be higher if acylation is used to replace the general alcoholic esterification. Pathway b is regarded as a transesterification process (Otera, 1993). This process is widely applied in laboratories and industries and serves as one of the most typical organic reactions. Comparing to esterification, this process is a convenient and practical way to obtain ester by using this reaction when the parent carboxylic acids are unstable or difficult to separate (Hoydonckx et al., 2004). Furthermore, the reactivity of transesterification depends mainly on the electrophilic and nucleophilic properties of the substrates. Thus, pathway b currently being adopted here to get MPC is to activate the substrates directly by [FeCl 4 ] − anion of the catalyst. By contrast, the pathway c is considered as an improved acid catalytic conversion process. As known to us, ILs with more Brønsted acid sites exhibit superior catalytic ability in transesterification reactions. Interestingly, structural rearrangement may influence the reduction of the reaction energy barrier. So, several factors responsible for the lignin transformation have been considered in the third path, such as the production of -FeCl 3 and free Cl − anion, intermolecular rearrangement. The three possible paths are summarized in Scheme 3. In the following, we discuss the mechanism of the transformation in terms of the three pathways.
Potential Reaction Pathways
Pathway a-Acyl Chlorination Process As shown in Scheme 3 (pathway a), acylation chlorination mechanism from PPC to MPC catalyzed by [Bmim][FeCl 4 ] is formed via three elementary steps: acyl chlorination, alcoholysis, and catalyst recovery. A detailed catalysis process corresponding to pathway a is also shown in Scheme S1. Figure 1 displays the calculated energy profile (G s ) with the optimized geometries of transition states corresponding to pathway a, and those of reactants, intermediates, and products are given in Figure 2.
The reactants (PPC and methanol) with an ionic pair of [Bmim] [FeCl 4 ] is taken as the zero reference point of relative energy in the following discussion. A clearer picture of all optimized structures of stationary points corresponding to pathway a is shown in Figure S1 and IRC calculations starting from a-1 to a-2, a-3 to a-4, and a-5 to a-6 are given in Figures S2-S4.
In a-1, the reaction starts with the nucleophilic attack of chlorine anion of [FeCl 4 It is noted that the phenoxy group in PPC has a strong electron-donating ability, which leads to the activation of the C-O group with a bond distance of 1.970 Å and promotes the reaction. This nucleophilic attack process involves a free energy barrier of 37.1 kcal/mol (a-ts1 relative to a-1). Once chlorine anion is introduced, the strong polarity of acyl chloride group in intermediate a-2 makes it easy to be replaced by other groups. Therefore, when methanol enters into the reaction system to attack acyl chloride, methoxy substitution is easy to occur with the elongation of C-Cl bond distance (from 1.899 to 2.573 Å) and the conversion of intermediate a-3 to a-4 is realized by alcoholysis under the catalysis of [Bmim] [FeCl 4 ]. It results in the formation of the product MPC through a-ts2 involving a relative free energy barrier of 13.3 kcal/mol (a-ts2 relative to a-3). The followed is the regeneration of the catalyst. HCl first approaches the phenoxy group and then forms another intermediate a-5. With the increase of H-Cl (from 1.312 to 1.683 Å) and Fe-O (from 1.845 to 1.985 Å) bond lengths and the decrease of O-H (from 1.827 to 1.103 Å) bond length, the catalyst is finally regenerated through a-ts3 with an energy barrier of 2.0 kcal/mol (a-ts3 relative to a-5) and releases 3.4 kcal/mol of energy.
By contrast to the energy barriers of the alcoholysis and catalyst regeneration steps only at 13.3 and 2.0 kcal/mol, the acylation process of that as high as 37.1 kcal/mol is determined as the rate-limiting step by the cleavage of the γ-O ester linkage in PPC. In this pathway, the -COOR functional group in PPC was first modified to -COCl by acyl chlorination reaction and then followed by the addition of methanol through an alcoholysis reaction to obtain the MPC product. This process is supported by Nese and Aysen's work (Kaynak et al., 2018) that -COCl is a very reactive functional group and can easily be transformed to -COOR functionality with a lower energy barrier. By a quantitative analysis of the mechanism of acylation chlorination, it is found that the high energy barrier indicates the formation of -COCl may be related to the steric hindrance of substrates and the coordination effect of Fe atom (Cunico and Pandey, 2005;Maslivetc et al., 2018).
Pathway b-Synergistic Effect of Cation and Anion
In pathway a, the initiation of the reaction begins with the attack of Cl atom of [FeCl 4 ] − at C=O group of PPC. Alternatively, Fe atom of [FeCl 4 ] − can also interact with the O atom of the C=O group because of the coordination effect of the iron atom. Consequently, the second possible pathway denoted as pathway b is proposed and shown in Scheme 3. A detailed catalysis process corresponding to pathway b is also shown in Scheme S2. A clearer picture of all optimized structures of stationary points corresponding to pathway b is shown in Figure S5 and IRC calculations starting from b-1 to b-2, b-3 to b-4, and b-5 to P+cat. are given in Figures S6-S8. Figure 3 illustrates the calculated free energy (G s ) profile with the optimized geometries of transition states corresponding to pathway b, and those of reactants, intermediates, and products are given in the same time, the catalyst is regenerated, making the completion of the whole catalytic cycle.
According to the above calculation results, it can be found that the coordination between Fe atom of [FeCl 4 ] − and O atom of C=O group leads to the activation of PPC, and the cation also promotes the coordination process by adjusting its position. In other words, the synergistic effects of the cation and anion make it easier for Fe atom of [Bmim] [FeCl 4 ] to attack the C=O group on PPC. Also, according to the literature (Lu et al., 2016), structural rearrangement is also beneficial to the reduction of the energy barrier. Therefore Figure 3, although the first several steps of the reaction proceed smoothly due to the synergistic effect of cations and anions of ionic liquid catalyst, the energy barrier of the last step is as high as 52.6 kcal/mol, which makes the whole pathway infeasible and needs to be excluded.
Pathway c-Acid Catalysis Process
All the above two pathways start with the activation of PPC by the catalyst. By contrast, we further proposed another reaction route in Scheme 3 denoted as pathway c, where the catalyst first interacts with methanol molecule and contains four stages: dechlorination, Fries-like rearrangement, alcoholysis, and catalyst recovery. A detailed catalysis process corresponding to pathway c is also illustrated in Scheme S3. The calculated free energy profiles (G s ) with schematic geometries along the reaction coordinate are shown in Figures 5, 6. A clearer picture of all optimized structures of stationary points corresponding to pathway c is shown in Figure S9 and IRC calculations starting from c-1 to c-2, c-3 to c-4, c-5 to c-6, and c-7 to c-8 are given in Figures S10-S13.
The ionic liquid catalyst first interacts with methanol molecule through forming hydrogen bond (O-H ... Cl, 2.533 Å) as illustrated in Figure 6 denoted as intermediate c-1. Subsequently, intermediate c-1 converts into intermediate c-2 through transition state c-ts1 with a relative energy barrier of 6.3 kcal/mol, suggesting that this interaction is feasible. In this step, the imaginary vibration of c-ts1 corresponds to the shrinking of H-Cl bond (from 2.533 to 1.794 Å) and stretching of Fe-Cl bond (from 2.336 to 4.123 Å), which indicates that the transfer of Cl − anion from [FeCl 4 ] − anion to H atom of -OH group in methanol and the dechlorinating stage is completed, thus forming -FeCl 3 with a concerted mechanism. Then, PPC participates in the reaction process, and complex c-3 is formed via hydrogen bond between H atom on the alkyl chain in the cation and O atom on the phenol group in PPC. Following the formation of c-3, the attack of -FeCl 3 to O atom on C-O bond in PPC results in the C-O cleavage process via transition state c-ts2. The relative free energy barrier for this step is calculated to be 25.1 kcal/mol (overall energy barrier is 32.4 kcal/mol), implying that the cleavage of C-O bond in such a way is more feasible than pathways a and b under 420 K with certain pressure. The process may be explained by the Fries-like rearrangement (Ucar et al., 1998;Guenadil and Aichaoui, 2003), which leads to the formation of complex c-4 containing an acyl chloride group. Moreover, this step is also the rate-determining step of the reaction. After the dissociation of Fe-Cl bond in the complex c-2, free Cl − anion is formed, which further attacks complex c-4 with the help of methanol, resulting in the formation of the complex c-5 with an acyl chloride group. From c-5, the acyl chloride complex reacts with methanol to produce the final product MPC by alcoholysis via c-ts3 with a relative energy barrier of 16.0 kcal/mol. The final step is similar to the process corresponding to pathway a, which completes the regeneration of the catalyst with a relative energy barrier of 2.0 kcal/mol and releases 3.4 kcal/mol of energy, also indicating the end of the reaction.
The above mechanism calculation corresponding to pathway c shows that the conversion of c-1 to c-2 by the production of -FeCl 3 and free Cl − anion is a very critical step for the whole reaction. The production of Lewis acid, -FeCl 3 , can promote the occurrence of Fries-like rearrangement reaction. The specific steps of this Fries-like rearrangement reaction are revealed as follows: complex c-3 forms typical intermediate c-4 as expected through an intermolecular rearrangement reaction, which emphasizes the dissociation of the phenoxy group. However, the completion of transesterification cannot be determined by the above steps. The subsequent acyl chloride complex c-5 completes the transfer of alkoxy group through the transition state c-ts3 and obtains the target product MPC. In this process, when only -FeCl 3 or [Bmim][Cl] catalyst is used, it would fail to obtain complex c-5 containing phenoxy ion and acyl chloride group, although the depolymerization of lignin is also an acid catalytic process, which means the complex c-5 is a crucial intermediate to produce MPC. Therefore, [Bmim][FeCl 4 ] is a useful medium for this improved acid catalytic process. It can act as both a Lewis acid catalyst for providing -FeCl 3 and a chlorination reagent for providing free Cl − anion.
Further Discussion
Based on the above studies, we concluded that the activation of C-O ester bond is the key to produce the target MPC product and the reaction might proceed by three possible pathways with different probability, depending on their energy barriers. Among them, pathways a and c need to be converted to the product through -COCl, while pathway b can be achieved by the direct cleavage of the ester C-O bond. Besides, our proposed mechanism can rationally explain to a certain extent that the cleavage of -COCl (pathways c) is more accessible than direct cleavage of ester C-O bond. The fact is that the acyl chloride complex c-5 has better reactivity than other intermediates (Qu et al., 2015). The most obvious advantage of this pathway is that it is a nucleophilic catalytic process. The substitution of Cl for phenoxy moiety to activate carbonyl is beneficial to the consequent attack by methanol molecule.
The overall energy barriers of the three pathways are calculated to be 37.1, 52.6, and 32.4 kcal/mol, respectively (see Figure S14), indicating that the pathway c is the energetically most favorable process than the other two for lignin depolymerization. These fairly high energy barriers also mean that the cleavage of C-O ether bond in the lignin is not smooth. A similar result was reported previously by Zhang's group (Jing et al., 2019). The results also demonstrate that pathway c has much lower activation energy in the initial step, revealing that pathway c may be the most optimal process. It can be said that pathway c integrates all the advantages of the other two pathways to some extent, but the effect of each elementary step is different from the previous mechanism. For example, carbocation intermediate can be obtained by using the Lewis acid -FeCl 3 produced by [FeCl 4 ] − anion to complete the Fries-like rearrangement. At the same time, the free Cl − anion is more favorable to the acyl chloride reaction compared to [FeCl 4 ] − . Therefore, [Bmim] [FeCl 4 ] is an efficient catalyst that combines the advantages of acid catalysis and acyl chlorination.
Solvation Effect
The above calculations have provided a mechanism understanding of the depolymerization of lignin model compound PPC catalyzed by [Bmim] [FeCl 4 ], and the catalytic properties of MBILs were explained to a certain extent.
According to previous studies (Sánchez-Sanz and Trujillo, 2018;Zhang et al., 2019), the solvent effect is of great importance for biomass conversion due to their impacts on the whole reaction. Figure 7 shows the relative Gibbs free energies of reactants, transition states, intermediates, and products corresponding to the reaction pathway c, which is the most energetically favorable path, in the gas phase and with solvation model. The Gibbs free energy profiles corresponding to the reaction pathways a and b both in the gas phase and with solvation model are shown in Figures S15, S16. Moreover, the specific energy barriers corresponding to the transition states are summarized in Table 1.
It is noted that the solvent effect hardly influences the reaction paths but the relative energies. The energy barriers associated with c-ts1, c-ts2, c-ts3, and c-ts4 are 9.8, 32.4, 31.7, and 7.6 kcal/mol with the SMD model, and 10.5, 35.9, 32.6, and 7.6 kcal/mol in the gas phase, respectively (see Table 1). There is a slight solvent effect for the first three steps, c-ts1, c-ts2, and c-ts3, where the energy barriers are reduced by 0.7, 3.5, and 0.9 kcal/mol, respectively, while the last step c-ts4 has almost no difference, which may be ascribed to the different solvation behavior of methanol to the intermediates and transition states. On the one hand, methanol molecules can be either a hydrogenbond donor or a hydrogen-bond acceptor (He et al., 2013), which will increase the probability of methanol to form hydrogen bond with [FeCl 4 ] − , as well as increase the hydrogen-bonding interactions between anions and methanol, thus the energy barrier to form c-2 is decreased. On the other hand, the solvation effect of implicit methanol was found to induce a polarization of the transition structure and enhance the electron-donating ability (Domingo et al., 1999;Kong and Evanseck, 2000;Domingo and Andres, 2003), and the protic solvent is beneficial for proton transfer in c-ts2 and c-ts3, which can lower the energy barriers of c-ts2 and c-ts3. Additionally, methanol molecules can solvate c-3 to form c-4 (Fries-like rearrangement step), leading to lower energy barrier of c-ts2. Note that the Fries-like rearrangement is the essential step in PPC catalysis (Pitchumani et al., 1996), and the reaction energy barrier can be reduced to a certain
CONCLUSION
In summary, by performing DFT calculations, we systematically investigated the mechanism of MBIL-catalyzed cleavage of γ-O ester linkage of the lignin model PPC to MPC. Three reaction pathways, including acyl chlorination process (pathway a), transesterification processes (pathway b), and Lewis acid catalyzed conversion (pathway c) were proposed, and the [FeCl 4 ] − anion of catalyst is proved to play an essential role in activating PPC or methanol molecule to promote the reaction. It is found that the overall energy barrier of pathway c is much lower than that of pathways a and b, indicating that Lewis acid catalysis is an energetically more favorable process with the most probability. This path takes the advantages of the enhanced polarity by acyl chlorination and Fries-like rearrangement by Lewis acid, leading to lower energy barriers in the initial and the rate-determining steps. Additionally, methanol serves as both solvent and reactant, which contributes to this reaction. Overall, these results suggest that MBIL could be an effective catalyst for lignin valorization, and we hope that this work will provide more profound insights for current studies and inspire further development of efficient catalysts for biomass transformation.
DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files. | v3-fos-license |
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} | pes2o/s2orc | The rate of cellular hydrogen peroxide removal shows dependency on GSH: Mathematical insight into in vivo H2O2 and GPx concentrations
Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPxr, as expected, but is a function of both GPxr and GSH when the initial GPxr is less than H2O2. This simulation is supported by the biological observations of Li et al.. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity × GSH (effective GPx activity). The predicted cellular average GPxr and H2O2 for their study are approximately GPxr ≤ 1 μm and H2O2 ≈ 5 μm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.
( Figure 1). There are several families of enzymes that remove H 2 O 2 . This network has at least three nodes for peroxide-removal: i. Catalase is the longest known enzyme for removal of H 2 O 2 ; it requires no cofactors in its catalytic mode [6]; ii. the six members of the peroxiredoxin family of enzymes remove H 2 O 2 by reducing it to water and are in general recycled by gathering reducing equivalents from thioredoxin [7,8]; and iii. the glutathione peroxidases rely on glutathione (GSH) for the necessary reducing equivalents.
This study focused only on the effects of GPx and GSH levels on H 2 O 2 removal, assuming the catalase and peroxiredoxin levels were unchanged.
GPx and GSH in removal of H 2 O 2
In 1957 the family of glutathione peroxidases (GPx) was discovered [9]. Currently, at least four members of this family of enzymes are known [10Á12]. They all reduce H 2 O 2 to water (organic hydroperoxides are reduced to water and the corresponding alcohol) with the electrons coming from GSH, a necessary and specific cofactor. The kinetic behaviour of GPx-1 in dilute aqueous solution is best explained by a sequence of simple bimolecular reactions [13Á15]: [GS-GPx]'GSH À! k 3 GPx r 'GSSG'H ' yielding the overall reaction, For bovine GPx-1, the kinetics of this reaction have been well studied and are considered to be a 'pingpong' mechanism with indefinite Michaelis constants, indefinite maximum velocities and no significant product inhibition [10,16Á22]. For this system the effective rate constants are given in Table I. The observations in dilute, buffered solutions lead to the paradigm that in most circumstances, the rate of peroxide removal in vivo is essentially independent of the concentration of GSH [16,18,23]. This assumes low levels of H 2 O 2 (i.e. H 2 O 2 BGPx r BGSH) and, thus, the rate of recycling of GPx r by GSH (equations 2 and 3) is rapid compared to the rate of the reaction of GPx r with H 2 O 2 . Thus, GPx would predominantly exist in its reduced form, which is highly reactive with hydroperoxides (equation 1). However, recent observations by Li et al. [24] in a cell culture model are not in agreement with the above paradigm. When human cytosolic GPx-1 cDNA was transfected into a set of MnSOD-overexpressing U118 cells (a glioma cell line), they observed that: a. The GSSG content of these cells had a linear direct relation to the product of (GPx activity)) GSH, referred to as effective GPx activity. This is consistent with a higher rate of removal of H 2 O 2 leading to an increase in GSSG; b. Intracellular ROS (oxidation within the cell), as measured by the change in fluorescence of intracellular dichlorofluorescin, had a linear inverse relationship to effective GPx activity. This is consistent with a higher steady-state level of H 2 O 2 ( Figure 2); c. The cell population doubling time had a linear inverse relationship to effective GPx activity, i.e. the greater the effective GPx activity, the faster the cells grew. This observation is coupled to the assumption that a higher effective GPx activity will lower the steady-state level of H 2 O 2 and lead to a more reduced cellular redox environment and increased rate of growth [25]; and d. Most striking is that when the tumourigenicity of this set of cells with varying GPx activity was tested in nude mice, the growth rate of the tumours had a direct, linear relationship to effective GPx activity [24] (Figure 2). This is consistent with the in vitro observations, (aÁc) above, and points to a fundamental role of H 2 O 2 in setting the biological status of cells and tissues [5,25].
GSSG
In the above study of Li et al. [24], over-expression of MnSOD and genetic modifications with respect to GPx-1 resulted in higher fluxes of H 2 O 2 and various levels of GPx-1 in the cells. Because of the linear relationships with respect to [GPx][GSH] seen in Figure 2, these modifications appear not to have caused any significant changes in catalase or peroxiredoxin. Thus, the work of Li et al. serves as a reference for our modelling efforts to understand the GPx1-GSH-H 2 O 2 system.
Objective
The objective of this work is to examine the rate of removal of H 2 O 2 with respect to the kinetic rate behaviour of GPx-1 and GSH. Justification of the kinetic model is possible by using the in vivo observations of Li et al. [24] to: (1) determine when the rate-results from the kinetic models are consistent with the observed effective GPx activity dependency; and (2) estimate the probable range of average cellular GPx and H 2 O 2 in the cell lines investigated. To do this, we employed both the generalized and the classical approaches to express the kinetic rate behaviour involved in the GPx1-GSH-H 2 O 2 -system (equations 1Á3) and extract concentration dependency from the overall system time constant, t (also termed turnover time or biological 'average life' [26]). Finally, the variation of the classical model results from those of the general model was examined within this framework.
Generalized mathematical description of the removal of H 2 O 2 by GPx
Often in determining the rate of removal of hydrogen peroxide, the concentration of GSH is assumed to be constant [27]. Invoking this approximation and assuming spatial independence, the transient behaviour of species described by equations (1Á3) are a set of non-linear ordinary differential equations (ODEs) that describe the rates of change in the concentration of each species, equations (5Á10). Here C i represents the concentration of species i. . Effective GPx activity is 'GPx-activity' (or GPx) as measured by standard activity assay [44] multiplied by the concentration of GSH. The units are somewhat arbitrary (AU); using typical expressions of the activity of GPx (mU/mg protein) and for GSH levels (nmol/mg protein) units for effective GPx activity would be mU?nmol (mg protein) (2 . Figure adapted from [55]. From a mathematical viewpoint, the experimental observations of Li et al. [24] can now be compared to the concentration dependency of the rate of removal of H 2 O 2 for initial masses of H 2 O 2 , GPx and GSH introduced to the system (termed impulse response). These masses are described as equivalent initial concentrations. Since effective GPx activity proposed by Li et al. is the GPx activity coupled with GSH, we represent this as the product of initial GPx r and GSH concentrations, [GPx r ] 0 )[GSH] 0 . This approximation is used to represent effective GPx activity for the purpose of investigating our kinetic rate models.
Classical approximation of the rate of removal of H 2 O 2 by GPx
Because of the inherent non-linearity of the generalized expressions for the rate of removal of H 2 O 2 , a traditional kinetic rate law approximation (the classical model) is typically used. The classical model, in fact, is derived from the generalized rate expressions. Using a steady-state approximation, assuming that the enzyme concentration is lower than the substrate concentration, the rate of change of all substrateenzyme intermediates are negligible, the relationship between the initial rate, n 0 , total enzyme concentration, e, and initial substrate concentrations, S i , for an enzymatic reaction with two substrates is approximated as [28]: where F i 's are functions of reaction rate constants, k i 's. This approximation can be obtained from the general model (equations 5Á10) by invoking several approximations for the kinetic rate model for the GPx1-GSH-H 2 O 2 system. Starting with equations (5Á10), by assuming constant concentrations of intermediates (equations 7 and 9, set to zero) and manipulating equation (6), one can obtain the classical rate expression for removal of H 2 O 2 , [16,29]: where, And This classical expression results in a rate that is constant and depends only on the initial concentrations.
In this study, both the generalized and classical models are used to evaluate the rate of H 2 O 2 removal. A comparison of relevant similarities and differences are provided.
Parameters: Initial concentrations and reaction rate constants
In developing the model, we first need a range of concentrations that bracket expected physiological values. Using the data of Li et al. [24], we estimate the range of GSH in the five cell lines ( Figure 2) to be 0.12Á0.44 mM. Thus, we used the initial concentrations of 0.1Á0.6 mM for GSH (Table II) Most GPx is determined to be in its reduced form (!99%) from both in vivo studies [18] and mathematical simulations [27]. Therefore, we assumed all GPx in our model to be initially in the reduced form, GPx r . Estimated cellular concentrations of GPx vary from 0.2 mM in red blood cells [18] to values of 2.5 mM and 6.7 mM derived from mathematical models [27,30]. Rat liver cytosolic GPx-1 has been estimated to be 5.8 mM from Se of 0.46 ppm [31]; total GPx (monomer) in mitochondria and in the luminal space of endoplasmic reticulum is estimated to be 10 mM and 0.32 mM, respectively [32]. These values may be an over-estimate as we now know Table II. Initial concentrations used for the GPx model.
Rate constants for equations (1Á3) have been determined in dilute buffer solutions [16,18,23]. These rate constants vary depending on conditions such as the buffer-salt and pH of the solution. Rate constants used (Table I) represent estimates of the effective intracellular rate constants for the three principal steps of the GPx catalytic cycle [30].
Time constant for the removal of H 2 O 2
In order to search for ranges of possible physiological GPx r and H 2 O 2 for cell lines under conditions used by Li et al. [24], time-dependent numerical solutions given by our model of the GPx1-GSH-H 2 O 2 system are correlated to the observations of Li et al. As shown in Figure 2, the data of Li et al. present a linear relation between the effective GPx activity and the relative cellular H 2 O 2 . This biological observation can be compared to the concentration dependency of the rate of removal of H 2 O 2 . The dependency is generally reflected in an analytical solution for the overall system time constant, t (turnover time), provided that the model is linear. The overall rate by which the system evolves is dominated by this approximated time constant in the system. Thus, the functional dependency of t will allow us to understand the kinetic behaviour of the GPx1-GSH-H 2 O 2 system.
However, because of the non-linearity of the rate equations associated with the removal of H 2 O 2 (due to the coupling of time-dependent concentrations of species in the terms on the right-hand side of each expression (equations 5Á10), a closed-form solution does not exist. For non-linear systems, t can be approximated.
Relating overall system time constant to effective GPx activity
To meet our objectives, we have determined the dependency of effective GPx activity on t for the chosen range of initial GSH, GPx r and H 2 O 2 concentrations. Specifically, this is when t is inversely proportional to effective GPx activity, consistent with the observations of Li et al. [24], Then, comparing these values to acceptable physiological conditions for the genetically-modified cells used by Li et al. [24], we will pose possible ranges of average cellular GPx and H 2 O 2 . The initial conditions for variables held constant are shown in Table II given by the classical approach is independent of time, t can be directly calculated by integrating equation (12).
Numerical methods
All equation-sets were solved with initial concentrations and rate constants, listed in Tables I and II. Species rate expressions, shown in equation (5Á10), are therefore numerically integrated by using the IMSL (International Mathematical and Statistical Library) DIVPAG (double-precision initial value problem solver using either Adam-Moulton's or Gear's method) coded using Fortran [40Á42].
Results and discussion
Mathematical ranges of concentrations demonstrating effective GPx activity dependency In Figure 3 Based on our generalized mathematical model, there exist sets of initial GPx r and GSH concentrations within all ranges studied where t is generally inversely proportional to [GPx r ] 0 [GSH] 0 for the removal of H 2 O 2 , agreeing with the findings of Li et al. [24] shown in Figure 2 and the relationship expressed in Equation (16). This linear relationship between t and [GPx r ] 0 [GSH] 0 is clearly visible for the following cases: [24]. Reported levels of GSH and activities of GPx of other cells are compared with those of the U118 cells.
Typical levels of GSH in cells range from 1Á10 mm [25]. From the data of Li et al. [24] on the level of GSH in U118 cells and a cellular volume of 2.4 pL (F.Q. Schafer, unpublished), we estimated the range of GSH in the five cell lines of Figure 2 to be 0.12Á 0.44 mm. This is 10-times smaller than concentrations typically observed in proliferating cells.
The measured activity of GPx in the set of cells studied ranged from 15Á65 mU/mg protein (using the assay and unit definition of [43]).
[GPx] is considered to be at lower levels in tumour cells and cancer [6,44Á48]. These values are comparable to the range of values published for other cancer cell lines, e.g. PC-3 cells, 18 mU/mg protein [49]; MCF-7, 38 mU/mg protein; MDA-MB231, 98 mU/mg protein; and MCF-10A, 218 mU/mg protein [50]. These comparisons point to the low levels of GSH in U-118 cells as being a contributor to Li et al.'s [24] observation that peroxide levels and tumour growth are a function of (GPx activity))[GSH].
The time constant results provided by the general model indicate that if the possible intracellular concentration of H 2 O 2 is in the range of 5Á50 mM, then the physiological concentration of GPx is likely to be between 0.1Á10 mM. However, as mentioned above, the upper limit for intracellular [H 2 O 2 ] in normal cells is proposed to be Â700 nm [37,38]. However, the genetically-modified glioma cells used by Li et al. [24] over-expressed MnSOD by as much as 5-fold. This increase in MnSOD will likely increase the steady-state concentration of H 2 O 2 [1] It should be noted that actual concentrations may vary from those proposed by our model. This is because the modelling results are a consequence of the selected reaction rate constants and initial concentrations used in Equations (1Á3).
Finally, it is important to recognize that, in our modelling of the removal of H 2 O 2 by the GPx-GSH-H 2 O 2 system, spatially dependent concentrations were not considered and cellular averages were used. However, gradients in the intracellular concentrations clearly exist [6,37,51] and can result in local dominance of the rate of removal of H 2 O 2 that can alter our predicted cellular average concentrations.
Deviations of the classical model from the general model results ] profiles for both the general (solid lines) and classical (dotted lines) models are presented on a semi-log plot (Figure 4(a)). The [H 2 O 2 ] from the classical model is calculated by integrating the rate expression shown in Equation (12). The time taken for 63% decay (which is  t) in both models agrees relatively well for the three cases where [GPx r ] 0 is 0.1, 0.5 and 1 mM (as also shown in Figure 3(d)). For example, in the case where [GPx r ] 0 is 1 mM, although t's given for both models are close, the times predicted for 10% decay by the two models are more than an order of magnitude different. The rates of removal of H 2 O 2 at 1 ms given by the two models, as shown in Figure 4 (2) and (3), are much slower compared to the H 2 O 2eliminating step. The reaction rate constant for Equation (2) is three orders of magnitude smaller than the rate constant for equation (1); the rate constant for Equation (3) is very near that of equation (1). Thus, Equation (2) would be a rate-limiting reaction in the recycling of GPx r . In cases with lower [GPx r ] and [GSH], the slow recycling effect becomes more significant at earlier times during the process.
Nevertheless, these discrepancies are based on the set of initial concentrations used, as illustrated in Figure 4 Since the classical rate expression is derived by invoking the steady-state approximation on GPx o and GS-GPx, the rate given by the classical model should be in agreement with this steady-state rate given by the general model, as seen in Figure 4(b).
Finally, modelling the removal of H 2 O 2 by the GPx-GSH-H 2 O 2 system is a multi-scale problem and is spatially dependent. The time scale for removal of H 2 O 2 is on the order of milliseconds [27,52] whereas cell growth is on the order of days. Therefore, small differences in modelling solutions could significantly impact long-term predicted behaviour. For this reason, the classical approach to expressing the rate of enzymatic reactions should be used with caution, especially when addressing more complex systems.
Conclusions
With the use of kinetic modelling, we have investigated the removal of H 2 O 2 by GPx. Our goal was to examine the concentration dependency of intracellular H 2 O 2 removal to understand the anomalies in the findings of Li et al. [24]. They observed that biochemical parameters related to the removal of H 2 O 2 in genetically-modified U118-9 cells were a function of effective GPx-activity; most striking was their observation that the rate of tumour growth in an animal model was directly related to effective GPx activity. Using mathematical modelling, with sets of reaction rate constants and initial species concentrations taken from the literature, we found that: . . but, while offering useful simplicity, under certain conditions, the classical approach can result in substantial differences from the more general form over long time periods.
In the future, to further examine this system, the current lumped parameter mathematical model should be refined to include spatial dependency and H 2 O 2 generation. Issues of transport properties, such as species diffusivities and membrane permeability, and reaction rate constants, perhaps due to the crowded environment [53,54], need to be investigated. A direct coupling of cell growth to H 2 O 2 residence time is required to connect mathematical simulation to biological observations.
Mathematical modelling made it possible to quantitatively study the time constants (turnover time) associated with the removal of H 2 O 2 by GPx, providing insight into a biological observation that could not be approached experimentally. Finally, modelling demonstrates that the paradigm established from the kinetic-observations in dilute aqueous buffer do not always hold in the complex milieu of the cell. | v3-fos-license |
2017-09-14T14:58:00.666Z | 2007-12-05T00:00:00.000 | 40735577 | {
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} | pes2o/s2orc | Increase in metal extractability after liming of sacrificial sewage sludge disposal soils
A sandy and a sandy clay-loam soil from two dedicated (sacrificial) sewage sludge disposal sites were incubated with a total lime equivalent of 45 Mg·ha-1. Both these soils were acidified (pH 4.0 to 4.2) and had a significant accumulation of organic material (organic C of 2.9 to 3.7 %) compared to non-polluted soils (organic C of 0.6 to 1.0 %). The limed soils did not attain the desired pH of 6.5 after 6 months’ incubation due to a high buffer capacity. After incubation, soil samples were taken from the incubated pots and the levels of Al, Fe, Mn, Cu, Zn, Pb and Cd were determined by atomic absorption spectrophotometry (AAS) after extraction with NH4-EDTA and BaCl2. Most of the metals extracted with BaCl2 (except Mn in the sandy soil and Cd in both soils) decreased after liming. The EDTA-extractable Mn, Fe, and Cd in both soils and Cu and Pb in the sandy clayloam soil increased after liming, whereas Al and Zn, decreased in extractability (Statistically significant differences could not be determined for the trial due to the trial not having been designed for the results that were obtained). Similar results were reported in the literature for EDTA metal extraction but the phenomenon was not elaborated upon, except for Cr. The increased extractability of some of the metals after liming could negatively influence the use of EDTA as an extracting agent in proposed heavy-metal guidelines for similar sacrificial soils. Should liming be considered as a strategy to decrease metal mobility in sacrificial soils, the observed increase in extractability becomes a cause for concern and should receive attention in further research.
Introduction
In recent years, the trend has been to express heavy-metal levels in soils as plant-or potentially plant-available metal levels (Beckett, 1989;McLaughlin, et al., 2000) rather than "total" concentrations.This implies that "weaker" extractants or chelating agents be used in heavy-metal studies.Ethylenediaminetetraaceticacid (EDTA), in either the di-sodium or di-ammonium salt form, has been used extensively in a host of studies as an extractant of potentially plant-available heavy metals.
In some trials, EDTA was found to give a very good indication of the pollution hazard of heavy metals in soils as well as being a reliable test for predicting plant-available metals (Hooda et al., 1997;Cajuste and Laird, 2000).Earlier, Bruemmer and Van der Merwe (1989) stated that the NH 4 -EDTA-extractable heavy metal concentration gives a good estimate of those potentially plant-available, and therefore suggested it to be used in the establishment of preliminary threshold values for heavy metals in South African soils.Currently, however, there are no guidelines stipulating the maximum EDTA-extractable metal levels in South African soils.
Neutral salt extractants are generally weaker extractants than EDTA and give an indication of the immediately exchangeable (therefore immediately plant-available) metals (Beckett, 1989;McLaughlin et al., 2000).Examples of such extractants are BaCl 2 , NH 4 NO 3 , NH 4 -Acetate buffered at pH 7, and more.The BaCl 2 method (Hendershot and Duquette, 1986) gives an indication of the effective cation exchange capacity (ECEC) of the soil at un-buffered pH levels.This is particularly relevant in studies where the pH dependence of metal extractability is one of the parameters of investigation.
In a preliminary study it was found that soils from some sacrificial sewage sludge disposal sites can be acidified (pH 4.0 to 4.2) and also have very high pH-buffering capacities due to relatively high organic carbon levels (organic C % of 2.9 to 3.7%).Coupled to this is a significant increase in total heavy metal content of the soil.The aim of this study was to determine the BaCl 2 and NH 4 -EDTA extractability of a range of metals in two acid soils after liming to near-neutral pH levels and incubation in pots.
Materials and methods
The buffer capacity of the two acid soils (here referred to as Soils 1 and 2) was determined with a Ca(OH) 2 buffer (Van der Waals and Claassens, 2002).The required amount of a commercial dolomitic lime (according to the buffer determination) for a pH of 6.5 was added to each soil in 7.5 kg pots (with 4 repetitions) and the soil incubated for 3 months with regular watering and mixing.After sampling the soil and finding only a slight change in pH it was decided to add an equal amount of lime and incubate the soil again for the same period of time.The total amount of lime added amounted to the equivalent of 45 Mg•ha -1 .
After the second incubation period, a representative sample was taken from each pot and the pH determined according to the method described by the The Non-Affiliated Soil Analysis Work Committee (1990).On the limed soil and a sample from the original soil a BaCl 2 extraction was done.The method was adapted from Hendershot and Duquette (1986) by agitating 5 g of soil with 50 mℓ 0.1 M BaCl 2 in a glass bottle on a horizontal shaker for 1 h.After filtering the solution, the Al, Mn, Fe, Cu, Zn, Pb, and Cd contents were determined through AAS.Further, an EDTA extraction (The Non-Affiliated Soil Analysis Work Committee, 1990) was done on the same samples and Al, Fe, Mn, Cu, Zn, Pb and Cd again determined by AAS.The samples were tested at the same time to minimise experimental error differences.The metals Cr and Ni were not determined due to the termination of the testing after the first results had yielded unexpected increases upon liming, as discussed below.
Results and discussion
Table 1 gives the pH results of the two soils before and after liming.The pH values increased by 1.4 pH units for Soil 1 and by 1.8 pH units for Soil 2 after the addition of the equivalent of 45 mg•ha -1 lime.This indicates a massive buffering capacity brought about by organic material and complexes stable at pH 4.2 and 4.0 for Soil 1 and 2 respectively.The buffer determination as described by Van der Waals and Claassens (2002) is therefore considered inadequate for soils with high buffer capacities such as these.
Table 2 indicates the influence of the lime addition and incubation on the BaCl 2 extractable metals from the two soils.The metals Mn and Cd in Soil 1 and Cd in Soil 2 did not decrease in extractability after liming as was expected.The expected decrease was found for Al, Cu, Fe, and Zn.The large coefficients of variation for the limed soils are indicative of the low values determined for some of the metals that approached their detection limit with AAS.
Table 3 indicates the EDTA-extractable metals for the two soils before and after liming.Here most of the tested metals (Cu, Mn, Fe, and Cd in both soils and Pb in Soil 2) did not decrease in extractability and in some cases even increased.Aluminium, Zn, and to a lesser extent Pb in Soil 1, indicated the expected decrease in extractability.
In a study by Bloomfield and Pruden (1975) an increase in the EDTA extractability of Cd, Cu, Cr, Ni, Pb, and Zn in limed sludge samples and an increased extractability of Cr in limed "sludge + soil samples" was reported.Lake et al. (1984) attributed this increase in Cr levels to the chemistry of the Cr(VI) form in soils.In the Bloomfield and Pruden (1975) study, no explanation was given for the increased extractability of metals other than Cr from the limed sludge samples.Although Zn decreased in extractability in the present study, the effect of increased extraction was still observed for Cu, Mn, Fe, Pb, and Cd, even though the samples are not exactly of the same type as those in the Bloomfield and Pruden (1975) study.
Conclusions and recommendations
The results in this trial have led to a number of questions concerning the extractability of the metals and the conditions in the pots.Firstly, although the lime used was a commercially available lime, the metals listed here did not occur at sufficiently high levels to lead to the resultant increase in extractability (data not presented here).It is also not clear whether the lime had reacted completely with the soil.Secondly, the possible fluctuation in pH during the incubation time is not known and it is therefore impossible to comment on its influence on the metal extractability.Thirdly, the possible mineralisation of organic material after liming could not be quantified due to the set-up of the trial (aerobic conditions in a greenhouse) and it is therefore not possible to comment on its influence on the increased extractability.
Although results similar to these have not been widely reported, the phenomenon warrants further investigation.These authors agree with the conclusions of Lake et al. (1984) concerning the increased Cr extractability in the Bloomfield and Pruden (1975) study, but consider the phenomenon of the increased extractability of the metals other than Cr as similar to those reported here.The metals that exhibited an increase in extractability do not occur in soil in the same state as Cr(VI) and therefore require a different explanation.
The trial, having generated a number of questions, was not designed to supply the required answers.It is therefore suggested that a dedicated trial be conducted to determine: • The change in pH over an extended incubation period with and without the addition of lime with regular sampling intervals • The change in metal extractability (complexed and exchangeable) over an adequate incubation period with and without the addition of lime with regular sampling intervals • The EDTA metal extractability as influenced by increasing pH (lime application rates) The increased extractability of the metals after the application of lime has a profound influence on the establishment of metal guidelines when EDTA is used.Before the use of EDTA as an extractant in guideline levels is advocated, this phenomenon has to be investigated further to determine the restrictions of the procedure -especially under the conditions experienced in this trial. | v3-fos-license |
2019-04-09T13:02:47.124Z | 2017-06-27T00:00:00.000 | 103495937 | {
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} | pes2o/s2orc | Fluid-solid coupling dynamic equations considering gas desorption contraction and coal motion deformations
There are enormous gas resources in coal mines, and so gas drainage has great important roles before and during coal mine excavation, such as to eliminate gas explosion, gas outburst and other gas accidents during coal mine exploitation, to reduce the air pollution caused by gas emissions, and to adjust and improve the traditional energy structures that are mainly composed of coals and natural gas. To study and understand the mechanism of gas seepage and migration in coals is the key to designing, evaluating and maintaining gas drainage [1, 2]. Coals are typical porous media, and so the process of gas drainage would result in two phenomena, to decrease gas pressure, increase the effective stresses of coals, compress coal cracks and pores, and reduce the gas flow channel; to promote coal gas desorption, produce a certain degree of coal matrix shrinkage, and increase the porosity of coals relatively [3]. While gas adsorption also occurs during the process of gas drainage, which always produces certain swelling stresses and then leads to swelling deformation of coals, and so gas seepage is a fluid-solid coupling dynamic process. In this paper, the fluid-solid coupling model is built that considers gas desorption, motion and deformation of coal solid skeletons, and the model is proved to be reasonable with a computational example of one coal mine in P. R. China.
Introduction
There are enormous gas resources in coal mines, and so gas drainage has great important roles before and during coal mine excavation, such as to eliminate gas explosion, gas outburst and other gas accidents during coal mine exploitation, to reduce the air pollution caused by gas emissions, and to adjust and improve the traditional energy structures that are mainly composed of coals and natural gas.To study and understand the mechanism of gas seepage and migration in coals is the key to designing, evaluating and maintaining gas drainage [1,2].
Coals are typical porous media, and so the process of gas drainage would result in two phenomena, to decrease gas pressure, increase the effective stresses of coals, compress coal cracks and pores, and reduce the gas flow channel; to promote coal gas desorption, produce a certain degree of coal matrix shrinkage, and increase the porosity of coals relatively [3].While gas adsorption also occurs during the process of gas drainage, which always produces certain swelling stresses and then leads to swelling deformation of coals, and so gas seepage is a fluid-solid coupling dynamic process.
In this paper, the fluid-solid coupling model is built that considers gas desorption, motion and deformation of coal solid skeletons, and the model is proved to be reasonable with a computational example of one coal mine in P. R. China.
Dynamic constitutive fluid-solid coupling model
Under the action of external forces, the coal solid skeletons would produce supporting roles, and the effective stresses are valued as the ratio of supporting forces produced in the solid skeletons to the cross section area.Coal gas is divided into absorbed gas and free gas based on the existing status, and the former occupies over 90% of the total content.The coals have large specific surface area, so the process of gas absorption belongs to physical absorption, and the absorbed gas would change to free gas under the action of disturbance of temperature, pressure and in-situ stress [4].Gas drainage from coal bed is actually a reverse process of adsorption expansion, which is also shrinkage desorption of coal matrix, and so the expression of gas drainage is a back-analysis of adsorption-swelling process.The gas seepage process in coals is usually treated as isothermal, so the influence of disturbance of temperature on gas seepage is ignored in relative studies of gas drainage.
Moving adaptability equations of coals
Coals are simplified as homogeneous media and gas as ideal fluid, and the moving adaptability equations of coals include 4 parts as follows [5].
Effective stresses
The coals are composed of solid skeletons of molecular-scale particles and inner pores, so the coals that contain gas can be considered as porous media, and the effective stresses of the solid skeletons can be directly depicted as Terzaghi principle of effective stresses: where ij are the total stresses, ij are effective stresses, is Biot's parameter, p is gas pressure, and ij is Kronecker's delta symbol,
Constitutive equations
Similar to the conventional elastic materials, the physical equations of coal solid skeletons can be given based on elastic theory: The geometric equations of coal solid skeletons can also be given directly based on elastic theory: where u is the displacement of the coal solid skeletons.The displacement u of coal solid skeletons is a dynamic variable and includes parameters of space and time, so u has the expression of ( , , , ) u u x y z t , and the equations of dynamic equilibrium of coal solid skeletons are: where w is relative displacement of coal solid skeletons comparative to gas, F is body stress, is the total physical density of coals and is the sum of solid skeletons and gas, , is porosity , and s and g are the densities of coal solid skeletons and gas respectively.Substituting Eqs. ( 1) and (2) into Eq.( 4), the equations of dynamic equilibrium are , , , According to the relevant expressions of , Eq. ( 5) can be rewritten in vectors: 2.2.Gas seepage equations that consider deformation of coal solid skeletons
Dynamic porosity
The pores such as holes, cracks and fissures in coals provide pathways for gas migration, so the coal porosity is one of the key physical parameters to study the characteristics of gas transportation.Porosity is the ratio of volume of pores to the total volume of coals, it changes with the deformation of coals, so it is a relative variable influenced by the loads that applied on the coals, and the equation of during the isothermal process is given as follows [6]: where 0 is the initial porosity of coals under certain fiducial temperature and without any outer loads, s K is bulk modulus of soil solid skeletons and
Dynamic permeability
Similar to porosity of coals, permeability of coals is also a dynamic parameter that changed with porosity, and permeability k can be simplified as follows based on Krozeny-Carman equation in fluid mechanics [7]: where 0 k is the initial permeability of coals under certain fiducial temperature and without any outer loads.
Gas flow in coals
Gas in coals is assumed as ideal fluid, and then gas density and pressure should satisfy with [8]: where is the compression coefficient of gas, M is the molecular weight of gas, R is ideal gas constant, and T is absolute temperature.
Gas content Q is composed of free gas content f Q and absorbed gas content where f Q and a Q are calculated with Langmuir's formula, L P is Langmuir's pressure, under which the absorption capacity of gas can reach 50 % of the maximum value, L V is Langmuir volume that reflects the maximum adsorption capacity, a is gas density that measured under standard conditions and aa p that is obtained from Eq. ( 9), and a p is the standard atmospheric pressure with constant value of 1.013*10 5 Pa.
Substituting Eqs. ( 9), ( 12) and (13) into Eq.( 11), the total gas content is obtained: Under pressure gradient p , the process of gas flow in coals satisfies with Darcy's law where q is the velocity vector of gas seepage and is dynamic viscosity of gas.
Gas seepage in coals meets with quality conservation law, as to the unit volume of coals, the seepage equation is: (16)
Volumetric strain of coals
Deformation of coals caused by gas pressure and gas desorption is considered, and the volumetric strain v of coal solid skeletons is composed of 3 parts: where s v is the volumetric strain that caused by the deformation of solid skeletons, and it can be calculated from Eq. (3) as follows: , The volumetric strain is caused by gas pressure increment p , and can be calculated as follows: and the volumetric strain 2 g v is caused by gas desorption, and it can be calculated as follows [9]: 17) is obtained: 2.3.Fluid-solid coupling dynamic equations Gas seepage velocity vector q is the sum of absolute velocity of soil solid skeletons and relative velocity of gas: so Eq. ( 15) can be rewritten in the vector form: Substituting Eqs. ( 7), ( 14), ( 15) and (19) into Eq.( 16), 2 p can be determined in Eq. ( 22), then substituting Eq. (21) into Eq.( 6), and dynamic equilibrium of coals are built in Eq. ( 23).
Eqs. ( 22) and ( 23) are the final fluid-solid coupling dynamic equations that contain gas seepage, motion of the coal skeletons and shrinkage deformation of coals, and they are named as model I.
If the parameter of is valued as 0 in Eq. (18c), the deformations caused by the motion of the solid skeletons are ignored and the right parts of Eq. ( 4) are valued as 0, and model I is degenerated to the traditional model [7], which is named as model II, and the relative fluid-solid coupling dynamic equations are built in Eqs. ( 24) and (25).
All the variables in Eqs. ( 22) ~ (25) are implicit solutions, and they can be solved with COMSOL software:
Engineering example
The gas drainage engineering in the 2 nd coal bed of one coal mine in Henan province of P. R.China is taken as a computational example.The diameter of the gas drainage hole is 100.0 mm, the coal seam is about 6.0 m Fig. 1 Simplified 2-dimensional computational model thick, the length of gas drainage hole and coal thickness are all much larger than the diameter of the gas drainage hole, and so the gas drainage process can be simplified as a 2-dimensional plane strain problem.
The computational model is given in Fig. 1, which is a rectangle with length of 100.0 m and height of 6.0 m, the origin of rectangular coordinate system is located at the center of gas drainage hole, and axis x and y are along horizontal and vertical direction respectively.t .The coal roof and floor are all mudstones with negligible permeability, and so the up and bottom boundaries of the computational model are assumed as impermeable layers.The coal bed is horizontally distributed and the length of calculation model is built too long to exceed the influence scope of gas drainage process, and so the left and right boundaries of the computational model are also assumed to have no gas seepage.The outer and inner boundary conditions in Fig. 1 The coals are assumed as homogeneous isotropic media, and the main physical and mechanical parameters of coals and gas of model I are given in Table 1.As to model II, only αg is valued as 0 and other parameters are valued same to model I.
Gas seepage calculation
The curves of radial displacement r u at the inner side of the drainage hole changing with distances from the gas drainage hole center are given in Fig. 2.
Fig. 2 shows that the elastic radial displacement r u of the two computational models of I and II all increase during the gas drainage process, while r u of model I is larger than model II, that is to say that if the gas desorption shrinkage and moving adaptability are considered, the gas drainage hole would turn to be valid earlier than the traditional calculation model II.And r u of model I reaches 16.2 mm at t=50 d, which is about 0.8 mm larger than that of model II at the same time, and the former is 32.4% of the original radius of 50 mm.
The line at section of y=0 m and x=0~50 m is selected, and the curves of gas pressure p at t=50 d changing with distances from the drainage hole center are given as shown in Fig. 3. Fig. 2 Curves of radial displacements at the gas-drainage hole Fig. 3 shows that the gas pressure of coal seam calculated by the model I is about 7.6% less than model II, and this phenomenon can be interpreted by the mechanism of gas extraction as follows: during the gas drainage process, the solid skeletons move towards the drainage hole caused by the gas flows, some expansion deformation of gas flow would occur, which can also be found in Fig. 2, desorption and contraction of coal solid skeletons would also occur, and then coal porosity increase and gas permeability is improved correspondingly.And at the actual gas drainage process of this mine in Henan Province of P. R. China, the drained gas contents usually increase about 8% after the beginning drainage for 10 days, which just meets with the calculation results of model I.The calculated gas pressure of model I is less than that of model II near the gas drainage hole at the same time, while they turn to be same at farer place where the distance is larger than 40 m from the drainage hole center, where the gas pressure is just equal to the original gas pressure of 1.40 MPa that is given in Table 1.Figs. 2 and 3 show that model I is more reasonable than the traditional model II, and so model I is selected for the further analyses about gas pressure and gas permeability during the gas drainage process in this paper.
The drainage time of t=50 d is selected, and cloud figure of gas pressure around the gas drainage hole in the location of r≤0.8 m is given in Fig. 4.
Fig. 4 shows that the shapes of gas pressure are regular circular rings and change from small value at the inside locations near the gas drainage hole to large value at the outside locations, which meets with the fact that the more the gas are drained out and the less the gas pressure would be.Fig. 6 Curves of gas pressure changing with distance at different drainage time Fig. 5 shows that gas pressure at positions near drainage hole is less than the farer position, and the gas pressure decreases with the ratio of about 0.0012 MPa/d.Fig. 6 shows that gas pressure in the farer place is mainly influenced by the gas drainage date, the place of x=20 m is taken for example, the reduced gas pressures are 0, 0, 0.11 and 0.25 MPa for t=10, 50, 100 and 150 d respectively, and these principles and characteristics meet with the relative curves in Ref. [4].Gas pressure would reach the initial value at the positions farer from the gas drainage hole center such as x=40 m.
Similar to Fig. 4, the drainage time of t=50 d is also selected, and cloud figure of gas permeability at the scope of r≤0.8 m is given in Fig. 7. Fig. 7 Cloud figure of gas permeability around the gasdrainage hole (t=50 d) Fig. 7 shows that when the gas near the drainage hole is drained in large amount, the gas permeability increases obviously, and this phenomenon can be explained as follows.When gas is drained out, coal solid skeletons have radial displacement towards the gas drainage hole as shown in Fig. 2, and so coal porosity and gas permeability near the drainage hole all increase.The maximum permeability is about 1.15*10 -16 m 2 near the drainage hole, which is about 1.5 times of the initial permeability of 7.8 *10 -17 m 2 that is given in Table 1.
Similar to Figs.Coals that contain gas are assumed as isotropic and homogeneous elastic media, the shrinkage deformation caused by gas desorption and motion deformation caused by gas flow are considered, and the fluid-solid coupling dynamic equations about gas drainage are built, which are mainly composed of gas seepage, motion of the coal skeletons and shrinkage deformation of coals.And the model built in this paper is proved to be more reasonable than the traditional model that ignores gas desorption and shrinkage.
2. The radial displacement around the gas drainage hole reaches 16.2 mm at t=50 d, which is 32.4% of the original radius of 50 mm.
3. Gas pressure and permeability that influenced by the gas drainage process are mainly focused at the locations near the gas drainage hole, and they are equal to the initial values at the farer locations.
is the changed gas pressure from the initial gas pres-
is the thermal expansion coefficient of gas adsorption and it is equal to Langmuir's volumetric strain L in numerical value.And v Eq. (
3. 1 .
Initial and boundary conditionsDisplacement constraints are applied in x direction on the left boundary (x=-50.0m) and right boundary (x=50.0m) and in y direction on the bottom boundary (y=--3.0m), and uniform pressure of 5.0 MPa is applied on the top boundary (y=3.0m), which is the pressure caused by the total weights of soils and rocks that just overly on the computational coal bed.The model has no initial dis- boundary of the gas drainage hole, in which w p is the gas drainage pressure, and the initial gas pressure of the model is
Fig. 3
Fig. 3 Comparison curves of gas pressure changing with distances
Fig. 4 Fig. 5
Fig. 4 Cloud figure of gas pressure around the gas drainage hole (t=50 d) Curves of gas pressure curves at different position (x=1, 5, 10 and 15 m) changing with drainage time t and at different drainage time ( t =10, 50, 100 and 150 d) changing with distance x are given in Figs. 5 and 6 respectively.
Fig. 8 Fig. 9
Fig.7shows that when the gas near the drainage hole is drained in large amount, the gas permeability increases obviously, and this phenomenon can be explained as follows.When gas is drained out, coal solid skeletons have radial displacement towards the gas drainage hole as shown in Fig.2, and so coal porosity and gas permeability near the drainage hole all increase.The maximum permeability is about 1.15*10 -16 m 2 near the drainage hole, which is about 1.5 times of the initial permeability of 7.8 *10 -17 m 2 that is given in Table1.Similar to Figs. 5 and 6, curves of coal permeability at different position (x=1, 5, 10 and 15 m) changing with drainage time t and at different drainage time ( t =10, 50, 100 and 150 d) changing with distance x are given in Figs. 8 and 9 respectively. | v3-fos-license |
2020-10-07T13:06:56.073Z | 2020-10-07T00:00:00.000 | 222142398 | {
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} | pes2o/s2orc | Porous COS@SiO2 Nanocomposites Ameliorate Severe Acute Pancreatitis and Associated Lung Injury by Regulating the Nrf2 Signaling Pathway in Mice
Severe acute pancreatitis (SAP) is associated with high rates of mortality and morbidity. Chitosan oligosaccharides (COSs) are agents with antioxidant properties. We developed porous COS@SiO2 nanocomposites to study the protective effects and mechanisms of COS nanomedicine for the treatment of acute pancreatitis. Porous COS@SiO2 nanocomposites released COSs slowly under pH control, enabling sustained release and maintaining the drug at a higher concentration. This study aimed to determine whether porous COS@SiO2 nanocomposites ameliorate SAP and associated lung injury. The SAP model was established in male C57BL/6 mice by intraperitoneal injection of caerulein. The expression levels of myeloperoxidase, malondialdehyde, superoxide dismutase, nuclear factor-kappa B (NF-κB), the NOD-like receptor protein 3 (NLRP3) inflammasome, nuclear factor E2-related factor 2 (Nrf2), and inflammatory cytokines were detected, and a histological analysis of mouse pancreatic and lung tissues was performed. In the SAP groups, systemic inflammation and oxidative stress occurred, and pathological damage to the pancreas and lung was obvious. Combined with porous COS@SiO2 nanocomposites before treatment, the systemic inflammatory response was obviously reduced, as were oxidative stress indicators in targeted tissues. It was found that Nrf2 was significantly activated in the COS@SiO2 treatment group, and the expressions of NF-κB and the NLRP3 inflammasome were notably decreased. In addition, this protective effect was significantly weakened when Nrf2 signaling was inhibited by ML385. This demonstrated that porous COS@SiO2 nanocomposites activate the Nrf2 signaling pathway to inhibit oxidative stress and reduce the expression of NF-κB and the NLRP3 inflammasome and the release of inflammatory factors, thus blocking the systemic inflammatory response and ultimately ameliorating SAP and associated lung injury.
INTRODUCTION
Acute pancreatitis (AP) is a common acute abdomen presentation in the clinic with increasing morbidity in recent years. The severity of AP can be described as mild, moderate, or severe according to the local injury to the pancreas and systemic injury to other organs (Banks et al., 2013). Severe acute pancreatitis (SAP) is a serious illness with rapid onset and a high fatality rate. It is characterized by the systemic inflammatory response syndrome, the multiple organ dysfunction syndrome, sepsis, and other complications (Bi et al., 2015).
Acute lung injury (ALI) is one of the most serious and earliest complications of SAP. ALI is described as an important risk factor for death in the early stages of SAP with a high mortality rate in the range of 30-40% (Zhou et al., 2010).
Recent studies have shown that oxidative stress is one of the pathophysiological mechanisms for AP (Pérez et al., 2015;Xie et al., 2017). During the pathogenesis of AP, the injured pancreatic cells and the activated immune cells release abundant reactive oxygen species (ROS), which can lead to an imbalance between the oxidation system and the antioxidant system (Hackert and Werner, 2011;Esrefoglu, 2012). This imbalance results in tissue damage, including to the pancreas and other organs such as the lungs, liver, and so on (Fukumoto et al., 2013). Overall antioxidative stress therapy seems to be a key to ameliorating SAP and its associated lung injury.
Chitosan oligosaccharides (COSs) are the degraded product of chitin and chitosan and consist of glucosamine linked by β-1,4-glycosidic bonds. COSs are made from chitosan or chitin derived from shrimp and crab shells by chemical or enzymatic hydrolysis (Muanprasat et al., 2015). COSs are popular because of the variety of their functional biological activities; for example, they have antioxidant, anti-inflammatory, antibacterial, and immunomodulatory properties (Yuan et al., 2019). Several studies have shown that COSs can inhibit oxidative damage to the liver and lung (Junyuan et al., 2018;Tao et al., 2019). Because of their low molecular weight and high solubility, COSs degrade rapidly in vivo.
Porous silica nanoparticles with a specific structure and specific surface properties have good biocompatibility and are often used as inorganic non-metallic nanomaterials in biological applications (Cao et al., 2016b;Su et al., 2019). Porous silica nanoparticles are also often used in drug delivery systems (Liu et al., 2015;Su et al., 2019;Zhu et al., 2019). They can release the drug slowly by means of pH control, achieving sustained release and maintaining a higher concentration of the drug (Cao et al., 2016a;Zou et al., 2016;Zhang et al., 2017). In addition, porous silica nanoparticles have the advantage of targeted drug delivery, so that the drug can reach a higher concentration in the target tissue (Rosenholm et al., 2009;Shahbazi et al., 2012;Giret et al., 2015).
We used porous silica nanoparticles loaded with COSs. The porous structure gives COS@SiO 2 nanospheres the potential to be multifunctional. Thus, in this study, we used an animal model to evaluate the effects of porous COS@SiO 2 nanocomposites on SAP and associated lung injury.
The porous silica nanospheres were prepared according to a previously described method (Yang et al., 2012) with some modification. A total of 0.3 g of CTAB was dissolved in 60 ml deionized water, and 9 ml ethylene glycol and 4 ml ammonium hydroxide were added. The mixture was heated to 50 • C, and then 4 ml of TEOS was added dropwise with stirring. After reaction for 3 h, the white products were collected by centrifugation and were washed with water. Then the white products were dried, and calcined at 550 • C for 3 h in a muffle furnace to remove the surfactant. Finally, the porous silica nanospheres were obtained for further use.
A total of 20 mg of the porous SiO 2 nanospheres described above were added to the COS solution (5 mg/mL) with stirring. The COSs were loaded into the porous SiO 2 nanospheres through the porous structure. After stirring at room temperature for 12 h, the COS-loaded porous silica nanospheres (COS@SiO 2 ) were obtained by centrifugation and washed with water. Then, 5 mg COS@SiO 2 was dispersed in 5 ml phosphate-buffered saline with stirring. After 12 h, the supernatant was collected by centrifugation to measure the released COSs.
Animals and Treatments
All animal experiments were conducted according to the guidelines of the Animal Care and Use Committee of Shanghai Jiaotong University and were approved by the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine (SYXK 2013-0050, Shanghai, China.). Male C57BL/6 mice (6-8 weeks old, 20-22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (China) and maintained in standard cages in a humidity-controlled room with an ambient temperature of 23 ± 2 • C and a 12 h light-dark cycle.
The mice were randomly divided into four groups, as follows.
1. Caerulein-induced AP (SAP) group: mice were injected intraperitoneally with caerulein (100 µg/kg, with a 1 h interval between injections) 10 times; LPS (5 mg/kg) was administered by intraperitoneal injection immediately after the last injection of caerulein (Lerch and Gorelick, 2013). 2. Control (CON) group: mice were injected with normal saline instead of caerulein. 3. COS+SAP (SAP) group: mice were first injected with COS (20 mg/kg); after half an hour they were continually injected with caerulein and LPS to induce SAP. 4. COS@SiO 2 +SAP (COS@SiO 2 ) group: mice were injected with a corresponding dose of COS@SiO 2 and then SAP was induced.
In addition, we used another SAP model of mice: L-arginineinduced AP, which is also non-invasive and provides a rapid induction of SAP. To induce AP in mice with L-arginine, the mice were injected intraperitoneally with 8% L-arginine (dose 4.5g/kg) twice, with an interval of 1 h (Lerch and Gorelick, 2013). The grouping of mice with L-arginine-induced AP is the same as for mice with caerulein-induced AP. Next, we used ML385, an Nrf2 inhibitor, to treat mice in order to explore the specific mechanism of action. In this group (ML385+COS@SiO 2 +SAP group), mice were injected intraperitoneally with ML385 (10 mg/kg); after half an hour, they were continually treated as for the COS@SiO 2 +SAP group. In this group, we only used the caerulein-induced AP model.
Mice were sacrificed at the time points indicated in Figure 2A after being anesthetized with chloral hydrate. Blood samples were taken from the eyeball for each mouse, and the pancreatic tissue and lung tissue were collected and fixed in 4% paraformaldehyde for histological examination or stored at −80 • C after freezing in liquid nitrogen for other purposes.
Histological Examination
After sacrifice, pancreatic tissue and lung tissue were harvested, rinsed, and fixed in 4% paraformaldehyde at 4 • C overnight. The tissues were then processed with sequential clearing, rehydrating, and dehydrating steps, and embedded in paraffin blocks. Samples were sectioned into 4 µm slices and subjected to standard hematoxylin and eosin (H&E) staining. H&E images were captured using a light microscope (Leica, Germany) at a magnification of ×100 or ×200. Pancreatic histopathology scores were evaluated by the Schmidt criteria (Schmidt et al., 1992) for pancreatic tissue by edema, hemorrhage, necrosis, and inflammatory infiltration. Lung histopathology scores were evaluated by alveolar neutrophils, interstitial neutrophils, hyaline membranes, proteinaceous debris, and alveolar septal thickening, as described previously (Matute-Bello et al., 2011). The results of all experiments were analyzed blind by two pathologists.
Pancreas and Lung Wet-to-Dry Weight Ratio
The pancreas and lungs were sucked dry 1 and weighed, and then baked in an oven at 80 • C for 48 h until a constant weight was obtained as the dry weight. To evaluate tissue edema, the ratio of wet lung weight to dry lung weight was calculated. 1 dried by using blotting paper.
Serum Amylase and Lipase Assay
The serum activity of amylase and lipase was measured by enzymatic kinetic chemistry using commercial kits according to the manufacturer's instructions (Roche/Hitachi modular analysis system; Roche, Berlin, Germany).
Enzyme-Linked Immunosorbent Assay
The blood samples that were collected from the mouse eyeballs were centrifuged at 3,000 rpm for 20 min at 4 • C, and the upper serum was frozen at −80 • C. Inflammatory cytokines, including IL-6, IL-1β, IL-10, and tumor necrosis factor-α (TNFα), were detected by Luminex Screening Human Magnetic Assay (R&D Systems, Inc., Minneapolis, MN, USA), which was performed according to the manufacturer's instructions. The serum samples were diluted 1:2 using PH7.4 PBS for the assay, and 50 µl of the diluted samples was added to each well. Fluorescence intensity was measured using Microplate Reader and analyzed using software (xponent 3.1; Luminex, Austin, TX, USA).
Statistics
All measurement data are expressed as the mean ± standard deviation, and statistical analysis was performed using the GraphPad Prism 7.0 software. The t-test was used for the normal distribution and comparison between the two groups. One-way analysis of variance (ANOVA) was used for comparisons between the three groups, and the Kruskal-Wallis test was used for data that did not satisfy a normal distribution. p < 0.05 indicates that the difference was statistically significant.
Characterization of Porous COS@SiO 2 Nanocomposites
The porous SiO 2 was first synthesized using a sol-gel method (Yang et al., 2012). The size of the SiO 2 nanoparticles was ∼110 nm and they exhibited an obvious porous structure (Figure 1A), which could be used for loading COSs. When SiO 2 was mixed with the COS solution, the COSs were loaded into the SiO 2 spheres because of their abundant pores. After loading the COSs, COS@SiO 2 appeared as COS absorption peaks (Figure 1B), indicating that the COSs had been incorporated into the porous SiO 2 . Compared with the smooth curve of SiO 2 , the COS@SiO 2 curve showed an obvious peak around 305 nm in the ultraviolet-near-infrared absorption spectrum, which further proved successful loading of COS ( Figure 1C). The highest loading efficiency of COSs reached 10.2%. COS release was studied at pH 7.4 and pH 8.0. The COS release was much more rapid and the release rate was higher in an alkaline environment, which could be attributed to the enhanced aqueous solubility of COSs ( Figure 1D). In the model of AP induced by caerulein, caerulein can promote the contraction of the gallbladder, rupture of the pancreatic duct and acinar cells, and then the release of pancreatin and pancreatic juice into the pancreatic interstitium (Kaiser et al., 1995;Sata et al., 1996). The pancreatic juice is alkaline, and the release rate and amount of COSs increase in an alkaline environment, so COS@SiO 2 can reach a higher concentration in the local pancreas during AP. Porous silica nanoparticles give COSs the ability to collect in the pancreas and then be released in a high concentration. Hypothetical mode of action of COS was shown in supplement information (Supplementary Figure 1).
Porous COS@SiO 2 Nanocomposites Alleviated Pancreatic and Lung Injuries in Mice With Caerulein-Induced AP
The results showed that in the control group and the COS@SiO 2 -treated group (Supplementary Figure 2) there was no significant change in pancreatic and lung tissue. There was slight intralobular septal expansion, but no bleeding, necrosis, inflammatory cell infiltration, or other pathological changes. Compared with the control group, there was no obvious pathological damage to the pancreas or lung and no distinct change in serum amylase in the experimental group. The SAP model was successfully established by intraperitoneal injection of caerulein and LPS in mice (Figure 2A). Some pathological changes in the pancreatic tissue were observed, such as obvious edema (intralobular, interacinar, widened intercellular space), hemorrhage, acinar necrosis, and inflammatory infiltration. Compared with the SAP group, pathological injury to the pancreas in the COS and COS@SiO 2 groups was significantly reduced, and the pathological score was significantly lower (p < 0.001). Porous COS@SiO 2 nanocomposites displayed a higher capacity to attenuate injury to the pancreas induced by caerulein (Figures 2B,C).
We also observed the lungs under light microscope. We found that the lung tissue structure of the control group was clear, the alveolar wall was not significantly thickened, and there was no obvious inflammatory cell infiltration in the interstitium and alveoli.
In the SAP group, the alveolar wall was obviously thickened, the alveolar space was obviously widened, the alveolar cavities and interstitium were infiltrated by a large number of neutrophil inflammatory cells, and the alveoli were filled with protein fragments and had formed a hyaline membrane. Compared with the SAP group, pathological injury to the lung tissue in the COS and COS@SiO 2 treatment groups was significantly reduced, and the pathological score was significantly different (p < 0.001). The pathological injury in the COS@SiO 2 group was further reduced compared with the COS group, and the pathological score was statistically different (p < 0.05; Figures 2D,E). Hence, these results show that porous COS@SiO 2 nanocomposites can reduce lung histopathology injuries in caerulein-induced AP in mice.
In the SAP group, we also found out that the specific indices of AP (including serum amylase and serum lipase) were significantly increased compared with the CON group. The same indices in the COS and COS@SiO 2 groups were obviously lower than in the SAP group and were significantly different (p < 0.001; Figures 3A,B). In addition, we assayed the pancreatic and lung wet-to-dry weight ratio in each group. This showed the same trend as the serum amylase and lipase (Figures 3C,D). These data indicate that COS@SiO 2 can reduce pancreatic and pulmonary edema.
To verify the effectiveness of the porous COS@SiO 2 nanocomposites, we used another well-known SAP model induced by L-arginine injection. In the same way as in the model of caerulein-induced SAP, porous COS@SiO 2 nanocomposites also attenuated injury to the pancreas and lung induced by L-arginine in a dose-dependent manner (Supplementary Figure 3). These data show that COS@SiO 2 is effective in both caerulein-induced and L-arginine-induced AP.
All in all, these results proved that COS@SiO 2 ameliorated not only pancreatic injury but also lung injury.
Porous COS@SiO 2 Nanocomposites Reduced Inflammation in Mice With Caerulein-Induced AP
Compared with the CON group, the serum level of proinflammatory cytokines, including IL-6, IL-1β, and TNF-α, was significantly higher in the SAP group, while the serum level of IL-10, a type of anti-inflammatory cytokine, was lower (p < 0.001). We analyzed the same indices in COS-treated groups. ELISA showed that the serum level of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) decreased and that the level of anti-inflammatory cytokine (IL-10) increased significantly (p < 0.001; Figures 4A-D). These results suggest that COS@SiO 2 could alleviate AP-mediated systemic inflammation.
Porous COS@SiO 2 Nanocomposites Inhibited Oxidative Stress in Mice With Caerulein-Induced AP
Compared with the CON group, the level of oxidation-related indices, including MPO and MDA, in pancreatic and lung tissues was obviously increased in the SAP group, while the antioxidation related index (SOD) was decreased (p < 0.001). We also assayed the same indices in the COS-and COS@SiO 2 -treated groups. The results showed that the levels of MPO and MDA were lower and the level of SOD was higher than in the SAP group (p < 0.05). Furthermore, we found that the COS@SiO 2 group showed lower MPO and MDA levels and a higher level of SOD ( Figure 5). These results indicate that COS@SiO 2 could inhibit oxidative stress more than using COS alone.
Porous COS@SiO 2 Nanocomposites Suppressed NLRP3-Mediated Inflammasome and NF-κB Activation by Regulating the Nrf2 Signaling Pathway in Mice With Caerulein-Induced AP
The components of the NLRP3 inflammasome/IL-1β secretion axis and NF-κB were significantly upregulated in the pancreas and lung of the SAP group compared with the CON group (p < 0.01). These effects could be inhibited by the COS and COS@SiO2 treatment. Furthermore, we found that the expression of Nrf2 protein was markedly increased in the pancreas and lung of the COS@SiO 2 -treated group (Figure 6). These results indicate that COS@SiO 2 may upregulate Nrf2 to reduce the NLRP3 inflammasome and NF-κB activation.
ML385, a type of Nrf2 inhibitor, was used to inhibit Nrf2 signaling in mice with caerulein-induced AP. Compared with the COS@SiO 2 nanocomposite treatment group, the pathological injury to the pancreas and lung in the intervention group with ML385 was more serious, the pathological score was statistically different (p < 0.01), and the serum amylase and lipase levels were significantly higher (p < 0.001; Figure 7). This suggests that ML385 could reverse the protective effect of COS@SiO 2 . In other words, COS@SiO 2 played a protective role in AP through the Nrf2 signaling pathway.
DISCUSSION
In this study, we determined the protective effects of porous COS@SiO 2 nanocomposites on caerulein-induced SAP in mice. We demonstrated that COS@SiO 2 could significantly reduce pancreatic and lung pathological damage in SAP and was more effective than COSs alone. Further investigations showed that COS@SiO 2 inhibited the systemic and local (pancreas and lung) inflammation and oxidative stress by activating the Nrf2 signaling pathway.
COSs are oligosaccharides with a degree of polymerization between 2 and 20 that are obtained by degradation of chitosan. They are mixtures of β-1,4-linked D-glucosamine residues (Muanprasat and Chatsudthipong, 2017). Compared with high molecular weight chitosan, COSs have better water solubility; are more easily absorbed, transformed, and utilized in vivo; have higher reactivity; and have more important biological functions (Yoon et al., 2008). COSs are well-known for their antioxidative stress ability. Because of their biocompatibility, biodegradability, non-toxicity, and absorption properties, COSs The pancreatic histopathology scores were evaluated by Schmidt criteria for pancreatic tissue by edema, hemorrhage, necrosis, and inflammatory infiltration. (D,E) Lung histopathology scores were evaluated by alveolar neutrophils, interstitial neutrophils, hyaline membranes, proteinaceous debris, and alveolar septal thickening. The data are provided as the mean ± SEM (n = 6 per group). *p < 0.05; ***p < 0.001.
have been recommended as agents that are superior to other antioxidants (Nidheesh et al., 2016;Naveed et al., 2019). As a prebiotic with full solubility, COSs have been linked to various health-promoting potentials, including regulating glucose and lipid metabolism, promoting calcium absorption, and maintaining the integrity of the gut barrier (Jung et al., 2006;Bai et al., 2018). Moreover, COSs have been shown to reduce the effects of many diseases, such as LPS-induced ALI (Liu et al., 2018) and fatty liver disease (du Plessis et al., 2019) induced by a high fat diet, and so on.
Porous silica nanoparticles form a flocculent amorphous white powder that is non-toxic, odorless, and has good biocompatibility, water solubility, easy modification, thermal stability, high specific surface area, and a regular and uniform pore structure (Al-Sagheer and Muslim, 2010). In contrast, owing to the SiO 2 coating structure, porous COS@SiO 2 nanocomposites have obvious advantages in terms of economy and stability (room temperature storage).
In addition, our study proves that, in a weak alkaline environment as occurs in AP, the release rate of COSs from . ww, wet weight; dw, dry weight. Pancreatic and lung tissues were weighed and the W/D ratio was calculated. The data are provided as the mean ± SEM (n = 6 per group). *p < 0.05; **p < 0.01; ***p < 0.001. porous COS@SiO 2 nanocomposites is higher and the release speed of COSs is faster. This feature allows COSs to play a biological role at smaller doses, thereby reducing side effects.
All in all, porous silica nanoparticles give COSs the advantage of pH-controlled, sustained release to maintain a high concentration, overcome the shortcomings of easy degradation of COSs, and also give COSs the advantage of accumulating in the pancreas.
AP is an inflammatory reactive disease commonly found in the clinic. It has a rapid onset and develops rapidly. ALI in SAP is a common and early complication, and also one of the most important causes of death from SAP (Forsmark et al., 2016).
The levels of ROS increase significantly from the injured pancreatic acinar cells and activated immune cells in AP (Chvanov et al., 2005). ROS are a type of oxygen-containing active substance with high reactivity. Pancreatic injury is highly correlated with oxidative stress (Steinbrenner and Sies, 2009). On one hand, ROS directly cause lung cell damage and control intercellular signal transduction. On the other hand, ROS are also involved in polymorphonuclear activation, cytokine production, and disturbance of the endothelial barrier and microcirculation (Guice et al., 1989).
We used COS@SiO 2 to pre-treat mice with SAP and found that the treated groups showed reduced damage to the pancreas and lungs and decreased systemic and local inflammation compared with the untreated AP group. In addition, we also proved the anti-oxidative stress ability of COS@SiO 2.
During AP, the increased levels of ROS cause NF-κB, one of the most important proinflammatory factors, to be activated. NF-κB moves from the cytoplasm into the nucleus and this results in the release of downstream inflammatory cytokines, including TNF-α, IL-6, IL-1β, and so on, activating NF-κB (Huang et al., 2013;Zhang et al., 2018). As previous studies have shown, these inflammatory cytokines are closely related to the development of AP (Wirth and Baltimore, 1988). In addition, ROS activate the NLRP3 inflammasome. The NLRP3 inflammasome consists of NLRP3, apoptosis-associated specklike protein containing CARD (ASC), and cysteinyl aspartatespecific proteinase-1 (caspase-1). The NLRP3 inflammasome forms a molecular platform that activates caspase-1, which can catalyze the proteolytic process and secrete mature IL-1β, which is a powerful proinflammatory factor that can trigger a series of inflammatory reactions (Loukovaara et al., 2017).
In our study, we have shown that, in caerulein-induced AP, the level of oxidative stress-related indices (MDA, MPO) in the FIGURE 6 | Porous COS@SiO 2 nanocomposites suppressed the NOD-like receptor protein 3 (NLRP3)-mediated inflammasome and nuclear factor-kappa B (NF-κB) activation by regulating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway in mice with caerulein-induced AP. The protein levels of (A) the NLRP3 inflammasome and β-tubulin and (B) Nrf2, phospho-NF-κB (p-NF-κB), and histone in pancreas were analyzed by western blotting. Also, the protein levels of (C) the NLRP3 inflammasome and β-tubulin and (D) Nrf2, p-NF-κB, and histone in lungs were also analyzed by western blotting. The data are provided as the mean ± SEM (n = 6 per group). *p < 0.05; **p < 0.01; ***p < 0.001. FIGURE 7 | The protective effect of porous COS@SiO 2 nanocomposites in mice with caerulein-induced AP could be reversed by nuclear factor E2-related factor 2 (Nrf2) inhibition. ML385, a type of Nrf2 inhibitor, was used to determine whether it could reduce the protective effect of porous COS@SiO2 nanocomposites. (A) Pancreatic and lung samples from each group of mice were stained with H&E. Representative images of pancreas and lung are shown. Original magnification ×100. (B) Pancreatic histopathology scores were evaluated by Schmidt criteria for pancreatic tissue by edema, hemorrhage, necrosis, and inflammatory infiltration. (C) Lung histopathology scores were evaluated by alveolar neutrophils, interstitial neutrophils, hyaline membranes, proteinaceous debris, and alveolar septal thickening. (D) The level of serum amylase in each group mice. (E) The level of serum lipase in each group mice. The data are provided as the mean ± SEM (n = 6 per group). **p < 0.01; ***p < 0.001. pancreas and lung, the expression of the NLRP3 inflammasome, and the level of NF-κB protein in the pancreas and lungs as well as the levels of inflammatory cytokines (TNF-α, IL-6, IL-1β) in the serum and pancreatic tissues increase obviously, which could reflect the activation of oxidative stress and inflammation. As a result, we surmise that oxidative stress is the key to the pathogenesis of AP, which means that the increased ROS levels lead to an inflammatory cascade effect. In other words, COS@SiO 2 may be an effective treatment for AP because of its ability to inhibit oxidative stress. Furthermore, we explored the antioxidative stress mechanism of COS@SiO 2 .
Nrf2 is an important transcription factor in the leucine zipper family that regulates oxidative stress, and its specific receptor is Kelch-like ECH-related protein 1 (Keap1). Normally, Keap1 and Nrf2 are present in the cytoplasm in the form of compounds (Yamamoto et al., 2018;Qin et al., 2019). Under oxidative stress, Keap1 dissociates from Nrf2, and then Nrf2 is translocated into the nucleus and integrated with antioxidant response elements and regulated antioxidant proteins such as SOD, which could protect the body from ROS (Motohashi and Yamamoto, 2004).
In our study, western blot analysis showed that, combined with the treatment of COS@SiO 2 , the expression of Nrf2 protein was increased and the expression of NF-κB and the NLRP3 inflammasome protein was decreased in both lung and pancreatic tissue, which indicated that COS@SiO 2 might inhibit oxidative stress by upregulating the Nrf2 signaling pathway.
To prove whether Nrf2 plays an important role, we also used ML385, a type of Nrf2 inhibitor, in COS@SiO 2 -treated mice with AP. The results showed that the protective effects of porous COS@SiO 2 nanocomposites was reversed by inhibiting the Nrf2 signal.
CONCLUSION
In conclusion, we have demonstrated that porous COS@SiO 2 nanocomposites activate the Nrf2 signaling pathway to inhibit oxidative stress and reduce the production of NF-κB and NLRP3 and the release of inflammatory factors, thus blocking the systemic inflammatory response and ultimately ameliorating SAP and the associated lung injury.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.
ETHICS STATEMENT
All experiments involving animals were conducted under the principle for replacement, refinement, reduction (the 3Rs) and according to the legislation on the protection of animals and were approved by the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine (SYXK 2013-0050, Shanghai, China). | v3-fos-license |
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} | pes2o/s2orc | A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis
The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1 (FGFR1) in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis (OA). The goal of the current study was to investigate the effect of a novel non-ATP-competitive FGFR1 inhibitor, G141, on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA. G141 was screened and identified via cell-free kinase-inhibition assay. In the in vitro study, G141 decreased the mRNA levels of catabolic markers ADAMTS-5 and MMP-13, the phosphorylation of Erk1/2, JNK and p38 MAPK, and the protein level of MMP-13 in human articular chondrocytes. In the ex vivo study, proteoglycan loss was markedly reduced in G141 treated human cartilage explants. For the in vivo study, intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus (DMM) induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice. Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes, and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression.
Scientific RepoRts | 6:24042 | DOI: 10.1038/srep24042 that FGFR1 is a potential therapy target for treating OA and pharmacological FGFR1 antagonists may prevent cartilage degradation and/or improve cartilage homeostasis.
At present, several small molecules, such as PD173074, SU5402 and PD166866, have been used as FGFR tyrosine kinase inhibitors 10 . These inhibitors were designed based on their competitive inhibition of the ATP-binding domain of FGFR1. However, the ATP-binding sites are highly conservative among majority of the tyrosine kinases, these small molecules exhibit poor selectivity profile and their drug potency is easily affected by the high intracellular ATP concentration. The non-ATP-competitive inhibitors, which bind to the non-ATP binding site, possess the superior selectivity 11 . We have identified several non-ATP-competitive FGFR1 inhibitors via kinase inhibition assay of a chemical bank that containing 156 bisaryl-1, 4-dien-3-one compounds, and these inhibitors specifically target FGFR1 with weak effect on other tyrosine kinases 10,11 .
In this study, we analyzed the impact of a novel non-ATP-competitive FGFR1 inhibitor, G141, on FGF-2 or IL-1β -induced catabolic events in human articular chondrocytes and cartilage explants. Furthermore, we performed intra-articular injection of G141 into mouse knee joints in a DMM model of OA, to examine whether G141 inhibits cartilage degradation during OA. Our observations suggest that G141 reduces the catabolic events in FGF-2 or IL-1β treated human articular chondrocytes and human cartilage explants, and intra-articular injection of G141 protects articular cartilage from degradation after DMM in mice.
Results
G141 inhibits the activity of FGFR1 selectively in an ATP independent manner. Previously, we designed a library of bisaryl-1, 4-dien-3-one compounds to screen and identify FGFR1 inhibitors 11 . In this study, G141 was found to have high affinity for FGFR1 (IC 50 : 2.7 ± 0.54 μM) (Fig. 1A). To test the specificity of G141, we further measured the inhibitory effect of G141 on other receptor tyrosine kinases (RTKs), including VEGFR2, PDGFRβ , FGFR2 and FGFR3. As displayed in Fig. 1B, G141 showed a much lower activity against these RTKs compared to that of FGFR1. These data demonstrated that G141 selectively inhibited the activity of FGFR1.
Subsequently, we used caliper mobility shift assay to study the competitive relationship between ATP and G141. As shown in Fig. 1C, the increased concentration of ATP did not affect the rate of FGFR1 substrate phosphorylation at the various concentrations of G141. In other words, the inhibition of FGFR1 kinase activity by G141 did not depend on the concentrations of ATP. Thus, G141 suppressed FGFR1 in an ATP-independent manner.
G141 inhibits the expressions of catabolic markers in FGF-2 treated human articular chondrocytes.
As FGF-2 induced catabolic and anti-anabolic activities in human articular chondrocytes mainly via FGFR1, we first examined whether G141 could abolish the catabolic and anti-anabolic effect of FGF-2 on human articular chondrocytes. Primary human articular chondrocytes in monolayer were pre-incubated with G141 (5 μM, 10 μM) . G141 were tested with caliper mobility shift assay for RTKs inhibition, and the IC 50 values were calculated using conversion rates. The data were shown as a mean of 3 independent tests. (C) G141 inhibited FGFR1 through a mechanism that was independent of the concentrations of ATP. Selective ATP-competitive kinase assay of G141 with FGFR1 was carried out through caliper mobility shift assay. The conversion data were fitted with Graphpad for global fitting. followed by stimulation with FGF-2 (20 ng/mL) for 24 hours. Real-time qPCR was performed to examine the effects of G141 on markers of extracellular matrix synthesis and breakdown. The mRNA levels of ADAMTS-5 and MMP-13 were significantly increased after FGF-2 treatment. G141 treatment resulted in a marked reduction in these two catabolic markers in human articular chondrocytes treated with FGF-2. In contrast, after FGF-2 treatment, the mRNA levels of cartilage markers aggrecan and type II collagen were markedly reduced in human articular chondrocytes, which were partially recovered by G141 treatment (Fig. 2A).
Since FGFR1-Ras/PKCδ -Raf-MEK1/2-ERK1/2 signaling pathway plays a major role in the FGF-2-mediated stimulation of extracellular matrix degrading enzymes (ADAMTS-5 and MMP-13) in human articular chondrocytes 12 , we examined whether treatment with G141 could affect the signaling activity of ERK1/2 and protein levels of ADAMTS-5 and MMP-13 in human articular chondrocytes treated with FGF-2. We analyzed samples from total cell lysates of human articular chondrocytes cultured in the absence or presence of FGF-2 and G141, by immunoblotting with antibodies specific for phosphorylated and total ERK1/2, ADAMTS-5 and MMP-13. Following stimulation of FGF-2, activation of ERK1/2 MAPK signaling pathway was evident, and the protein levels of ADAMTS-5 and MMP-13 were largely induced. In the presence of G141, the degree of increase in the protein levels of phosphorylated ERK1/2, ADAMTS-5 and MMP-13 were attenuated in FGF-2-treated human articular chondrocytes (Fig. 2B).
IL-1β has been shown to play a prominent role in cartilage degradation by inhibiting ECM synthesis and promoting cartilage breakdown. We determined whether G141 treatment could affect the catabolic events in human articular chondrocytes initiated by IL-1β . G141 treatment resulted in a remarkable reduction in the IL-1β up-regulated mRNA levels of ADAMTS-5 and MMP-13, and a marked increase in the IL-1β down-regulated mRNA levels of aggrecan and collagen type II (Fig. 3A). Western blotting result showed that G141 attenuated the degree of increase in the protein levels of MMP-13 and phosphorylated JNK, ERK1/2 and p38 MAPK resulting from IL-1β treatment in chondrocytes (Fig. 3B,C).
G141 reduces the loss of proteoglycan in cultured human articular cartilage explants. To exam-
ine the effect of G141 on proteoglycan loss, we cultured human femoral head cartilage samples in the absence or presence of FGF-2 and G141 for 14 days. Safranin-O-fast green staining showed that FGF-2 (50 ng/ml) significantly induced proteoglycan depletion, which was partially rescued by G141 treatment (Fig. 4A). Culture medium was also collected to analyze the release of GAG using DMMB assay. G141 treatment markedly decreased the release of GAG into the culture medium from FGF-2-treated human femoral head cartilage samples (Fig. 4B).
G141 delays articular cartilage degradation in a mouse model of DMM. Surgical destabilization
of the medial meniscus (DMM) in mice is a well-established model of OA, which is characterized with articular cartilage degradation including abrasion of the articular surfaces, and up-regulated levels of pro-inflammatory cytokines (e.g. IL-1) and catabolic molecules (e.g. ADAMTS5, MMP13) 5,15 . This mouse OA model is commonly used to screen biological and pharmacological agents for OA treatment 13,14 . To examine the effect of G141 on the development of OA induced by injury, we performed DMM surgery in the right knee joints of 10-week-old male C57 mice. After DMM surgery, the mice received twice weekly intra-articular injection of 10 μM G141 in PBS or PBS alone for 2, 4 and 8 weeks. Total RNA and protein from mouse knee joints 2 weeks following DMM or sham surgery were extracted. The mRNA levels of IL-1β and FGF-2 were significantly increased in joints after DMM surgery compared with that in sham operated control joints. Intra-articular injection of G141 resulted in a marked reduction of the mRNA levels of IL-1β and FGF-2 in joints after DMM surgery (see Supplementary Fig. S1A). The level of phosphorylated FGFR1 was increased in joints after DMM surgery, which was attenuated by G141 treatment (see Supplementary Fig. S1B). These data demonstrated that G141 inhibited the activity of FGFR1 and IL-1β expression in mouse knee joints after DMM surgery. Safranin O-fast green staining results demonstrated significant reduction in proteoglycan loss, cartilage destruction and loss of articular chondrocyte cellularity in mice treated with G141 at 4 and 8 weeks after DMM surgery compared to the DMM mice treated with vehicle ( Fig. 5A,C). OARSI histologic scoring system was applied to quantitatively analyze the cartilage degradation after DMM surgery. The summed OARSI score demonstrated that G141-treated mice had a significantly lower score than the vehicle-treated mice at 4 and 8 weeks following DMM surgery. The summed OARSI score in the G141-treated mice at 8 weeks was increased compared to the score at 4 weeks. These findings suggested that intra-articular injection of G141 did not completely prevent but delayed cartilage degradation in mouse knee joint after DMM surgery (Fig. 5B,D).
G141 attenuates chondrocyte hypertrophy in knee joints of DMM mice.
To explore the mechanisms underlying the delayed progression of cartilage degradation in G141-treated mice with DMM, we performed immunohistochemical staining to examine the expressions of type X collagen and MMP-13, marker gene for hypertrophic articular chondrocytes. The results showed that G141 treatment significantly decreased the number of MMP-13 ( Fig. 6A,E) and type X collagen (Fig. 6B,E) positive cells by 66% and 52%, respectively, in the knee joints 8 weeks after DMM surgery compared to vehicle treatment. These results suggested that local intra-articular injection of G141 reduced articular cartilage damage, at least in part, through inhibition the hypertrophy process of articular chondrocytes.
Discussion
OA is a degenerative disease characterized with cartilage degradation, synovial inflammation and dysregulated subchondral bone remodeling. The mechanisms underlying OA is not well understood, and there is currently few effective treatment to prevent the development of OA, joint replacement is usually carried out in patients with severe OA. It is urgent to find effective therapies to prevent or slow down the progression of OA.
Decades of studies have demonstrated that fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate the development and maintenance of cartilage, and therefore play vital roles in cartilage homeostasis and OA development 5 . We previously demonstrated that conditional deletion of Fgfr1 in mature mouse articular chondrocytes delays the progression of cartilage degradation 8 . Here, we revealed that pharmacologically antagonize FGFR1 using G141, a novel non-ATP-competitive inhibitor, can attenuate the development of OA.
Four members of the FGF family, FGF-2, FGF-8, FGF-9, and FGF-18, have been reported as key factors regulating cartilage degradation and homeostasis. Recently, studies have begun to explore the potential therapeutic effect of these biological agents. FGF-18 is a well-established anabolic growth factor that induces cartilage ECM formation 18,19 . FGF-8 has been identified as a catabolic factor in rat and rabbit articular cartilage 20 . Local delivery of FGF-9 in a rat meniscal tear model of OA has been found to provide significant beneficial effect on the damaged cartilage 21 . On the other hand, results from studies investigating the therapeutic effects of FGF-2 have been conflicting. In human articular cartilage, FGF-2 plays a degenerative role in cartilage homeostasis 9,22 . However, in mouse joints, FGF-2 has been identified as an anabolic mediator as intra-articular injection of FGF-2 can delay cartilage degradation 23,24 . These discrepancy are thought due to distinctive expression patterns of FGFR1 and FGFR3 in mouse and human articular cartilage, and to the fact that FGF-2 mediates anabolic processes in mouse cartilage via its binding to FGFR3, while mediates catabolic activities via FGFR1 in human cartilage 5 .
It is reported that FGF-2 binds to FGFR1 to activate the FGFR1-Ras/PKCδ -Raf-MEK1/2-ERK1/2 signaling pathway, which leads to increased expressions of extracellular matrix degrading enzymes and down-regulated aggrecan synthesis 12 . Yan and colleagues found that blockade of Ras, PKCδ , and MAPK pathway in chondrocytes abolishes FGF-2-mediated catabolic events in vitro and ex vivo 12 . In present study, we, for the first time, provide evidence showing that a novel FGF-2/FGFR1 antagonist, G141, prevents cartilage degradation in a mouse model of OA.
Over the past three decades, several FGFR1 inhibitors, such as PD173074, SU5402 and PD166866 have been developed as candidates for the treatment of FGF signaling related diseases 11 . Most of these compounds are ATP-competitive FGFR1 inhibitors, and majority of them have failed to enter clinical application for their low-specificity and toxicity. The non-ATP-competitive inhibitors, which bind to the non-ATP binding site, possess the superior selectivity 11 . In this study, we identified a novel FGFR1 inhibitor, G141, via cell-free kinase-inhibition assay. Unlike PD173074 and other ATP-competitive FGFR1 inhibitors, G141 inhibited the activity of FGFR1 selectively in an ATP independent manner. We found that G141 had significantly stronger inhibitory effect on FGFR1 compared to that of other RTKs, such as VEGFR2, PDGFRβ , FGFR2 and FGFR3, and the inhibition of FGFR1 kinase activity by G141 did not depend on the concentration of ATP.
Next, we demonstrated that G141 inhibited the FGF-2 or IL-1β -induced upregulation of ADAMTS-5 and MMP-13 and downregulation of aggrecan and collagen type II, in human articular chondrocytes, via its inhibition on the activities of ERK1/2, JNK and p38 MAPK signaling pathway. As these 3 pathways have been found to be directly involved in the upregulation of MMP-13 in articular chondrocytes after stimulation with IL-1β or FGF-2 25,26 . Selective inhibition of p38 and ERK1/2 MAPK pathway has also been found to attenuate cartilage degradation in rabbit OA model and in ex vivo organ culture model treated with IL-1β 27,28 .
We further demonstrated that G141 inhibited proteoglycan degradation in FGF-2-treated human cartilage explants cultures. Most importantly, we found that the severity of cartilage degradation in a mouse model of surgically induced OA was attenuated by intra-articular injection of G141. To further investigate the cellular mechanism of the effects of G141 on articular cartilage homeostasis and OA, we examined chondrocyte hypertrophy, as chondrocyte hypertrophy can result in increased metabolic activity of articular chondrocytes and trigger unbalanced cartilage homeostasis favoring degenerative changes 29 . We found that G141 treatment reduced the expressions of type X collagen and MMP-13, most widely used markers for identifying hypertrophic chondrocytes 14,30 , in comparison to mice treated with vehicle after DMM surgery. These findings revealed that intra-articular injection of G141 prevented articular chondrocytes from hypertrophy in this surgical model of OA, which contributed to the inhibitory effect of G141 on OA development. The inhibitory effect of G141 on chondrocyte hypertrophy is consistent with our previous findings showing that conditional knockout of Fgfr1 in chondrocytes decreased chondrocyte hypertrophy in articular cartilage 8 .
Chondrocyte apoptosis is believed to play an important role in the pathogenesis and progression of OA 16,17 . Inhibition of chondrocyte apoptosis is shown to alleviate the extent of OA in a rabbit model of surgically induced OA 31 . FGF signaling pathway is highly associated with chondrocyte apoptosis 32 . Gain-of-function mutation of FGFR3 promotes chondrocyte apoptosis in thanatophoric dysplasia (TD) mice 33 . FGF-2 transgenic mice exhibit chondrodysplasic phenotype resulting from both reduced proliferation and increased apoptosis of growth plate chondrocytes 34 . FGF18 markedly reduces chondrocyte apoptosis and enhances the repair response of cartilage following cartilage insult 35 . In current study, we showed that pharmacologically inhibiting FGFR1 by G141 decreased chondrocyte apoptosis partially through its down-regulation of cleaved caspase 3 in a surgically induced mouse OA model. Our findings demonstrated that G141 positively maintains cartilage homeostasis by preventing chondrocyte apoptosis.
In conclusion, in this study, we found a novel non-ATP dependent specific FGFR1 inhibitor, G141, and for the first time, we showed that pharmacologically antagonize FGFR1 using G141 protects the knee joint cartilage from degradation in a DMM model of mouse OA, probably by suppressing the production of matrix-degrading enzymes MMP-13 and ADAMTS-5 and preventing articular chondrocytes from hypertrophy and apoptosis.
G141 Synthesis and kinase inhibition assays. G141 (1-methyl-4-(4-methoxyphenyl) pyrrolo (spiro-
2′ ]-5′ -(4-methoxyphenyl)methylidenecy-clopentanones) was synthesized and identified via kinase inhibition assay as previously reported 11,36 . Briefly, a mixture of a bisaryl-1, 4-dien-3-one analogue (0.348 g, 1 mmol), acenaphthenequinone (0.182 g, 1 mmol), and sarcosine (0.089 g, 1 mmol) was dissolved in methanol (10 mL) and refluxed for 1 h. After completion of the reaction as evident from thin-layer chromatography (TLC), the mixture was cooled to room temperature and poured into water (50 mL). The precipitated solid was filtered and washed with water to obtain crude product, then purified by silica-gel column chromatography (petroleum ether -ethyl acetate) to give the pure product as yellow solid. The general chemical structure of G141 is shown in Fig. 1A. The kinase inhibition assay was performed using Caliper Mobility Shift Assay on EZ Reader (Caliper Life Sciences, MA) with ATP concentration at its Km value (262 μM). The compounds were tested in duplicate at 10 concentrations (5 nM-100 μM) to determine the IC 50 . In the experiments for testing the relationship between the compounds and ATP, the concentration of the substrate was constant, while the concentrations of ATP was set at 5000, 2500, 1250, 625, 313, 156, 78, and 39 μM. The global competitive inhibition fit for the compounds was performed based on percent conversion = (Vmax*X)/{km*[(1 + I/Ki)n] + X},where X is the ATP concentration, and n is the Hill coefficient.
Isolation and culture of human articular chondrocytes. Human articular chondrocytes and cartilage explants were isolated from articular cartilage tissue harvested from patients undergoing total joint replacement surgery at Daping Hospital (Chongqing, China) because of traffic accident. Written informed consent was obtained from all subjects. Samples were collected according to protocols approved by the Institutional Review Board and Ethics Committee of Daping Hospital and the methods were carried out in accordance with the approved guidelines. Human articular chondrocytes were isolated from the cartilage according to previously described methods 8 . Isolated chondrocytes were plated into 6-well plates at a density of 1 × 10 6 cells/well and cultured in Dulbecco's modified Eagle's medium/F-12 (HyClone) containing 10% fetal bovine serum (Gibco) and 50 units/ml of penicillin and streptomycin (HyClone). At 90% confluence, human chondrocytes were cultured under serum-free conditions for 24 hours. The chondrocytes were incubated with G141 (5 μM and 10 μM) for 1 hour before treatment with 20 ng/ml FGF-2 (Peprotech) or 20 ng/ml IL-1β (Peprotech) for 24 hours.
Human articular cartilage explants culture. For cartilage explants culture, full thickness femur head cartilage tissue was cut into pieces of ~2 mm-3 mm. Following 48 hours of culture in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine serum and 50 units/ml of penicillin and streptomycin, explants were treated with FGF-2 (50 ng/ml) and G141 (5 μM and 10 μM) for 14 days under serum-free conditions (with ITS) 24 . Following 14 days culture, the medium was collected and the explants were fixed in 4% paraformaldehyde.
Dimethylmethylene blue assay. The DMMB dye binding assay was performed to analyze glycosaminoglycan (GAG) release of the cultured explants as previously described 13,37 . Briefly, 250 μl of DMMB reagent was added to 40 μl of culture medium and the absorbance was measured at 525 nm. Using different concentrations of chondroitin sulfate (Sigma-Aldrich) to plot a standard curve and then the amount of GAG released was determined by this standard curve. GAG released into the medium was normalized as mass of GAG per milliliter (ml) of culture medium.
Western blotting. Human articular chondrocyte cultures were extracted using RIPA lysis buffer containing protease inhibitors (Roche). Protein was extracted from mouse whole joints following removal of the skin and muscle bulk. Tissue was snap-frozen and then extracted using RIPA lysis buffer. Equal amount of protein samples (30 μg) were dissolved by 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred Scientific RepoRts | 6:24042 | DOI: 10.1038/srep24042 onto a polyvinylidene difluoride membrane. After being blocked with 5% nonfat milk in Tris buffered saline-Tween buffer, the membrane was probed with primary antibodies specific for phosphorylated and total ERK1/2 (CST), p38 (CST), JNK (CST), MMP-13 (Millipore), ADAMTS-5 (Abcam), and phosphorylated and total FGFR1 (Santa) followed by secondary antibodies. The signal was detected using chemiluminescent (Pierce) according to the manufacturer's instruction. The antibody specific for β -actin (Sigma) was applied to normalize the protein expression levels.
Mouse Surgically induced model of OA.
Animal experiments were performed according to protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (Chongqing, China) and the methods were carried out in accordance with the approved guidelines. Destabilization of the medial meniscus (DMM) surgery was made on the right knee joints of 10-week-old male C57BL/6 mice, as previously described 38 . Sham surgery were performed with medial capsulotomy only. All mice were then allowed to move freely and take food and water ad libitum after surgery.
Intra-articular injection of G141. DMM mice received intra-articular injection of 10 μl of 10 μM G141 in PBS twice a week for 4 and 8 weeks immediately after DMM surgery. The control group received intra-articular injection of 10 μl of PBS only. At 4 and 8 weeks post DMM, animals were sacrificed and the knee joints were harvested and fixed in 4% paraformaldehyde.
Histology. Knee joints of the mice were decalcified in 20% formic acid, and embedded in paraffin. 5-mm thick sections were cut sagittally through the medial knee joints and stained with stained Safranin O-fast green to assess cartilage destruction as previously described 8 . The Osteoarthritis Research Society International (OARSI) recommended subjective scoring system was used to histologically grade the severity of the cartilage destruction 39 . The score for an individual joint was expressed as a summed score for the medial femora and medial tibiae within each joint separately.
Immunohistochemistry. Immunohistochemistry was performed on sagittal sections of paraffin-embedded knee joints. After being deparaffinized using xylene and deprived of endogenous peroxidase activity with 3% H 2 O 2 , and antigen retrieval with 0.1% trypsin, sections were incubated with rabbit anti-MMP-13 polyclonal antibody (1:200 dilution; Abcam), rabbit anti-type X collagen polyclonal antibody (1:200 dilution; Millipore), rabbit anti-cleaved caspase-3 polyclonal antibody (1:100; Boster) overnight at 4 °C. After warming and cleaning, sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at 37 °C. Finally, sections were stained with diaminobenzidine (DAB) kit and counterstained with methyl green. TUNEL staining. In situ cell death detection kit (Roche) was used to detect apoptotic articular cartilage chondrocytes according to the manufacturer's instruction.
Statistical analysis.
The numeric data were expressed as the mean ± SD. Differences between 2 groups were evaluated using Student's t-test. Analysis of variance (ANOVA) was used for comparisons of 3 or more groups followed by Tukey post hoc test (SPSS program version 13.0). P < 0.05 were considered statistically significant. | v3-fos-license |
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} | pes2o/s2orc | Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence
Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles.
Introduction
The translocation of the transactivator of transcription (Tat) protein of the human immunodeficiency virus type 1 (HIV-1) into cells depends on a sequence that contains eleven amino acids (amino acid sequence: YGRKKRRQRRR) [1]. Using this basic amino acid-rich sequence as a guide, many small peptides were designed that possess a similar membrane penetrating potential [2]. These peptides, dubbed cell-penetrating peptides (CPPs), may be amphipathic, hydrophobic or cationic [3]. CPPs can facilitate the delivery of cargoes, including DNAs, RNAs, proteins and nanoparticles, into living cells [2,4,5]. More than 843 varieties of CPPs have been catalogued on a CPP site (http://crdd.osdd.net/ raghava/cppsite/) [6].
Although CPPs have recently gained much attention as powerful tools to introduce exogenous molecules into cells, their cellular uptake pathways and subsequent intracellular trafficking are still not fully understood. Studies have suggested that CPPs utilize multiple pathways for cellular entry [7][8][9]. Endocytosis and direct membrane translocation appear to be two major uptake mechanisms for CPPs. Endocytosis is an energy-dependent pathway that includes two major categories: phagocytosis involving uptake of large particles, and pinocytosis involving solute uptake [7]. Pinocytosis can be further subdivided into macro-pinocytosis, and clathrin-dependent, caveolin-dependent and clathrin/caveolin-independent pathways [10]. Endocytosis involves binding to membranes, accumulation in membrane-sunken vesicles, transfer to early and late endosomes, and fusion to become late endosomes/lysosomes [11][12][13]. The progress from binding to transport into early endosomes can be accomplished within 30 min [12,13]. Direct membrane translocation, also known as direct cell penetration, includes a variety of energyindependent pathways, such as pore formation, inverted micelle formation, carpet-like alternations and membrane thinning [7,9]. Various physical and pharmacological endocytic inhibitors can be used to identify pathways of CPP-mediated transduction. For instance, low temperature (4uC) treatment arrests all energydependent movement across the cell membrane [14]. The endocytic inhibitor cytochalasin D (CytD), an F-actin polymerization disrupter, perturbs endocytic processes that involve clathrin-, caveolae-dependent endocytosis and macropinocytosis [15,16]. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) specifically inhibits macropinocytosis by inhibiting Na + /H + exchange proteins [16,17]. Filipin inhibits lipid raft dependent caveolae endocytosis, while nocodazole inhibits clathrin-dependent endocytosis [15,16]. The destination of materials internalized by endocytosis is acidic lysosomes, where proteins and other molecules may be degraded by hydrolytic enzymes [18].
Since endocytosis is one of the primary membrane translocation mechanisms of CPPs, escape from endocytic vesicles is essential to preserve biological activity of endocytosed cargoes [7][8][9]11]. Chloroquine, a lysosomotropic agent, is commonly used to circumvent this problem [19,20]. Alternatively, peptides with certain sequences can be effective. For instance, the penetration accelerating sequence (Pas) is a synthetic peptide (FFLIPKG) derived from the cleavable sequence (GKPILFF) of cathepsin D enzyme, a lysosomal aspartyl protease [21,22]. Addition of Pas to octa-arginine (R8), denoted as PasR8, enhances the efficiency of intracellular delivery of bioactive peptides by promoting endosomal escape [21].
The aims of this study were to (1) demonstrate Pas nonaarginine (PR9)-mediated cellular internalization of QDs, (2) elucidate the cellular uptake mechanism and subcellular localization of PR9/QD complexes, (3) identify the molecular mechanisms of intracellular trafficking of PR9/QD complexes and (4) identify the physical properties of PR9 and PR9/QD complexes that affect uptake. To achieve these goals, we synthesized PR9 and examined transduction pathways and intracellular shuttling of PR9/QD complexes using flow cytometry and live cell imaging. To identify the cellular uptake mechanisms of PR9 and PR9/QD complexes, pharmacological and physical inhibitors were used to block specific uptake pathways. Transmission electron microscopy (TEM) was used to monitor endocytic progress. Certain physical properties of PR9 and PR9/QD complexes were characterized using zeta-potential analysis and circular dichroism (CD) spectroscopy. The toxicity of PR9 and PR9/QD complexes was assessed using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) dye reduction assay. This study provides valuable mechanistic insights into how PR9s promote escape from endocytic vesicles and provides a basis for the design of optimized cargo delivery in cells.
Protein Transduction and Mechanistic Assay
To analyze the kinetics of protein transduction, A549 cells were seeded at a density of 1610 5 per 35-mm petri dish and then incubated overnight in 1 ml of complete growth medium [33]. Six mM CPPs was mixed with 100 nM QDs (i.e., at a molecular ratio of 60) at 37uC for 2 h to form CPP/QD complexes. Cells were washed with 1 ml of phosphate buffered saline (PBS) twice. The cells were then treated with PBS as a control, 100 nM QDs only, or CPP/QD complexes in RPMI 1640 medium supplemented with 1% serum at 37uC for a period of 0-6 h. Cells were washed with PBS five times to remove free CPP/QD complexes before analysis.
To determine the cellular uptake mechanism of PR9/QD complexes, cells were treated with PBS as a negative control, 100 nM QDs alone, or PR9/QD complexes prepared at a molecular ratio of 60 in RPMI 1640 medium supplemented with 1% serum at 37uC for 30 min in the absence or presence of various pharmacological and physical inhibitors [16,33]. Cells were incubated for 30 min at 4uC to arrest energy-dependent movement across the cell membrane [14], then treated with 100 nM QDs or PR9/QD complexes at 4uC for 30 min. After this treatment, the cells were washed with PBS twice to remove free QDs or PR9/QD complexes, followed by flow cytometric analysis. The influence of modulators on uptake processes was investigated by treating cells with 100 nM QDs or PR9/QD complexes in the absence or presence of 100 mM EIPA, 10 mM CytD, 5 mg/ml filipin, 10 mM nocodazole at 37uC for 30 min. Non-transduced QDs or PR9/QD complexes were removed from cell surface by washing with PBS five times. To assess lysosomal escape, cells were treated with QDs or CPP/QD complexes as described above in the absence or presence of 25 mM chloroquine for 2 h. Free QDs or CPP/QD complexes were excluded by washing with PBS five times before uptake was determined by flow cytometry.
Flow Cytometric Analysis
Cells were seeded at a density of 1610 5 per well in 24-well plates and incubated overnight in 500 ml/well of complete culture medium. Cells in the control and experimental groups treated with QDs or CPP/QD complexes were harvested and counted using a Cytomics FC500 flow cytometer (Beckman Coulter, Fullerton, CA, USA) [33]. To detect green fluorescent proteins (GFP), excitation was set at 488 nm and emission at 515-545 nm with a FL1 filter. Data were analyzed using CXP software (Beckman Coulter). Results are expressed as the percentage of the total cell population that displays fluorescence.
Subcellular Colocalization Analysis
Endocytic vesicles appear within 30 min, and transport of vesicles toward lysosomes is completed in approximately 2 h [12,13]. To examine subcellular localization of the delivered PR9/ QD complexes, cells were treated with 100 nM green fluorescent QDs alone or CPP/QD complexes in RPMI medium with 1% serum at 37uC for either 30 min or 2 h. Cells were washed with PBS five times to remove free CPP/QD complexes, followed by staining with organelle-specific fluorescent trackers [16,33]. Treatments with organelle trackers included 16.2 mM Hoechst 33342 (Invitrogen; in blue) at 37uC for 40 min, Texas Red-X phalloidin (Invitrogen; in red) at 37uC for 20 min, 50 nM LysoTracker DND-99 (Invitrogen; in red) at 37uC for 30 min, 50 nM MitoTracker Deep Red FM (Invitrogen; in red) at 37uC for 30 min, and 1 mM ER-Tracker Red (Invitrogen; in red) at 37uC for 30 min to visualize subcellular colocalization with nuclei, actins, lysosomes, mitochondria and endoplasmic reticula (ER), respectively. To investigate intracellular trafficking of PR9/QD complexes, cells were treated with PR9/QD complexes at 37uC from 30 min to 5 h, and then stained with 1,0006 diluted rabbit anti-human early endosome antigen 1 protein (EEA1) antibody and goat Alexa Fluor 647-conjugated anti-rabbit antibody fragment (Cell Signaling Technology, Danvers, MA, USA) at 37uC for 12 and 2 h, respectively, to visualize subcellular colocalization with early endosomes.
Confocal and Fluorescent Microscopy
Fluorescent and bright-field live cell images were recorded using a BD Pathway 435 bioimaging system (BD Biosciences, Franklin Lakes, NJ, USA) equipped with Olympus 206 and 606 oil objectives (Olympus, Tokyo, Japan) [33]. This system includes both confocal and fluorescent microscopy sets. Excitation filters were set at 377/50 nm, 482/35 nm and 543/22 nm for blue (BFP), GFP and red (RFP) fluorescent proteins, respectively. Emission filters were set at 435LP (long-pass), 536/40 nm and 593/40 nm for BFP, GFP and RFP, respectively. Bright-field microscopy was used to assess cell morphology. Intensities of fluorescent images were quantified using BD Pathway software (BD Biosciences).
Intracellular Trafficking of Different PR9/cargo Complexes
A549 cells were seeded at a density of 1610 4 per well in 96-well plates. Cells were treated with PR9/QInP complexes prepared at a molecular ratio of 30 in RPMI 1640 medium supplemented with 1% serum at 37uC from 30 min to 5 h (i.e., all PR9/QD complexes were prepared at a molecular ratio of 60, while all PR9/QInP complexes were formed at a molecular ratio of 30). The solution was then removed, and the cells were washed three times with PBS. The cells were stained with Hoechst 33342 and LysoTracker DND-99 followed by observation using a BD Pathway 435 bioimaging system.
To determine whether PR9 can deliver functional genes, cells were treated with either 3 mg the pEGFP-N1 plasmid DNA alone (control) or a pEGFP-N1 plasmid DNA mixed with PR9 (27 nmole) at a nitrogen (NH 3 + )/phosphate (PO 4 -) (N/P) ratio of 3 in RPMI 1640 medium supplemented with 1% serum for 30 min to 24 h at 37uC. The solution was then removed, and the cells were washed three times with PBS. The cells were supplemented with 100 ml full growth medium and incubated at 37uC for 48 h. After two days, the cells were stained with Hoechst 33342 and observed using a BD Pathway 435 bioimaging system.
Transmission Electron Microscopy (TEM)
Morphological examination of PR9/QD-and PR9/QD-transduced cells was performed using a Hitachi H-7500 transmission electron microscope (Hitachi, Tokyo, Japan). PR9/QD complexes were dropped on Formvar/carbon coated copper grids with 300 mesh and dried at room temperature. Cells were treated with PR9/QD complexes, washed five times with PBS to remove free CPP/QD complexes, and then pre-fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h. The cells were washed with 0.1 M phosphate buffer twice at a 15-min interval. The cells were post-fixed with 1% osmium for 1 h and washed with 5% sucrose. The cells were stained with or without 2% uranyl acetate, dehydrated in a graded ethanol-acetone series, embedded in Spurr's resin (Electron Microscopy Sciences, Hatfield, PA, USA) [42], and then sliced using a Ultracut-R ultramicrotome (Leica, Wetzlar, Germany). Finally, the cell sections were immobilized on single-well copper grids for TEM analysis.
Zeta-potential and Particle Size Measurements
QDs (100 nM), PR9 (6 mM) or PR9/QD complexes at a molecular ratio of 60 was dissolved in double deionized water at pH 7 or 5, representing physiological or endosomal conditions, respectively [43,44]. Each solution was temperature-equilibrated at 25uC for 2 min in a zeta cell. Sizes and zeta-potentials of complexes were measured using a Zetasizer Nano ZS and analyzed using Zetasizer software 6.30 (Malvern Instruments, Worcestershire, UK) [34,45].
Circular Dichroism (CD) Spectroscopy
Twenty mM PR9 was dissolved in doubly deionized water with pH 7 or 5 at room temperature. CD spectra of PR9 were analyzed in a cylindrical quartz cuvette with a 1 mm path-length using a Jasco J-715 CD spectrometer (Jasco, Easton, MD, USA) at a scan speed of 50 nm/min [46]. The contents of secondary structures were calculated using CDPro software.
Cytotoxicity Assay
Cells were seeded at a density of 1610 4 per well in 96-well plates and incubated overnight in 200 ml/well of full growth medium. Cells were treated with PBS as a negative control, treated with 100% dimethyl sulfoxide (DMSO) as a positive control or treated with 25 nM-5 mM QInP in RPMI medium with 1% serum at 37uC for 24 h, washed with PBS, and cultured in full growth medium at 37uC for 48 h. The cells were treated with various concentrations of PR9, individual or combinations of 6 mM PR9, 100 nM QDs, 25 mM chloroquine and 15 mM QInP, as indicated, in RPMI medium with 1% serum at 37uC for 24 h. The cells were washed with PBS and cultured in full growth medium at 37uC for 24 h. Cell viability was measured using the MTT assay [16,47].
Statistical Analysis
Data are expressed as mean 6 standard deviation (SD). Mean values and SDs were calculated from at least three independent experiments of triplicates per treatment group. Comparisons between the control and treated groups were performed by the Student's t-test using levels of statistical significance of P,0.05 (*) and 0.01 (**), as indicated.
PR9 Displays Cellular Internalization at a Relatively Longer Time
To investigate the efficiency of PR9 mediated transport of QDs into cells, a time course experiment was conducted in human A549 cells. QDs were premixed with or without CPPs SR9, HR9 or PR9 for 2 h, added to cells for up to 6 h, and then analyzed by flow cytometry. At 1 h, the extent of uptake displayed the following order: HR9/QD . SR9/QD . PR9/QD (Figure 1), and uptake of all CPP/QD complexes was essentially complete by 4 h. The differences in uptake kinetics may reflect the involvement of different uptake mechanisms.
Endocytosis is the Main Pathway for Intracellular Delivery of PR9/QD Complexes
Various pharmacological and physical inhibitors were used to shed light on the uptake mechanism of PR9/QD complexes. A549 cells were treated with PBS (negative control), QDs alone or PR9/ QD complexes in the absence or presence of endocytic inhibitors, followed by flow cytometric analysis. The fraction of cells containing PR9/QD complexes (positive cells) was reduced to 47.5% by incubation at 4uC, to 66.2% by CytD, to 60.7% by filipin, and to 71.7% by nocodazole. In contrast, EIPA treatment (a macropinocytosis inhibitor) did not decrease uptake (Figure 2A). These results indicate that classical energy-dependent endocytosis is the major route for cellular internalization of PR9/QD complexes. To further validate our findings, cells were treated with QDs or CPP/QD complexes with a lysosomotropic agent chloroquine. Chloroquine increased the cellular delivery of SR9/ QD and PR9/QD complexes, but not HR9/QD complexes ( Figure 2B). This further supports the notion that cellular internalization of PR9/QD complexes involves endocytosis.
Colocalization of PR9/QD Complexes with Actin Filaments and Lysosomes
To determine the subcellular localization of CPP/QD complexes, cells were treated with QDs or CPP/QD complexes and then stained with organelle-specific fluorescent markers, including Hoechst 33342, Texas Red-X phalloidin, LysoTracker DND-99, MitoTracker Deep Red FM and ER-Tracker Red to visualize nuclei, actins, lysosomes, mitochondria and ER, respectively. Merged images revealed that some PR9/QD complexes colocalized with actins ( Figure 3A) and lysosomes ( Figure 3B) at 30 min and 2 h. SR9/QD complexes colocalized with actins at 2 h ( Figure 3A) and with lysosomes ( Figure 3B) at 30 min and 2 h. HR9/QD complexes did not colocalize with any organelles examined ( Figure 3A-D). Furthermore, CPP/QD complexes were not associated with mitochondria ( Figure 3C), ER ( Figure 3D) or nuclei ( Figure 3A-D) at any time. These results indicate that cellular internalization of PR9/QD complexes involves classical endocytosis, in agreement with the results presented in Figure 2.
Intracellular Trafficking of Different PR9/cargo Complexes
The data indicate that PR9/QD complexes are endocytosed. We investigated intracellular trafficking and the fate of the complexes over a period of five hours using signal colocalization of fluorescent CPP/QD complexes with organelle-specific markers.
Cells were treated with PR9/QD complexes for 30 min, 1, 2, 3, 4, and 5 h, followed by staining with anti-human EEA1 antibody, LysoTracker DND-99 and Hoechst 33342. CPP/QD complexes and early endosomes showed limited colocalization at 30 min, and colocalization gradually increased from 1 to 5 h ( Figure 4A). Figure 4B indicates that PR9/QD complexes enter lysosomes (see insets 1, 2, 3 obtained at 30 min), and most complexes are trapped in lysosomes at 2-3 h (see insets 1, 2, 3 at 2-3 h). Partially overlapping images between PR9/QD complexes and lysosomes were obtained following longer treatments (insets 1-3 obtained at 4 and 5 h of Figure 4B), suggesting an escape of PR9/QD complexes from the acidic vesicles. High green fluorescent intensity was noticed at the periphery of the nucleus following the longer incubation, indicating an accumulation of PR9/QD complexes in this region (GFP channel at 5 h of Figure 4B and 4 h of Figure 4C). Colocalization intensity of PR9/QD complexes and lysosomes was maximal following a 2 h incubation, and then started to decrease ( Figure 4B and D), indicating lysosomal escape. Figure 4C and E show trafficking of PR9/QD complexes toward nucleus. The complexes spread out near plasma membrane at an early stage and later condense near the nucleus. Localization of PR9/QD complexes in the nucleus was observed from 3 to 5 h ( Figure 4B, C and E). Together, these data indicate that after being endocytosed, CPP/QD complexes move sequentially from endosome to lysosome to cytoplasm to nucleus.
To study intracellular trafficking of different PR9/cargo complexes, A549 cells were treated with PR9/QInP complexes for 30 min to 5 h, followed by staining with LysoTracker DND-99 and Hoechst 33342. Trafficking routes of PR9/QInP complexes were recorded at different times. Merged confocal images reveal a colocalization of PR9/QInP complexes with lysosomes and nucleus ( Figure 5A). Most PR9/QInP complexes were trapped in lysosomes at 2 h, but exited the lysosomes and translocated toward the nucleus at 2-5 h ( Figure 5A). To demonstrate delivery of PR9/cargo complexes to the nucleus, a functional gene assay was carried out. Cells were treated with pDNA alone or PR9/ DNA complexes for 30 min, 1-5 and 24 h, followed by staining with Hoechst 33342. After a 24 h incubation, no fluorescence was detected in the cells treated with pDNA only ( Figure 5B). In contrast, green fluorescence was apparent in cells treated with PR9/DNA complexes at later times ( Figure 5B and C), indicating that plasmid DNA delivered by PR9s can be expressed. These results demonstrate that different cargoes delivered by PR9 follow a similar trafficking pattern in cells. The functional gene assay confirmed the colocalization of cargoes with the nucleus at a later stage.
TEM
The morphology of PR9/QD complexes and PR9/QDtransduced cells was observed using a Hitachi TEM. PR9/QD complexes were spherical with an average diameter of 2.060.1 nm (Figure 6A left). Electron-dense cores of PR9/QD complexes were observed associated with the membranes of PR9/ QD-transduced cells following a 30-min incubation ( Figure 6A middle). PR9/QD complexes were observed in a lysosome after a 2-h incubation (Figure 6A right). The endocytic progress of PR9/ QD complexes was indicated by the appearance of labeled macropinosomes and lysosomes in PR9/QD-transduced cells ( Figure 6B). PR9/QD complexes on the membrane were observed enclosed in a clathrin-coated pit ( Figure 6C), and later enclosed within a vesicle (early endosome) ( Figure 6D). PR9/QD complexes-containing macropinosomes, which ultimately fuse with lysosomes ( Figure 6E), were noted in lamellipodia-like membrane protrusions ( Figure 6F). These results indicate that PR9 transports QDs into cells by an endocytic pathway.
Zeta-potential, Particle Size Measurements and CD Spectroscopy
To characterize the physical properties of PR9 and PR9/QD complexes, zeta-potential, particle size and secondary structure of PR9 were determined. PR9 and PR9/QD complexes were 16.961.4 and 17.162.6 nm at pH 7 and 5, respectively, while PR9/QD complexes were larger than QDs ( Figure 7B). These results agree with our recent report that the electrostatic interactions of CPP/cargo complexes can be a predictor of transduction efficiency within the charge range tested [48], emphasizing the significance of zeta-potential for the transduction of PR9/QD complexes. To determine whether conformational changes of PR9 in acidic vesicles could be a factor in lysosomal escape, the secondary structure of PR9 was analyzed using CD spectroscopy. PR9 conformations were very similar at pH 7 and 5 ( Figure 7C). Collectively, these results indicate that zeta-potentials of CPP/QD complexes are a key factor of transduction efficiency [48], reflecting the importance of electrostatic interactions of PR9/ QD complexes with plasma membranes and endomembrane system.
Cytotoxicity
The MTT assay was used to determine the effect of PR9mediated cargo delivery on cell viability. Cells were treated with either individual or a combination of PR9s, QDs, chloroquine and QInP, as indicated. None of the materials used in this study caused cytotoxicity (Figure 8), indicating that arginine-rich PR9 would be a safe vehicle to carry cargo into cells.
Discussion
In this study, we demonstrate that classical energy-dependent endocytosis is the major route for cellular internalization of PR9/ QD complexes, and that chloroquine exerts a lysosomotropic effect on PR9/QD complexes, allowing them to escape from endocytic vesicles into the cytoplasm. PR9/QD complexes colocalize with actins, lysosomes, early endosomes and nucleus. TEM analysis revealed the endocytic trafficking of PR9/QD complexes in cells. A reporter gene assay confirmed that plasmid DNA delivered by PR9s can be actively expressed by cells [49]. Zeta-potentials of CPP/cargo complexes correlated with trans-duction efficiency [48], emphasizing the importance of electrostatic interactions of PR9/QD complexes with plasma membranes. Cell viability assay confirmed that none of the components of the PR9/QD complexes are cytotoxic.
CPP transduction of QDs into stem cells with high transduction efficiency and low cytotoxicity has been demonstrated [37,50,51]. Moreover, we have shown that PR9 and PR9/cargo complexes are relatively nontoxic in A549 cells by SRB [33] and MTT [52,53] assays. Our present results with PR9/QD complexes in human cells are consistent with these earlier results. It was reported that more than 80% of adipose tissue-derived stem cells could be labeled by R8/QD complexes prepared at a ratio of 10,000 within 1 h, and that the consequent fluorescent staining was maintained at least for 2 weeks [37]. No cytotoxicity was observed in cells transduced with less than 16 nM of QDs. In addition, the transduced cells could differentiate into adipogenic and osteogenic cells, indicating that the transduced cells maintained their stem cell potency [37].
Research on CPPs has focused on improving transduction efficiency. The hybrid PasR8 peptide markedly enhanced the translocation efficiency of active peptides by permitting endosomal escape in cells [21]. For instance, Pas conjugated with flock house virus (FHV)-derived arginine-rich peptide was attached to the p53 C-terminal 22-amino-acid peptide (p53C'), a retro-inverso peptide that induces p53-dependent autophagic cell death [54]. In another study, the growth of malignant glioma cells was inhibited by the triplex D-isomer peptides (dPasFHV-p53C'). Recently, the importance of hydrophobic sequences in the Pas segment, especially phenylalanine residues, in promoting cellular uptake of R8 was demonstrated [55]. Attachment of aromatic moieties, such . Zeta-potential and particle size of PR9 and PR9/QD complexes and the secondary structure of PR9. (A) Zeta-potentials of PR9 and PR9/QD complexes. PR9 or PR9/QD complexes prepared at a molecular ratio of 60 were dissolved in doubly deionized water at pH 7 or 5. Each solution was equilibrated at 25uC for 120 sec in a zeta cell and then analyzed using a Zetasizer Nano ZS. (B) Particle size of QD or PR9/QD complexes. PR9/QD complexes were dissolved in doubly deionized water with pH 7 or 5 and then analyzed using a Zetasizer. Significant differences between PR9/QD complexes and QDs at P,0.01 (**) are indicated. Data are presented as mean 6 SD from seven independent experiments. (C) Secondary structure of PR9. All CD spectra were recorded in millidegree (mdeg). doi:10.1371/journal.pone.0067100.g007 as Pas, to a R8 segment may increase peptide-proteoglycan interactions, thereby stimulating macropinocytosis. PasR8 working in a serum-containing medium was an additional advantage of the Pas segment, since serum-binding often decreases cytosolic internalization of CPPs. The promotion of cellular uptake by Pas addition is prominent when the molecular weight of cargoes is relatively small. Finally, the total hydrophobicity of PasR8 conjugates appears to be crucial for efficient cytosolic translocation [55].
TEM is a valuable tool for the morphological characterization of biological and nonbiological materials at high resolution [56]. Direct information on the intracellular distribution of transduced material comes from TEM, which reveals electron-dense cores of PR9/QD complexes associated with plasma membrane and in the cytoplasm of PR9/QD-transduced cells ( Figure 6). While there are multiple types of endocytic pathways [15], the endocytic progress of transport vesicles of the widely studied clathrin-dependent endocytosis of nanoparticles is from early endosomes to multivesicular bodies/late endosomes and finally to lysosomes. TEM images of PR9/QD-transduced cells obtained in the present study were generally in accord with this endocytic progression. PR9/QD complexes were somewhat larger than QDs alone ( Figure 7B), suggesting that positively charged PR9s form stable complexes with carboxyl-functionalized QDs by electrostatic interactions [16,33].
Zeta-potential is a useful measure in nanoparticle applications that indicates the interaction energy on the particle-carrier surface [57,58]. Zeta-potential depends on nanoparticle size, methods of production and treatment, surface structure and the pH value of the environment [37]. The combined effects of both zeta-potential and particle size on nanoparticles provide insight into the stability of particles in solution [59,60]. We found that more electropositive zeta values of CPP/cargo complexes correlate well with protein transduction efficiency, presumably due to increased electrostatic interactions of PR9/QD complexes with plasma membranes. In this study, the more electropositive PR9/QD complexes had a higher transduction efficiency than PR9s or QDs.
Qualitative secondary structure assignments of CD spectroscopy were based on the following: minima at both 208 and 222 nm, and maximum at 190 nm for a-helix; minimum at 218 nm and maximum at 195 nm for b-sheet; minimum at 198 nm and no positive peak for random coil [61]. We found that the secondary structural contents of PR9 have very similar conformations in aqueous buffers at pH 7 and pH 5 ( Figure 7C). These two patterns (minimum at 198-222 nm and maximum) of PR9 are similar to those of R9, which is mostly unstructured in solution [62]. Binding of poly-L-arginine composed of 293 (PLA239) and 554 (PLA554) arginine-residues to an anionic phospholipid large unilamellar vesicle (LUV) was accompanied by a transition from random coil to a-helix structure; however, a similar structural change was not observed with PLA69 and R8 [63].
Subcellular colocalization analyses revealed that HR9/QD complexes do not colocalize with any organelles tested; these complexes stay in the cytosol most of time ( Figure 3). This result is consistent with our earlier demonstration that HR9/QD complexes enter cells by direct membrane translocation [33,34]. In contrast, endocytosis appears to be the main route for intracellular delivery of PR9/QD and SR9/QD complexes [34]. However, SR9/QD complexes entered cells by multiple pathways [64]. Among them, macropinocytosis, a lipid raft-dependent form of endocytosis, is a prominent route for SR9/QD entry [65]. Actin forms microfilaments, one of key components of the cytoskeleton, participating in many cellular processes, including endocytosis. Macropinocytosis and classical endocytosis, such as clathrin-, caveolae-dependent, and clathrin-and caveolae-independent pathways, involve actin rearrangements. Therefore, the observed colocalization of PR9/QD with actin, lysosomes and early endosomes, indicates that these complexes enter cells through an endocytic pathway.
Numerous factors, including experimental conditions, physicochemical properties of CPPs and their cargoes, cell type, temperature and serum level in the medium can influence the pathway of cellular uptake [7,11,[66][67][68][69][70]. R9, antennapedia peptide and Tat peptide use a combination of three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis [66]. It seems likely that PR9s use the same three endocytic pathways (Figure 2A and 3-6). The chemical properties of the cargo molecules are a contributing factor of dodeca-arginine (R12) peptide-mediated translocation [69]. R12 attached to hydrophobic cargoes stimulate dynamic morphological alternations in plasma membranes, and these structural changes allow R12 to permeate plasma membranes [69].
Conclusions
PR9/QD complexes comprised of a cell penetrating peptide (nona-arginine; R9) and a peptide penetration accelerating sequence (Pas) that promotes lysosomal escape noncovalently complexed with a quantum dot (QD) probe were evaluated as a cell transduction system. Histological results demonstrate that endocytosis is the main pathway for cellular uptake of PR9/QD complexes. PR9/QD complexes initially colocalize with actins, lysosomes and early endosomes, and later with the nucleus.
Plasmid DNA delivered by PR9s was expressed by cells. Zetapotential analysis revealed the importance of electrostatic interactions of PR9/QD complexes with plasma membranes. PR9/QD complexes were not toxic to the cells. Thus, PR9 may be an efficient and safe delivery vector for biomedical applications. | v3-fos-license |
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} | pes2o/s2orc | Comparison of Structural Stabilities of Prostaglandin H Synthase-1 and -2*
There are two known isoforms of prostaglandin H synthase (PGHS), a key enzyme in the conversion of arachidonic acid to bioactive prostanoids. The “constitutive” isoform, PGHS-1, is thought to have housekeeping functions, and the “inducible” isoform, PGHS-2, has been implicated in cellular responses to cytokines. The two isoforms have high sequence conservation in the cyclooxygenase active site and quite similar crystallographic structures, but differ markedly in their interactions with many cyclooxygenase substrates and inhibitors. We have evaluated the stability of the overall folding, and of the active sites of ovine PGHS-1 and human PGHS-2 using denaturation with guanidinium hydrochloride (GdmHCl). Changes in hydrodynamic and cross-linking properties indicated a dimer 3 mon- omer transition for both isoforms between 0.5 and 2 M GdmHCl; the monomers unfolded at higher GdmHCl levels. Changes in overall secondary and tertiary structure, measured by tryptophan fluorescence and circular di-chroism, occurred in two phases for each isoform, with the transition between the phases at 0.2–0.5 M GdmHCl. Disruption of active site functions (cyclooxygenase, peroxidase, and cyclooxygenase inhibitor binding activities) began at GdmHCl levels below 0.2 M . The structural and functional changes were completely reversible up to about 2 M GdmHCl, they were more pronounced at lower protein levels, and they required lower GdmHCl levels for PGHS-2 than for PGHS-1. The results are consistent with a m M subunit) were recorded at a scan speed of 50 nm/min with a response time of 2 s and a 2.0 nm slit setting, in a 1-mm pathlength quartz cuvette at room temperature using a Jasco J700 spectropolarimeter. Estimations of secondary structure used the standard algorithm included in the polarimeter software. Quantitation Solvent-exposed and-2— Exposure of tryptophan residues to solvent in the two PGHS isoforms and lysozyme was evaluated by solvent perturbation difference spectroscopy, using 20% glycerol as the perturbant (20). All solu- tions were prepared in high purity (Milli Q) water and passed through a 0.45- m m filter. The spectrophotometer cuvettes were carefully washed with a cotton swab moistened with dilute Tween 20 and then rinsed with high purity water. UV absorbance spectra were recorded for each protein (16.7 m M for oPGHS-1 and hPGHS-2 or 20 m M for lysozyme) in 0.1 M potassium phosphate, pH 7.2, containing the desired level of Tween 20 and either 0 or 20% (v/v) glycerol. The slit width was 2 nm, and the cuvettes were thermostatted at 25 6 0.1 °C. For each Tween level, the control spectrum was subtracted from the spectrum obtained with glycerol present to obtain a difference spec- trum, which had a maximum near 292 nm for each protein. A similar procedure was followed to obtain a difference spectrum with the same buffer for the model tryptophan compound, NAc-Trp-OEt (72.8 m M , based on an e 282 of 5.55 m M 2 1 cm 2 1 (21)). The maximum in the NAc- Trp-OEt difference spectrum was found at 292 nm. The fractional exposure of tryptophan residues to solvent in each protein at each level of Tween 20 was then calculated using the equation below. intensity. Equilibration with excess (3 eq) indomethacin produced only small red shifts, but did decrease the fluorescence intensity by 47% in oPGHS-1 and by 42% in hPGHS-2. A similar effect of indomethacin on the hPGHS-2 fluorescence was reported recently by Houtzager et al. (29) and attributed to quenching by inhibitor bound in the vicinity of the tryptophans. To assess complex formation by flurbiprofen and indomethacin, PGHS samples were pre- incubated with the desired level of GdmHCl at room temperature for 30 min before addition of 3 eq of flurbiprofen or indomethacin. The incu- bation was then continued for an additional 40 min before measurements of the fluorescence intensity at 338 nm were made. This length of time was found to maximize cyclooxygenase inhibition, reflecting maximal complex formation. The intrinsic fluorescence intensities of un- bound flurbiprofen and indomethacin were found to be constant over the range of 0–2 M GdmHCl.
Prostaglandin H synthase (PGHS) 1 catalyzes two key steps in prostanoid biosynthesis: oxygenation and cyclization of arachidonic to form PGG 2 and the reduction of the hydroperoxide of PGG 2 to form PGH 2 . Two isoforms of PGHS have been described. PGHS-1 is believed to be a housekeeping enzyme, whereas PGHS-2 is thought to play important roles in cell proliferation and inflammation (1). Both isoforms have cyclooxygenase and peroxidase activity, with comparable specific activities for the purified recombinant enzymes (2). PGHS-1 and PGHS-2 have about 60% amino acid identity, with much higher conservation of active site residues (3). Crystallographic data for oPGHS-1 and hPGHS-2 revealed similar dimeric structures for the two isoforms, with a root-mean-square deviation of only 0.9 Å for backbone atoms (4 -6).
Despite the similarities in the crystallographic structures, the two PGHS isoforms have been found to have different selectivities for fatty acid substrates and cyclooxygenase inhibitors (1). These specificities have been ascribed in part to a larger cyclooxygenase pocket in PGHS-2 (7). The observations that PGHS-2, but not PGHS-1, retains oxygenase activity and native tyrosyl radical after acetylation by aspirin (8 -10) also indicate that PGHS-2 has a more accommodating cyclooxygenase active site. The presence of a smaller valine residue at position 509 in PGHS-2, instead of the isoleucine at the corresponding position (residue 523) in PGHS-1, does provide a significantly larger accessible volume in the upper part of the cyclooxygenase channel (5,6). However, results from mutagenesis studies in both PGHS-1 and PGHS-2 indicate that the difference between valine and isoleucine at position 509/523 does not account for all the differences in inhibitor specificity between the isoforms (11)(12)(13).
As a first step in examining more dynamic aspects of the structural properties of the two isoforms, we have evaluated their susceptibilities to unfolding by denaturant. Controlled denaturation has proven a powerful approach to characterizing the folding stabilities of proteins in solution (14) and the contributions of oligomeric arrangements to overall structure (15). Results from present chemical denaturation studies indicate that the PGHS-2 structure, both overall and in the peroxidase and cyclooxygenase active sites, is less stable than in PGHS-1.
in insect cells 2 using the methods described for PGHS-2, and solubilized from the membrane fraction by extraction with 1% Tween 20. oPGHS-1 was purified to homogeneity from sheep seminal vesicles (16). Protein concentrations were determined by the modification of the Lowry assay described by Peterson (18). The cyclooxygenase specific activity ranged from 58 to 147 mol of O 2 /min/mg for oPGHS-1 and from 30 to 56 mol of O 2 /min/mg for hPGHS-2. The apoenzymes of both PGHS isoforms were used in these studies; heme was added only for assay of activity.
Activity Assays-Cyclooxygenase activity was determined with an oxygen electrode (16) at 30°C. The standard reaction mixture contained 0.1 M potassium phosphate, pH 7.2, 5 mM D-tryptophan, 0.1 mM arachidonate, and 1 M heme. Peroxidase activity was measured spectrophotometrically at 436 nm in a 2-ml reaction mixture containing 0.1 M Tris-HCl, pH 8.0, 0.5 mM guaiacol, 0.4 mM hydrogen peroxide, and 1 M heme (19). Samples treated with GdmHCl were diluted at least 200-fold into the assay mixtures for determination of cyclooxygenase or peroxidase activity; the residual denaturant was found not to affect the activity significantly.
Spectral Measurements-Electronic absorbance spectra were recorded in 1-cm pathlength quartz cuvettes with a Shimadzu model UV-2101PC spectrophotometer thermostatted at 25 Ϯ 0.1°C. For critical measurements, the spectrophotometer was warmed up for 30 min before use and the base-line spectrum was scanned repeatedly to verify stable performance.
Fluorescence measurements were carried out with a SLM Aminco model SPF-500 spectrofluorometer at room temperature. Inner filter effects were minimized by diluting protein solutions to reach an A 280 of less than 0.05.
CD spectra of the PGHS apoenzymes (1.0 M subunit) were recorded at a scan speed of 50 nm/min with a response time of 2 s and a 2.0 nm slit setting, in a 1-mm pathlength quartz cuvette at room temperature using a Jasco J700 spectropolarimeter. Estimations of secondary structure used the standard algorithm included in the polarimeter software.
Quantitation of Solvent-exposed Tryptophan Residues in PGHS-1 and-2-Exposure of tryptophan residues to solvent in the two PGHS isoforms and lysozyme was evaluated by solvent perturbation difference spectroscopy, using 20% glycerol as the perturbant (20). All solutions were prepared in high purity (Milli Q) water and passed through a 0.45-m filter. The spectrophotometer cuvettes were carefully washed with a cotton swab moistened with dilute Tween 20 and then rinsed with high purity water. UV absorbance spectra were recorded for each protein (16.7 M for oPGHS-1 and hPGHS-2 or 20 M for lysozyme) in 0.1 M potassium phosphate, pH 7.2, containing the desired level of Tween 20 and either 0 or 20% (v/v) glycerol. The slit width was 2 nm, and the cuvettes were thermostatted at 25 Ϯ 0.1°C.
For each Tween level, the control spectrum was subtracted from the spectrum obtained with glycerol present to obtain a difference spectrum, which had a maximum near 292 nm for each protein. A similar procedure was followed to obtain a difference spectrum with the same buffer for the model tryptophan compound, NAc-Trp-OEt (72.8 M, based on an ⑀ 282 of 5.55 mM Ϫ1 cm Ϫ1 (21)). The maximum in the NAc-Trp-OEt difference spectrum was found at 292 nm. The fractional exposure of tryptophan residues to solvent in each protein at each level of Tween 20 was then calculated using the equation below.
Fraction exposed ϭ ͑⌬A 292 /A 292 ͒ protein /͑⌬A 292 /A 292 ͒ NAc-Trp-OEt (Eq. 1) ⌬A 292 and A 292 are the difference and direct absorbance values at 292 nm, respectively, for the protein or the model compound. The number of exposed tryptophans was obtained by multiplying the fractional exposure calculated from Equation 1 by the total number of tryptophans in each protein.
GdmHCl Denaturation of PGHS Isoforms-Stock solutions of 6 or 8 M GdmHCl were prepared in 0.1 M potassium phosphate, pH 7.2. The pH of the stock solutions was adjusted as necessary with KOH. Gdm-HCl concentrations were determined from refractive index measurements as described by Nozaki (22). Concentrated solutions of enzyme were diluted into the phosphate buffer containing 0.1% Tween 20 and the desired level of GdmHCl and incubated at room temperature for at least 30 min (unless otherwise noted) before removal of aliquots to assess intrinsic fluorescence, CD, enzymatic activity, inhibitor binding, cross-linking, or hydrodynamic properties.
Size Exclusion Chromatography-The chromatographic system consisted of a Rainin Dynamax pump set and controller connected to a 0.46 ϫ 25-cm Rainin Hydropore 5 SEC column and a Shimadzu model RF-535 fluorescence detector (excitation at 282 nm, emission at 335 nm). The column was eluted isocratically at a flow rate of 0.25 ml/min with 0.1 M potassium phosphate, pH 7.2, containing 0.1% Tween 20 and 0 -6 M GdmHCl. The detergent concentration in the elution buffer was about 14 times the critical micelle concentration; silica-based column matrix interactions with detergent-solubilized proteins have been found to be minimal under these conditions (23). The denaturant concentration was set by varying the proportions of solvent delivered from Pump A (buffer alone) and Pump B (buffer with 6 M GdmHCl). Equilibration of the column at each denaturant level was confirmed by refractive index measurements of the eluate. The column was calibrated in the buffer alone with a series of standards (thyroglobulin, apoferritin, catalase, transferrin, cytochrome c, and glycyl-tyrosine) using published values for the Stokes radii (24).
Cross-linking Studies-The procedures were adapted from those of White et al. (25). oPGHS-1 was diluted to 0.15 or 1.5 M in 0.1 M potassium phosphate, pH 7.2, containing 0.1% Tween 20 and 0 -3 M GdmHCl (total volume, 250 l) and preincubated at room temperature for 30 -45 min before addition of 20 l of 25% glutaraldehyde. The reaction was quenched after 15 min by addition of 12 l of 2 M NaBH 4 and incubation for 20 min. The mixture was diluted by addition of water (0.75 ml), and the protein was precipitated by addition of 100 l of 0.3% sodium deoxycholate and 100 l of 72% trichloroacetic acid. The precipitate was collected by centrifugation at 13,000 rpm in a microcentrifuge for 15 min, washed by resuspension in 0.75 ml of ice-cold acetone, and reisolated by centrifugation at 13,000 rpm for 10 min. The precipitated proteins were dissolved in 20 -40 l of electrophoresis sample buffer and separated by electrophoresis under denaturing conditions in an 8% polyacrylamide gel (26). Protein bands were stained with Coomassie Blue R250 and quantitated by densitometry.
Cyclooxygenase Inhibitor Binding-Flurbiprofen and indomethacin are competitive, tight-binding, stoichiometric cyclooxygenase inhibitors for purified PGHS-1 and -2 (2,4,27,28). The two PGHS isoforms exhibited emission maxima near 337 nm when excited at 282 nm. Equilibration of oPGHS-1 or hPGHS-2 with excess (3 eq) flurbiprofen led to a blue shift of 7-8 nm in the emission maximum and a 20% decrease in fluorescence intensity. Equilibration with excess (3 eq) indomethacin produced only small red shifts, but did decrease the fluorescence intensity by 47% in oPGHS-1 and by 42% in hPGHS-2. A similar effect of indomethacin on the hPGHS-2 fluorescence was reported recently by Houtzager et al. (29) and attributed to quenching by inhibitor bound in the vicinity of the tryptophans. To assess complex formation by flurbiprofen and indomethacin, PGHS samples were preincubated with the desired level of GdmHCl at room temperature for 30 min before addition of 3 eq of flurbiprofen or indomethacin. The incubation was then continued for an additional 40 min before measurements of the fluorescence intensity at 338 nm were made. This length of time was found to maximize cyclooxygenase inhibition, reflecting maximal complex formation. The intrinsic fluorescence intensities of unbound flurbiprofen and indomethacin were found to be constant over the range of 0 -2 M GdmHCl.
Solvent Exposure of Tryptophan Residues in oPGHS-1 and
hPGHS-2-oPGHS-1 has nine, and hPGHS-2 six, tryptophan residues (3,30), which are potentially useful in fluorimetric monitoring of unfolding in these proteins. Solvent perturbation difference spectroscopy was used to determine how many of these tryptophan residues are exposed to solvent, and thus not likely to contribute significantly to the decrease in intrinsic fluorescence during denaturation. It was anticipated that the detergent used to solubilize the enzymes might give a variable level of masking of some tryptophan residues, particularly those in the putative membrane anchor region (4), so the tryptophan exposure was checked at several detergent levels. Approximately 1.2 tryptophan residues in oPGHS-1 and 0.5 tryptophan residues in hPGHS-2 were found to be exposed to solvent at 0.05% Tween 20 (Fig. 1). Higher detergent levels decreased the tryptophan exposure somewhat for both PGHS isoforms, indicating a small increase in masking of the tryptophans by detergent. Lysozyme was examined as a control, and found to have about four tryptophans exposed to solvent, with less exposure as the Tween concentration was raised (Fig. 1), in accord with the report that four of the six tryptophans in lysozyme are exposed (20). Because few of the tryptophan residues in either isoform are exposed to solvent, the proteins' intrinsic fluorescence is likely to include contributions from many buried tryptophans distributed throughout the proteins. These results are consistent with oPGHS-1 crystallographic data (4), which show four of the nine tryptophan residues (at positions 75, 77, 98, and 100) on the surface. All four are in the proposed membrane anchor domain and are thus probably partially shielded by bound detergent. Only one of the oPGHS-1 surface tryptophans is conserved in hPGHS-2: residue 85, corresponding to residue 100 in oPGHS-1 (3). On the other hand, the five buried oPGHS-1 tryptophans are all conserved in hPGHS-2 (3).
Intrinsic Fluorescence of the Native and Partially Denatured PGHS Isoforms-The fluorescence excitation and emission spectra of oPGHS-1 and hPGHS-2 were examined to determine appropriate wavelengths for monitoring denaturation. The two PGHS isoforms exhibited similar excitation spectra, with maxima near 282 nm; the emission maxima were at 337 nm for oPGHS-1 and 335 nm for hPGHS-2 (Fig. 2). The similar emission intensities in Fig. 2 from oPGHS-1, with nine tryptophan residues, and hPGHS-2, with six tryptophans, presumably reflects the higher concentration of hPGHS-2 used and the greater exposure of tryptophans to solvent in oPGHS-1 (Fig. 1). The spectral characteristics observed for hPGHS-2 are similar to those recently reported by Houtzager et al. (29). Incubation for 30 min in buffer containing 4 M GdmHCl shifted the emission maxima of both isoforms to near 353 nm, with considerable decreases in intensity. These changes are consistent with unfolding of the proteins by the denaturant, with consequent exposure of normally buried tryptophan residues to the polar solvent. Excitation and emission wavelengths of 282 and 335-340 nm, respectively, were chosen to monitor GdmHCl-induced denaturation of the two isoforms. Over 90% of the fluorescence from hPGHS-2 under similar excitation and emission settings has been found to originate from tryptophan residues (29). This disproportionate contribution from tryptophan in a protein with 27 tyrosine residues and 6 tryptophan residues is counterintuitive, but it is in line with experience with other proteins (31). The fractional fluorescence contribution from tryptophan residues in oPGHS-1 is expected to be even greater, because it has the same number of tyrosine residues as hPGHS-2, but 50% more tryptophan residues.
Kinetics of Fluorescence Changes during Reaction with GdmHCl-To establish the time required to reach equilibrium, the tryptophan fluorescence intensity of oPGHS-1 was monitored during incubation in buffer containing several levels of GdmHCl (Fig. 3). In the control and the incubation with 0.5 M GdmHCl, the fluorescence intensity drifted slowly downward, reaching a plateau at about 90% of the initial value after 1200 s. With GdmHCl levels of 1.5 M and above, however, there was a rapid decrease in fluorescence over the first 60 s or so, followed by a more gradual decrease. The magnitude of the fluorescence decrease was proportional to the GdmHCl concentration used. In each case, relatively stable fluorescence readings were reached after about 1600 s, indicating that equilibration of the native and unfolded states was essentially complete at that time. Accordingly, a 30-min preincubation period with GdmHCl was routinely used in subsequent denaturation experiments.
Dependence of Intrinsic Fluorescence of oPGHS-1 and hPGHS-2 on GdmHCl Concentration-Fluorescence intensities were measured for both isoforms equilibrated at a variety of denaturant levels between 0 and 6 M (Fig. 4A). Biphasic changes in fluorescence intensity were observed for both enzymes as the denaturant level was increased. The fluorescence increased very slightly at GdmHCl levels below 0.5 M, and then decreased markedly in a monotonic fashion as the denaturant was increased to 6 M. No further change was seen at GdmHCl levels up to 7 M for either oPGHS-1 or hPGHS-2 (data not shown), indicating that complete unfolding of both proteins was essentially complete at 6 M GdmHCl.
The second phase of the fluorescence changes was clearly sensitive to protein concentration for both oPGHS-1 and hPGHS-2, with a more rapid decline in fluorescence at lower enzyme level (Fig. 4A). This dependence on protein concentration is consistent with coupling of the tertiary structure changes to dissociation of dimeric enzyme into monomers for both PGHS isoforms (15). For a two-state system where dissociation of a dimer is coupled to unfolding of the monomers, the equilibrium constant for the process is given by Equation 2. P t is the total protein concentration (as monomer) and f U is the fraction of protein unfolded (15). The data for the higher concentration of oPGHS-1 and hPGHS-2 at 1-4 M GdmHCl (where the values of f U could be measured most precisely) were used to calculate K values as a function of the GdmHCl level (Fig. 4A, inset). The data diverge from straight lines for both enzymes, indicating that the two-state model is an incomplete description of the unfolding process for these two proteins, and that the K values calculated from Equation 2 must be regarded as rough estimates. However, the curves for PGHS-1 and -2 run parallel, with the approximate K value for PGHS-2 greater than that for PGHS-1 throughout. This indicates that the hPGHS-2 structure was less stable than that of oPGHS-1 regardless of the denaturant level.
The reversibility of the denaturant-induced decreases in tryptophan fluorescence was examined for both isoforms (Fig. 4B). For enzyme preincubated with 6 M GdmHCl and subsequently diluted to lower denaturant levels, considerable fluorescence intensity was recovered after 30 min for both isoforms (Fig. 4B, solid symbols). However, the recovery of fluorescence was limited to slightly more than half the initial value for both oPGHS-1 or hPGHS-2, indicating that complete denaturation was not readily reversible for either isoform. When the PGHS isoforms were initially equilibrated with only 2 M GdmHCl, subsequent dilution was able to restore the fluorescence intensities to essentially the control levels ( Fig. 4B, open symbols). This demonstrated that the early part of the denaturation process was completely reversible for both isoforms.
CD Changes in oPGHS-1 and hPGHS-2 upon Denaturation with GdmHCl-CD measurements were used as a second indicator of bulk structural changes during denaturation of the PGHS isoforms. In the native state, oPGHS-1 and hPGHS-2 had very similar CD spectra, with troughs at 209 nm and shoulders near 222 nm (Fig. 5). The ␣-helical contents predicted from these spectra were 38% for oPGHS-1 and 36% for hPGHS-2. These values are very close to the value of 38% helix calculated from secondary structure assignments based on the oPGHS-1 crystal structure (4); detailed secondary structure assignments were not reported for PGHS-2 (5, 6). Little organized -sheet was reported for either PGHS-1 or PGHS-2 (4 -6). Incubation for 30 min with 4 M GdmHCl greatly decreased the intensity of the CD trough for both isoforms (Fig. 5), indicating a considerable loss of helical structure (32). The CD intensity at 222 nm thus was chosen to monitor the helical content of both isoforms in subsequent denaturation experiments.
Dependence of oPGHS-1 and hPGHS-2 Secondary Structure on GdmHCl Concentration-Biphasic changes in the CD intensity at 222 nm with increasing denaturant were seen for both isoforms (Fig. 6). For oPGHS-1, the CD increased slightly by 0.5 M GdmHCl, then decreased monotonically with higher levels of denaturant, reaching minimal values near 6 M. For hPGHS-2, the initial increase was smaller, and the peak occurred at 0.2 M GdmHCl. The decrease in CD intensity for hPGHS-2 was larger than that for oPGHS-1 at all GdmHCl Fig. 6 to show approximate values of K (calculated using Equation 2) as a function of the GdmHCl level produced parallel curves for PGHS-1 and -2 (not shown), much like the pattern obtained for the fluorescence data in the inset to Fig. 4A. The results from the CD measurements thus indicate that the secondary structure of hPGHS-2 was less stable than that of oPGHS-1, independent of the denaturant level. The overall patterns of CD changes with GdmHCl concentration (Fig. 6) were similar to those for the intrinsic fluorescence changes (Fig. 4A). This correlation between losses of secondary structure, monitored by CD, and increases in tryptophan exposure, monitored by fluorescence, support the use of the more convenient fluorescence measure-ments as an indicator of the overall folding status of the PGHS isoforms.
Dependence of oPGHS-1 and hPGHS-2 Cyclooxygenase Activity on GdmHCl Concentration-The cyclooxygenase activities were measured after incubation of concentrated or dilute solutions of each PGHS isoform with 0 -3 M GdmHCl (Fig. 7A). For oPGHS-1, a monotonic loss of activity was seen for both levels of the enzyme, with an IC 50 Fig. 7A thus indicate that the hPGHS-2 active site structure is less stable than that of oPGHS-1. The influence of enzyme concentration on the sensitivity to inactivation for both oPGHS-1 and hPGHS-2 indicates that loss of activity is coupled with dissociation of the native dimers into monomers for both isoforms.
For an overall appraisal of the reversibility of the denaturant-induced loss of cyclooxygenase activity, the activity was measured after a 50-fold dilution of oPGHS-1 and hPGHS-2 preincubated with 0 -6 M GdmHCl (Fig. 7B). Considerable cyclooxygenase activity was recovered from both isoforms exposed to the lower GdmHCl levels. However, no activity was recovered from oPGHS-1 preincubated with denaturant levels over 3 M, or from hPGHS-2 exposed to levels above 2 M, indicating that irreversible inactivation of both isoforms occurred at higher denaturant levels.
The reversibility of the activity losses in oPGHS-1 and hPGHS-2 exposed to lower levels of denaturant was examined in more detail (Fig. 7C). Here, each isoform was preincubated with sufficient GdmHCl to produce essentially complete inactivation (2.5 M for oPGHS-1, 1.5 M for hPGHS-2) and then equilibrated at a lower denaturant level before cyclooxygenase assay. Lowering the denaturant level resulted in a progressive recovery of activity in both isoforms, with essentially complete recovery of activity when the denaturant levels were reduced below 0.1 M. Thus, the inactivation of cyclooxygenase activity observed at lower GdmHCl levels is completely reversible in both PGHS isoforms. A larger fraction of the activity was recovered for oPGHS-1 than for hPGHS-2 at each GdmHCl level (Fig. 7C), consistent with the unfolding results in Fig. 7A, again indicating a higher active site structural stability in oPGHS-1.
Dependence of oPGHS-1 and hPGHS-2 Hydrodynamic Properties on GdmHCl Concentration-The effects of denaturant concentration on the hydrodynamic properties of the two PGHS isoforms were characterized by size exclusion chromatography (Figs. 8 and 9). Native oPGHS-1 and hPGHS-2 eluted in nearly symmetrical peaks with retention times of about 9.15 min, indicating a Stokes radius of about 5.5 nm (Fig. 9A). This is roughly equivalent to a globular protein with a mass of 250 kDa. This estimated mass is consistent with previous reports for the PGHS dimers with bound Tween 20 detergent (33,34). The ratios of peak width at half-height to elution volume were 0.16 for oPGHS-1 and 0.14 for hPGHS-2. These are comparable to values calculated from previous reports: 0.18 for oPGHS-1 (33) and 0.16 for hPGHS-2 (35). The present chromatographic behavior of the two PGHS isoforms is thus consistent with results obtained by other laboratories.
Preincubation with GdmHCl up to 1.25 M increased the retention times for both isoforms, to maxima of just over 10 min. These retention times are consistent with Stokes radii of about 3.5 nm (Fig. 9A). This large decrease in Stokes radius indicates either dissociation of the oPGHS-1 and hPGHS-2 dimers to monomers or a large shape change to much more compact dimer species in this denaturant range. The chromatographic profiles did not change when the samples were preincubated with denaturant for 45 min instead of 30 min (data not shown), confirming that equilibrium had been reached.
The species with Stokes radii of 5.5 and 3.5 nm were not resolved, but the transition between the two was reflected in the changes of peak width at half-height (Fig. 9B). For both PGHS isoforms, the peak width increased at GdmHCl levels above 0.5 M, peaked at 1.25 M, and declined to a stable value above 2 M denaturant. Conversion from the 5.5-nm species to the 3.5-nm species thus occurred over the range of 0.5-2 M GdmHCl. The significant peak widening indicates the presence of both species at intermediate denaturant levels. Maximal peak width is expected when the amounts of the two species are equal, putting the midpoint of the transition near 1.25 M GdmHCl for both isoforms.
Further increases in GdmHCl level above 2 M did not change the peak width for either oPGHS-1 or hPGHS-2 (Fig. 9B), indicating the presence of a single species or multiple rapidly equilibrating species. However, the retention times continued to decrease as the GdmHCl level was raised above 2 M (Fig. 9A), with minima reached at 4 M for hPGHS-2 and near 6 M for oPGHS-1. Such an increase in the hydrodynamic size is consistent with complete unfolding of a monomeric species and has been observed with other proteins (36 -38).
Dependence of Subunit Cross-linking on GdmHCl Concentration-The two isoforms underwent very similar changes in hydrodynamic properties as the denaturant level was raised (Fig. 9). oPGHS-1 was chosen to evaluate the relationship between the hydrodynamic changes and the oligomeric state. The effect of denaturant level on the proportion of oPGHS-1 in the dimeric state was assessed by examining the efficiency of monomer cross-linking by glutaraldehyde at two different protein concentrations. Electrophoretic results for cross-linking of 0.15 M oPGHS-1 are shown in Fig. 10A. In the absence of denaturant, some of the protein migrated as a band with an apparent molecular mass of about 62 kDa, corresponding to the monomer. The remaining protein was in several ill-defined bands with apparent molecular masses of 150 kDa and above, presumably reflecting cross-linked dimers and higher oli- (Fig. 9B).
A transition in efficiency of subunit cross-linking by glutaraldehyde was found at a much higher GdmHCl level, around 5 M, for transthyretin (39), showing that the oPGHS-1 crosslinking transitions are not due to effects of the denaturant on glutaraldehyde reactivity. The cross-linking efficiency in the absence of denaturant was little affected by the 10-fold change in protein concentration, with 47% as monomer with 0.15 M enzyme and 41% as monomer with 1.5 M enzyme. This indicates that the observed loss of monomer is mostly due to cross-linking within dimers, rather than between dimers or between dissociated monomers, because the latter two reactions require bimolecular collisions and are consequently very sensitive to protein level. Cross-linking of monomers within dimers thus was less efficient with increasing denaturant. The straightforward interpretation is that the denaturant increases dissociation of dimers to monomers. This interpretation is supported by the observation that increasing the protein level, which shifts the dimer 7 monomer equilibrium in favor of the dimer, raises the midpoint GdmHCl level for the transition in cross-linking efficiency (Fig. 10B). Overall, the cross-linking results provide independent evidence that the very similar transitions in hydrodynamic properties observed for PGHS-1 and -2 at 0.5-2 M GdmHCl (Fig. 9B) reflect dimer 3 monomer transitions.
Comparison of Cyclooxygenase and Peroxidase Active Site Stabilities in oPGHS-1 and hPGHS-2-The stabilities of the native structures of the active sites in oPGHS-1 and hPGHS-2 were further assessed by monitoring decreases in peroxidase activity and cyclooxygenase-inhibitor complex formation after equilibration of the proteins with various levels of GdmHCl; the results are shown in Fig. 11. Inhibition of the peroxidase activity of both isoforms was observed, even at the lowest levels of denaturant tested, and progressed as the levels were increased (Fig. 11A). The IC 50 values for peroxidase activity were 0.4 and 0.2 M GdmHCl for oPGHS-1 and hPGHS-2, respectively, showing that the PGHS-2 activity was more easily disrupted than that of PGHS-1. The inhibitory effects on the peroxidase activities were essentially identical with those on the cyclooxygenase activity at the same enzyme concentrations.
Flurbiprofen and indomethacin are tight-binding, stoichiometric cyclooxygenase inhibitors for purified PGHS-1 and -2; both compete for the arachidonate binding site (2,4,27,28). The action of these agents involves an initial reversible binding, followed by a time-dependent conversion of the initial enzyme-inhibitor complex to a much higher affinity complex (27,40,41). Complex formation can be monitored by changes in the intrinsic fluorescence of the proteins (see Ref. 29 and "Experimental Procedures"). The ability to form the tight complex with these inhibitors thus furnishes a convenient way to assess the overall structural integrity of the cyclooxygenase active site. Preincubation of PGHS-1 and -2 with low levels of Gdm-HCl led to loss of their ability to form complexes with indomethacin and flurbiprofen (Fig. 11B). The IC 50 values for indomethacin binding were 0.45 and 0.25 M GdmHCl for oPGHS-1 and hPGHS-2, respectively. For flurbiprofen binding, the IC 50 values were 0.45 and 0.2 M GdmHCl for oPGHS-1 and hPGHS-2, respectively. Thus, results with both probes indicated that the cyclooxygenase active site in hPGHS-2 was considerably more sensitive to disruption than the site in oPGHS-1. For each isoform, the effects of denaturant on inhibitor complex formation were almost the same as those on peroxidase and cyclooxygenase activity (Fig. 11, A and B). Replots of the data in Fig. 11 to show approximate values of K (calculated using Equation 2) as a function of the GdmHCl level produced parallel curves for PGHS-1 and -2 (not shown), much like the pattern obtained for the fluorescence data in the inset to Fig. 4A. Thus, the results in Fig. 11 indicate that both the cyclooxygenase and peroxidase active sites were less stable in hPGHS-2 than in oPGHS-1.
Comparison of Stabilities of Cyclooxygenase Activities in the Two Human PGHS Isoforms-Partially purified samples of hPGHS-1 and hPGHS-2, with comparable concentrations of the recombinant proteins, were equilibrated with various GdmHCl levels before assay of cyclooxygenase activity (Fig. 12). A monotonic loss of activity with increasing GdmHCl was seen with both isoforms, with more extensive loss of activity for hPGHS-2 than for hPGHS-1 at each denaturant level below 2 M. The IC 50 values for hPGHS-1 and hPGHS-2 were near 0.5 M and 0.2 M GdmHCl, respectively. The results of this comparison of hPGHS-1 with hPGHS-2 ( Fig. 12) were similar with those in the comparison of oPGHS-1 with hPGHS-2 (Fig. 7A). Thus, the cyclooxygenase activities of both hPGHS-1 and oPGHS-1 are more stable than the hPGHS-2 activity.
DISCUSSION
Previous hydrodynamic and cross-linking studies established that both oPGHS-1 and hPGHS-2 are dimers after solubilization with Tween 20 (33,34,42). The stabilizing effects of quaternary structure in oligomeric proteins increase the potential complexity of the denaturation process (15). Nevertheless, in many oligomers dissociation destabilizes the monomers to such an extent that these systems can be considered as twostate equilibria between native oligomers and completely unfolded monomers, permitting detailed thermodynamic analyses (15). With both oPGHS-1 and hPGHS-2, however, the complex patterns observed for loss of higher order structure with increasing denaturant level (Figs. 4, 6, 8, and 9) show that the two-state model does not completely describe the unfolding process for either PGHS isoform.
For both isoforms, the major loss of overall secondary and tertiary structure occurs above 0.5 M GdmHCl, with its onset overlapping with the transition from dimers to monomers monitored by hydrodynamic and cross-linking properties (Figs. 8 -10). It is important to emphasize that the dimer dissociation expected from the protein concentration-dependence of the fluorescence and activity denaturation curves (Figs. 4 and 7) is corroborated by the hydrodynamic and cross-linking observations (Figs. 8 -10). Dissociation does not totally disrupt the monomer structure in either isoform, because the monomers retain considerable structure and appear to be relatively compact until the denaturant level is raised considerably higher (Figs. 4A, 6, and 9). The later part of the decline in secondary and tertiary structure observed in fluorescence and CD measurements (Figs. 4A and 6) thus reflects the unfolding of these compact monomeric intermediates. Several examples are known of dimeric proteins dissociating to relatively stable monomers with considerable secondary and tertiary structure (36 -38, 43).
It is interesting that the loss of quaternary structure and activity is completely reversible for both isoforms, whereas unfolding of the monomers is not readily reversible (Figs. 4B, 7B, and 7C). This suggests that disordered PGHS monomers may not be able to properly fold to a compact intermediate by themselves. Once the compact monomer fold is reached, however, it seems likely that assembly into dimers can proceed spontaneously. The observed recovery of activity from PGHS-1 and -2 treated with GdmHCl levels sufficient to cause dissociation of the dimers into monomers (Figs. 7-10) raises the possibility of isolating undamaged monomers by judicious denaturant treatment. Such monomers would be very useful in evaluating the nature and functional consequences of monomer-monomer interactions in homodimers and heterodimers of the two PGHS isoforms.
Disruption of cyclooxygenase activity by GdmHCl was also sensitive to the protein concentration for both isoforms (Fig. 7A), demonstrating that loss of activity is coupled to dissociation of the dimers. However, significant loss of activity occurred at GdmHCl levels which produced little change in the fluorescence signal or in the hydrodynamic and cross-linking behavior (Figs. 4A, 7A, and 9 -11). This indicates some localized loss of active site structure occurs without disruption of the overall secondary or tertiary structure in the subunits, or of the interactions between the subunits. Thus, dimeric structure is necessary, but not sufficient, for activity in both PGHS isoforms.
A simple model consistent with the present data has two intermediates between native dimers (M-M) and completely unfolded monomers (U) (Reaction 1).
Here, dimer with disrupted active site structure is represented by M*-M*, and compact monomer by M c . For simplicity, the process is shown as a series of equilibria, although in practice only the first two steps appear fully reversible on the time scale tested (Figs. 4B, 7B, and 7C). The complexity of the denaturation process and the inability of denatured monomers to return to native dimers makes it difficult to calculate thermodynamic parameters for either isoform. However, the midpoint for dissociation to monomers was near 1.25 M GdmHCl for both oPGHS-1 and hPGHS-2 ( Fig. 9), indicating that the two isoforms have similar K 2 values. Because of this, if PGHS-1 and PGHS-2 are compared at the same protein concentration, the relative stabilities of their active site structure (i.e. the M-M 7 M*-M* process in Reaction 1) can be gauged from their sensitivities to inactivation by denaturant. To simplify this comparison, the IC 50 values for cyclooxygenase, peroxidase, and cyclooxygenase inhibitor binding activities calculated from the data in Figs. 7A, 11, and 12 are presented in Table I. It should be noted that these IC 50 values are not equivalent to the C m , Gdn 50 , or midpoint ⌬G values used in reference to thermodynamically better defined systems (15).
Two major points emerge from the data in Table I. First, the separate measurements of peroxidase and cyclooxygenase active site stability produce convergent values. The cyclooxygenase activity of both PGHS isoforms requires initiation via peroxidase intermediates (17,44,54). Because of this mechanistic linkage, disruption of the peroxidase site would be expected to lead to indirect loss of peroxidase activity. Thus, it is not surprising that the cyclooxygenase and peroxidase activities were lost in concert as the denaturant concentration was increased ( Figs. 7A and 11A). However, the binding of cyclooxygenase inhibitors, which provides a direct assessment of cyclooxygenase site integrity, displayed about the same sensitivity to denaturant as the enzymatic activities in both isoforms (Fig. 11). This indicates that the folding stability at the peroxidase site is comparable to that at the cyclooxygenase site. Although distinct from one another, the two active sites are parts of the same folded domain in both isoforms (4 -6), and it may be that disruption of one site is physically coupled to disruption of the other.
The second pattern evident from Table I is that all measurements of active site function indicate that the PGHS-1 active sites are considerably more stable than those in PGHS-2. It remains to be seen whether the lower stability of the PGHS-2 active sites to denaturant reflects a higher flexibility in the native state, as shown experimentally by measurement of con- formational mobility for several other enzymes (45). Higher flexibility in the active site might well be a major factor in the ability of PGHS-2 to accommodate the relatively bulky isoform-2 specific inhibitors, and it is consistent with the more expansive cyclooxygenase site observed in crystal structures of PGHS-2 with bound inhibitors (5,6). A lower cyclooxygenase active site flexibility in PGHS-1 would also help explain why acetylation of the active site serine by aspirin completely blocks fatty acid oxygenase activity in that isoform, whereas the steric impediment of the acetyl group is tolerated in the PGHS-2 cyclooxygenase active site (8,9,46).
The two amino acid differences between the isoforms in the immediate cyclooxygenase pocket, at positions 499 and 509 in hPGHS-2, have been shown to affect the interactions with specific inhibitors (11)(12)(13) and thus may contribute to the difference in active site stability between the isoforms. These substitutions are subtle, but addition of a single methylene group can significantly alter a protein's folding stability (47). The peroxidase site residues are less well conserved between the two PGHS isoforms, and so it is more difficult to postulate a structural basis for the difference between the isoforms in the stability at that site. Curiously, the loop near Arg-277 in PGHS-1 (Pro-263 in PGHS-2), which lies near the peroxidase site, is protease-sensitive in PGHS-1 but protease-resistant in PGHS-2 (48), suggesting that this structural element is more stable in PGHS-2.
The active site structures of both isoforms are more easily destabilized than the overall folding, with cyclooxygenase and peroxidase activities dropping significantly at GdmHCl levels which caused little change in fluorescence or CD intensities (Figs. 4, 6, 7, and 11). Thus, the enzymatic activities serve as sensitive indicators of the early stages of unfolding. The fact that the levels of non-ionic detergents commonly used to maintain the isoforms in solubilized form do not lead to loss of activity indicates that these detergents do not induce a significant degree of unfolding. The PGHS-1 and -2 crystallographic data were obtained using enzyme solubilized with such nonionic detergents (4 -6), further demonstrating that the levels of detergents generally used with the enzymes are not structurally disruptive. It is possible that the unfolding equilibria are perturbed by changes in detergent level. This would not change the conclusions about the relative stability of the two isoforms drawn in the present study, because a fixed, low concentration of Tween 20 was used for all experiments.
As mentioned above, the hydrodynamic results indicate that dissociation of dimers to monomers occurs over similar denaturant levels for the two PGHS isoforms, indicating little difference in the second step of the denaturation scheme in Reaction 1. Several amino acid differences between the isoforms have been noted in the subunit-subunit interface (5), but these apparently are not enough to have a large impact on the strength of the interaction between the subunits. The interface area in the PGHS-2 dimer, 2700 Å 2 (5), is well below the value of about 4000 Å 2 expected for a subunit of 70 kDa (49). Assuming that the strength of quaternary interactions is proportional to the interfacial area, this would predict that the quaternary interactions in PGHS are weaker than in the average dimer. This prediction is consistent with observations indicating dissociation of both PGHS isoforms without complete destabilization of the monomers (Figs. 4, 6, and 9).
Fluorescence, CD, and hydrodynamic changes at GdmHCl levels above 2 M were more pronounced for hPGHS-2 than for oPGHS-1 (Figs. 4A, 6, and 9). Both isoforms are predominantly in a monomeric state at these denaturant levels (Fig. 9), so the third step in the denaturation process, the unfolding of compact monomers (Reaction 1), appears to occur more readily in hPGHS-2 than in oPGHS-1. This would suggest a lower stability in PGHS-2 than in PGHS-1 of the secondary and tertiary structure in isolated monomers. The situation is complicated, however, by the fact that loss of secondary and tertiary structure begins well before complete conversion to monomers, and the structural disruption is sensitive to the protein concentration (Figs. 4A, 6, and 9). This indicates that considerable changes in subunit folding accompany dissociation of the dimers, with the fraction of subunit unfolding occurring during and after dissociation difficult to estimate. In any case, the overall stability of secondary and tertiary structure is considerably higher in oPGHS-1 than in hPGHS-2.
The sensitivity of the denaturation process to the protein concentration for both PGHS-1 and PGHS-2 (Figs. 4A and 7A) raises the possibility that the differences in denaturant susceptibilities between the isoforms are due to different amounts of "silent," inactive PGHS protein in the electrophoretically pure oPGHS-1 and hPGHS-2 preparations. One way to assess the presence of such inactive protein is to monitor the stoichiometry of heme cofactor needed to restore full cyclooxygenase activity to the purified apoenzyme. For oPGHS-1, titrations with heme had an average end point of about 0.7 heme/subunit (50 -52); similar titration of recombinant hPGHS-2 expressed in the baculovirus system produced an end point near 0.9 heme/subunit (34). The difference between the end points indicates a potential difference of about 30% in the proportion of inactive protein. This potential difference is unable to account for more than a small fraction of the observed shifts between the PGHS-1 and -2 curves, which were roughly equivalent to a 10-fold change in protein level (Figs. 4 and 7). As noted earlier, the oPGHS-1 helical content calculated from CD measurements is indistinguishable from that calculated from the crystal structure, indicating that the secondary structure was largely intact even if some of the preparation was unable to bind heme.
The present studies used detergent-solubilized preparations of the PGHS isoforms. Both isoforms are intrinsic membrane proteins and are thought to be bound to the endoplasmic reticulum membrane via amphipathic helical segments on one face of the dimeric structures (4 -6). The structural changes that occur upon solubilization are not known, but they do not grossly affect the enzymatic activities or the responsiveness to inhibitors. In addition, the putative membrane anchor comprises a relatively small, poorly conserved portion of the PGHS proteins, well removed from the interface between the subunits; the bulk of the proteins are globular (4 -6). All of this makes it likely that the native lipid membrane has little influence on the folding stability of either PGHS isoform, and that the folding stabilities of the isolated, solubilized proteins will be similar to those of the cellular enzymes.
oPGHS-1 was used in the present studies because the ovine enzyme can readily be purified in the quantities required for biophysical studies. Recombinant hPGHS-1 can be expressed in the baculoviral system, but we have found the yields to be considerably lower than those for hPGHS-2. The human and ovine PGHS-1 amino acid sequences have 91% identity and 96% similarity; conservation in the cyclooxygenase active site is essentially complete (53). There are very few non-conservative residue substitutions between human and ovine PGHS-1 at the interface between the subunits, suggesting a similar dimer stability in the two enzymes. Human and ovine PGHS-1 also have quite similar protease susceptibilities (48), and the cyclooxygenase activity of hPGHS-1, like that of oPGHS-1, is more resistant to denaturation than the hPGHS-2 activity (Figs. 7A and 11). These consistently similar properties of the human and ovine enzymes indicate that the conclusions drawn from the present studies with oPGHS-1 will be valid for hPGHS-1 as well.
In summary, results from these denaturation studies show that the two PGHS isoforms have similar quaternary structure stabilities, but differ markedly in their secondary and tertiary structural stabilities, with PGHS-2 having a lower folding stability both in the cyclooxygenase and peroxidase active sites and in the subunit as a whole. This difference in the stability of the folding of the two PGHS isoforms may well be related to the observed differences in their functional interactions with substrates, activators, and inhibitors. | v3-fos-license |
2018-04-03T04:30:08.045Z | 2013-02-09T00:00:00.000 | 208930568 | {
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} | pes2o/s2orc | (Z)-N-[2-(N′-Hydroxycarbamimidoyl)phenyl]acetamide
The asymmetric unit of the title compound, C9H11N3O2, contains two molecules (A and B), which exist in Z conformations with respect to their C=N double bond. The dihedral angles between the benzene ring and the pendant hydroxycarbamimidoyl and acetamide groups are 28.58 (7) and 1.30 (5)°, respectively, in molecule A and 25.04 (7) and 27.85 (9)°, respectively, in molecule B. An intramolecular N—H⋯N hydrogen bond generates an S(6) ring in both molecules. Molecule A also features an intramolecular C—H⋯O interaction, which closes an S(6) ring. In the crystal, the molecules are linked by N—H⋯O, N—H⋯N, O—H⋯O, O—H⋯N, C—H⋯O and C—H⋯N hydrogen bonds and C—H⋯π interactions, generating a three-dimensional network.
Data collection: APEX2 (Bruker, 2009); cell refinement: SAINT (Bruker, 2009); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON (Spek, 2009 Amidoximes are bi-functional molecules exhibiting a rich and diverse chemistry and provides the shortest way to reach certain heterocycles, such as 1,2,4-oxadiazoles (Clapp, 1976(Clapp, , 1984Jochims, 1996). They are also considered interesting molecules in view of their biological applications (Fylaktakidou et al., 2008). Their ability to release NO or nitrites in vitro and in vivo experiments has recently attracted attention (Mansuy et al., 2004;Kontogiorgis et al., 2002). The discovery that nitric oxide (NO) acts as an important mediator of smooth muscle relaxation has led us to the preparation and testing of a wide variety of compounds with the aim of finding suitable new NO-donors. In the process, the title compound, (Z)-N-(2-(N′-hydroxycarbamimidoyl) phenyl)acetamide (Wang et al., 2002) was prepared.
The title compound consist of two crystallographically independent molecules (A and B) as shown in Fig. 1. The molecules exist in Z configuration with respect to the C7A ═N1A and C7B ═N1B double bonds. The intramolecular N3 -H3···N1 hydrogen bonds (Table 1) form S(6) ring motifs (Bernstein et al., 1995) in both molecules. Molecule A is stabilized by an additional intramolecular C2A-H2AA···O2A hydrogen bond (Table 1) which also generates an S(6) ring motif (Bernstein et al., 1995). The bond lengths (Allen et al., 1987) and angles are within normal ranges.
Experimental
Equimolar amount of N-(2-cyanophenyl)acetamide (10 mmol) and NH 2 OH.HCl (10 mmol) were dissolved in a minimum amount of methanol(10 ml)-water (5 ml) and followed by the addition of Na 2 CO 3 (5 mmol). The solution was refluxed for 2 h. The solid product formed was collected through filtration and then evaporated to dryness. The product was redissolved in MeOH for recrystalliziation as colourless plates. M. P.: 145°C.
Refinement
All N and O bound H atoms were located from the difference map and were refined freely [N-H = 0.857 (19)-0.90 (2) Å and O-H = 0.93 (2) and 0.95 (2) Å]. The remaining H atoms were positioned geometrically and refined using a riding model with U iso (H) = 1.2 or 1.5U eq (C) (C-H = 0.9500 and 0.9800 Å). A rotating group model was applied to the methyl groups.
Figure 1
The molecular structure of the title compound, showing 50% probability displacement ellipsoids. Dashed lines indicate the intramolecular hydrogen bonds. (Cosier & Glazer, 1986) operating at 100 (1) K. Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. | v3-fos-license |
2017-05-08T23:58:54.928Z | 2012-09-24T00:00:00.000 | 41380844 | {
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} | pes2o/s2orc | Radically Enhanced Molecular Switches
: The mechanism governing the redox-stimulated switching behavior of a tristable [2]rotaxane consisting of a cyclobis(paraquat- p -phenylene) (CBPQT 4+ ) ring encircling a dumbbell, containing tetrathiafulvalene (TTF) and 1,5-dioxynaphthalene (DNP) recognition units which are separated from each other along a polyether chain carrying 2,6-diisopropylphenyl stoppers by a 4,4 ′ -bipyridinium (BIPY 2+ ) unit, is described. The BIPY 2+ unit acts to increase the lifetime of the metastable state coconformation (MSCC) signi fi cantly by restricting the shuttling motion of the CBPQT 4+ ring to such an extent that the MSCC can be isolated in the solid state and is stable for weeks on end. As controls, the redox-induced mechanism of switching of two bistable [2]rotaxanes and one bistable [2]catenane composed of CBPQT 4+ rings encircling dumbbells or macrocyclic polyethers, respectively, that contain a BIPY 2+ unit with either a TTF or DNP unit, is investigated. Variable scan-rate cyclic voltammetry and digital simulations of the tristable and bistable [2]rotaxanes and [2]catenane reveal a mechanism which involves a bisradical state coconformation (BRCC) in which only one of the BIPY • + units in the CBPQT 2( • +) ring is oxidized to the BIPY 2+ dication. This observation of the BRCC was further con fi rmed by theoretical calculations as well as by X-ray crystallography of the [2]catenane in its bisradical tetracationic redox state. It is evident that the incorporation of a kinetic barrier between the donor recognition units in the tristable [2]rotaxane can prolong the lifetime and stability of the MSCC, an observation which augurs well for the development of nonvolatile molecular fl ash memory devices.
■ INTRODUCTION
The drive to achieve the miniaturization of electronic devices beyond the limits that "top-down" conventional lithographic techniques can provide has led 1 to the research and development of an array of "bottom-up" protocols. One approach invented and implemented by Hawker et al. 2 relies on the use of nanoscale lithographic templates made possible by the self-assembly of block copolymers into highly ordered hexagonal and rectilinear arrays. Another approach created and developed by Mirkin et al. 3 relies on the use of atomic force microscopy (AFM) tips within the context of what has become known as dip-pen nanolithography (DPN), which when used as massively parallel arrays of "ink"-coated tips can deliver soft organic substrates onto hard surfaces with remarkable degrees of order and complexity. In the realm of molecular electronics, 4 where the objective very often is to understand the electronic behavior of single molecules bridging the gap between two electrodes, some extraordinary advances have been made 5 in recent years. For example, Fujita et al. 5b have demonstrated the use of self-assembled nanocages in between a gold substrate and a scanning tunneling microscopy (STM) tip, which act as hosts for a relatively small number of π-stacked organic guests. By controlling the size of the cage, the researchers were able to investigate the conductance of single discrete stacks of two, three, and four π-electron-deficient anthracene-type moieties in a very precise manner. In collaboration with Heath, 6 we have relied on fabrication of nanoelectronic devices with two-dimensional "cross-bar" architectures, employing a superlattice nanowire pattern transfer 7 (SNAP) methodology, in which bistable [2]rotaxane 8 molecules by the hundreds or thousands are sandwiched as molecular monolayers between the top and bottom crossbar elements in a molecular switch tunnel junction (MSTJ). These bistable rotaxanes served as the active binary components 6f of this molecular memory device. This device has demonstrated 6f data density storage capacity predicted at the time (2007) by Moore's Law not to become available until at least the year 2020.
The bistable [2]rotaxanes that were incorporated into these memory devices were those whose redox-active switching is achieved by employing the π-electron-poor tetracationic cyclophane, cyclobis(paraquat-p-phenylene) 9 (CBPQT 4+ ), mechanically interlocked around a dumbbell component incorporating π-electron-rich 1,5-dioxynaphthalene (DNP) and redoxactive tetrathiafulvalene 10 (TTF) units. The CBPQT 4+ ring encircles the TTF unit predominantly in the ground state of such bistable [2]rotaxanes at equilibrium, leading to the naming 11 of this translational isomer as the ground-state coconformation (GSCC). The ring can be made to encircle the DNP unit, as a result of an oxidation/reduction cycle of TTF, to form a translational isomer known as the metastable-state coconformation 11 (MSCC). The fact that the MSCC represents a higher conductive state than the GSCC was exploited 6f to achieve single bits of memory in the context of the device, i.e., the GSCC represents a "0" and the MSCC represents a "1" for a collection of these bistable [2]rotaxanes possessing these particular states in monolayers trapped within an array of MSTJs. The memory implanted in these devices is volatile, however, and only lasts 6f for about an hour. One of the hypotheses which attempts to rationalize the volatile nature of these molecular memory devices is one that reasons that the relaxation of the MSCC back to the GSCC also occurs after about an hour within the MSTJ environment. Therefore, developing a strategy to increase the lifetime of the MSCC provides a means of testing this hypothesis, and also holds out the prospect of being able to construct nonvolatile molecular memory devices.
A strategy which has undergone significant scrutiny in order to increase the lifetime of the MSCC has been one focused on inserting kinetic barriers 12 between the TTF and DNP units that would act to slow down the shuttling motion of the CBPQT 4+ ring, trapping the MSCC kinetically once it is populated. Such kinetic barriers or "speed bumps" have included steric ones, 13 such as trans-azobenzene 13e,g units or even foldamers. 13f Another strategy employs the use of electrostatic barriers, 14 such as 4,4′-bipyridinium dicationic (BIPY 2+ ) units, 15 which, on account of Coulombic repulsion with the CBPQT 4+ ring, have also been shown 14c−e to increase the lifetime of the MSCC. Furthermore, once we have a way of trapping the MSCC, we must also have the means of releasing it in a timely manner. Such a capability would provide an erase mechanism in the context of molecular memory devices, allowing for the development 16 of bistable rotaxane-based molecular flash memory.
Herein, we describe a detailed mechanistic investigation of the redox-active switching behavior of a [2]rotaxane R3 6+ (Scheme 1) composed of the CBPQT 4+ ring, mechanically interlocked with a dumbbell component containing a BIPY 2+ unit flanked on each side by a TTF and a DNP unit. This investigation is supported by study of its related [2]rotaxanes R1 6+ and R2 6+ , wherein, respectively, either TTF or DNP is absent from the dumbbell component along with a bistable [2]catenane C1 6+ incorporating a macrocyclic polyether which contains DNP and BIPY 2+ units. We show that the BIPY 2+ unit in R3 6+ serves as such an effective electrostatic barrier that it is possible to employ a synthetic procedure for trapping and isolating the MSCC as a pure solid. In particular, we report the reduction-induced switching that makes use of favorable radical−radical interactions, 17 which occur between BIPY •+ radical cations in the ring and dumbbell/macrocycle components in these mechanically interlocked molecules (MIMs), using variable temperature and variable scan-rate cyclic voltammetry (CV) in combination with digital simulations and X-ray crystallography. Moreover, we show by variable scan-rate CV as well as by UV−vis and 1 H NMR spectroscopies that we can employ the dynamic nature of these radical−radical interactions to restore the GSCC from the kinetically trapped MSCC. Such a demonstration also outlines a strategy 18 for gaining a large degree of control over the relative mechanical motions of the components within a switchable MIM that relies on only one type of stimulus, i.e., redox chemistry.
Design (Scheme 1), which can be switched electrochemically between three thermodynamic states that alter the translational position of the ring. Upon oxidation of the TTF unit, the CBPQT 4+ ring is obliged to translate over the BIPY 2+ unit in order to encircle the DNP unit, in a pathway which is activated 19 by Coulombic energy. This Coulombic repulsion between the CBPQT 4+ ring and TTF •+/2+ unit is what enables the ring to pass over the BIPY 2+ electrostatic barrier rapidly, in a process which we hypothesize, 20 based on theoretical calculations, proceeds at a rate of approximately 10 8 s −1 . We have also demonstrated 11c,14c beforehand that thermal relaxation of the MSCC to the GSCC, that is, translation of the CBPQT 4+ ring from the DNP unit over the BIPY 2+ electrostatic barrier and onto the TTF unit, occurs at a rate governed by a free energy barrier 21 of 19 kcal mol −1 . The rate of relaxation is decreased 14c 100-fold as a consequence of the presence of the BIPY 2+ unit; therefore, we have defined (Scheme 2) the coconformation in which the CBPQT 4+ ring is positioned over the BIPY 2+ unit as the transition-state coconformation (TSCC) and, as the name implies, is not actually a thermodynamically stable state yet is a coconformation that must be considered in order for shuttling of the CBPQT 4+ ring to occur. The notion of the TSCC is a mechanistic feature which is necessary to consider when discussing (vide inf ra) the reduction-based mechanism of switching in all the MIMs.
In relation to the ability of the rotaxane R3 6+ to switch by virtue of BIPY •+ radical−radical interactions, we have shown 17h−j previously that upon a three-electron reduction in which two electrons are transferred to the CBPQT 4+ ring and one to the BIPY 2+ unit of the dumbbell, the resulting CBPQT 2(•+) ring encircles the BIPY •+ radical cation as the most thermodynamically stable coconformation. We label (Scheme 2) this translational isomer as the radical-state coconformation (RSCC). The stability of this state comes about through favorable radical−radical interactions, referred to previously as "pimerization", 17c that is, a process which occurs between two or more BIPY •+ radical cations. We reported 17j recently a detailed investigation of the supramolecular properties during the formation of the trisradical complex which occurs spontaneously between the CBPQT 2(•+) ring and the methyl viologen radical cation, MV •+ . In particular, the mechanism involves a bisradical intermediate CBPQT (•+)(2+) ⊂ MV •+ en route to reoxidation of the trisradical complex to its fully oxidized form. This bisradical intermediate occurs as a consequence of spin-pairing to a singlet state, such that only two BIPY •+ radical cations of the complex are paired at any one time, rendering the third unpaired BIPY •+ easier to oxidize in relation to the other two. We demonstrate in this paper that a similar bisradical intermediate is involved in the switching mechanism of these MIMs. As a consequence of the fact that the CBPQT (•+)(2+) ring component contains both BIPY 2+ and BIPY •+ units in the bisradical tetracationic redox state of these MIMs, the ring is both "donor-loving" and "radical-loving" at the same time. This dichotomy leads to the shuttling of the CBPQT (•+)(2+) ring from the BIPY •+ radical cation unit to the π-electron-rich donor unit, i.e., TTF or DNP. We label the coconformation (Scheme 2) when the CBPQT (•+)(2+) ring encircles the BIPY •+ radical cation as the bisradical state coconformation (BRCC). When the CBPQT (•+)(2+) ring encircles either the TTF or DNP unit, we define it as the bisradical donor state coconformation (BDCC). This shuttling of the CBPQT (2+)(•+) ring can be harnessed further in the case of the rotaxane R3 6+ so as to remove the dependence involved in restoring the GSCC from the MSCC by means of a thermally activated pathway, by providing a redox-activated one. We now proceed in our discussion by first of all considering the mechanistic features of the simpler cases of the two [2]rotaxanes R1 6+ and R2 6+ as well as those of the [2]catenane C1 6+ .
■ RESULTS AND DISCUSSION CV and Digital Simulations.
[2]Catenane C1 6+ . The synthesis of the [2]catenane C1 6+ was achieved using a previously reported 22 protocol. The electrochemical switching of the catenane C1 6+ was investigated by variable scan-rate CV and compared to digital simulations generated from the proposed mechanism. At the slow scan rate of 20 mV s −1 (Figure 1a, purple trace), an initial three-electron reduction process is observed (−0.3 V), with two electrons being transferred to the CBPQT 4+ ring and one to the BIPY 2+ of the macrocyclic polyether. Following this three-electron transfer, the RSCC becomes the predominant translational isomer in solution on account of the radical−radical interactions that ensue between the BIPY •+ radical cations of the ring and the macrocyclic polyether. This result has been previously confirmed 22 by spectroelectrochemistry.
A one-electron process is observed at −0.77 V (peak potential), assignable to the reduction of the unpaired BIPY •+ Scheme 2. Generalized Graphical Representations of the Assigned Coconformations for the Trisradical, Bisradical and Fully-Oxidized Electronic States a a In the case of the tristable rotaxane R3 6+ , the ground redox state can exist in three forms, as the thermodynamically stable GSCC, the MSCC, and the activated TSCC. The bisradical oxidation state has two forms, namely, the BRCC and the BDCC. It is important to note that, in the BDCC, one of the radicals is located on the CBPQT (•+)(2+) ring, while the other is still associated with the BIPY •+ unit in the dumbbell or macrocycle component, which are not shown. The trisradical state is only observed to have one form, defined as the RSCC. Shown below are specific examples of the rotaxane R3 6+ in its GSCC and MSCC and once reduced to its trisradical form, its RSCC. radical cation unit of the CBPQT 2(•+) ring, while a two-electron process observed at −1.10 V (peak potential) is the result of the simultaneous transfer of electrons to the remaining paired BIPY •+ radical cations. The return scan shows that these two redox processes are independent of scan rate (20−1000 mV s −1 ) and are totally reversible.
Journal of the American Chemical Society
Reoxidation of the trisradical RSCC displays, however, scanrate-dependent behavior similar to that observed previously 17j for a trisradical host−guest complex. On scanning at a slow rate, a single broad three-electron reoxidation peak is observed at −0.25 V. At faster and faster scan rates, it becomes apparent that reoxidation of the trisradical RSCC occurs via two different reoxidation processes. As the scan rate is increased, an oxidation peak at 0 V emerges. The oxidation process at −0.25 V is assigned to the one-electron oxidation of the unpaired BIPY •+ unit of the CBPQT 2(•+) ring, while the peak at 0 V is assigned to the simultaneous oxidation of the two spinpaired BIPY •+ units in a singlet state. In order to explain the scan-rate-dependent behavior, we have proposed a mechanism of switching involving the bisradical intermediate in which the catenane C1 6+ , its proposed mechanism for electrochemical switching, and the digitally simulated data generated from the proposed mechanism. The purple trace was obtained at a scan rate of 20 mV s −1 , while intermediate traces recorded at higher scan rates are shown in black leading up to a scan rate of 1000 mV s −1 , shown as the red trace. The reoxidation peak at 0 V increases in relative intensity in comparison to the reoxidation peak observed at −0.25 V with faster scan rates. (b) Variable scan-rate CV of the [2]rotaxane R2 6+ , proposed mechanism, and digitally simulated data. The purple trace was recorded at a scan-rate of 50 mV s −1 with intermediate traces taken at higher scan rates shown in black, leading up to a scan rate of 1200 mV s −1 shown as the green trace. The reoxidation peak at 0 V increases in relative intensity with faster scan rates. Both the catenane C1 6+ and R2 6+ were found to display the same basic mechanism underlying their electrochemical switching processes. In particular, the observed scan-rate-dependent nature of the CV traces of both MIMs is explained by an equilibrium between two coconformations in their bisradical electronic states. CBPQT (•+)(2+) ring undergoes shuttling from the BIPY •+ (BRCC) to the DNP (BDCC) unit. This shuttling motion occurs 23 on account of the fact that the CBPQT (•+)(2+) ring is capable of recognizing both radicals and π-electron-rich donors. Once encircled around the DNP unit as a consequence of donor−acceptor interactions, reoxidation of the resulting unpaired BIPY •+ radical cations shifts to more negative voltages, resulting in a single broad reoxidation peak. If insufficient time is allowed for shuttling to occur, reoxidation of the bisradical occurs as the BRCC, i.e., the two BIPY •+ radicals are still paired as they undergo oxidation. This oxidation results in formation of the TSCC for a very brief period before it relaxes back to the ground state, where the CBPQT 4+ ring encircles the DNP unit exclusively. X-Ray crystallography supports the existence of the BRCC intermediate in the solid state. Digital simulations modeled on the basis of the proposed mechanism agree well with the experimental data. Comparison with the simulated data leads to a rate of shuttling of 25 s −1 , a rate which corresponds to a free energy barrier to shuttling of 15.5 kcal mol −1 at room temperature.
Journal of the American Chemical Society
[2]Rotaxanes R1 6+ and R2 6+ . The syntheses of the [2]rotaxanes R1 6+ and R2 6+ were achieved by following previously reported procedures. 17h We also investigated R1 6+ and R2 6+ which are composed of the CBPQT 4+ ring mechanically interlocked with dumbbells containing BIPY 2+ and DNP or TTF, units, respectively, by variable scan-rate CV. Similar variable scan-rate-dependent behavior was observed (Figure 1b and SI) with these rotaxanes in comparison with the [2]catenane C1 6+ , an observation which indicates a similar electrochemical switching mechanism is operating within these different MIMs. For both R1 6+ and R2 6+ , at relatively fast scan rates, a reoxidation peak is observed at 0 V. At relatively slow scan rates, this second reoxidation peak is not observed, rather only a single broad reoxidation peak. In accordance with the proposed mechanism of switching for the catenane C1 6+ , the bisradical forms of R1 6+ and R2 6+ both involve shuttling of the CBPQT (•+)(2+) ring from the BIPY •+ unit (BRCC) to the πelectron-rich DNP or TTF units (BDCC), respectively. By comparing the experimental with the simulated data 24 based on the proposed mechanism, we have ascertained a barrier to shuttling of the CBPQT (•+)(2+) ring from the BRCC to the BDCC to be equal to 16.1 and 14.2 kcal mol −1 for R1 6+ and R2 6+ , respectively.
On comparing R1 6+ with R2 6+ , the difference in the barrier to shuttling (Figure 2) from the BRCC to the BDCC is 1.9 kcal mol −1 . This difference in the barriers to shuttling is approximately equal to the difference in binding energies 11c between the TTF-DEG ⊂ CBPQT 4+ and DNP-DEG ⊂ CBPQT 4+ (DEG = diethylene glycol) [2]pseudorotaxanes when they are measured in MeCN. We hypothesize, that the affinity of the π-donor in the dumbbell component for the dicationic BIPY 2+ unit of CBPQT (•+)(2+) ring has a stabilizing influence on the transition state involved in the shuttling of the CBPQT (•+)(2+) ring from the BRCC to the BDCC, a situation which we will discuss in more detail in the case of the rotaxane R3 6+ .
Tristable [2]Rotaxane R3 6+ . With the discussion of the relaxation of the bistable [2]rotaxanes R1 6+ and R2 6+ complete, we will now turn our attention toward a consideration of the tristable [2]rotaxane R3 6+ incorporating both TTF and DNP recognition sites as well as the BIPY 2+ unit located between the two π-electron-rich donor sites. It is important to note the significance of the BIPY 2+ unit being located centrally between the TTF and DNP units. Similar scan-rate-dependent behavior was also observed (see SI) in the case of this rotaxane. Upon a one-electron oxidation of the trisradical RSCC of the rotaxane R3 6+ to form the BRCC, a priori, the CBPQT (•+)(2+) ring has two options: it can either undergo translation (i) to encircle the TTF unit or in the opposite direction (ii) to encircle the DNP unit. In the knowledge that the barrier to relaxation is significantly lower in the rotaxane R2 6+ incorporating TTF compared to that of the rotaxane R1 6+ incorporating DNP, we would expect the shuttling of the CBPQT (•+)(2+) ring in the BRCC of the tristable rotaxane R3 6+ to favor translation predominantly to the TTF unit, rather than to the DNP unit. The variable scan-rate behavior of R3 6+ is illustrated in the SI. By simulation, the barrier to shuttling of the CBPQT (•+)(2+) ring was measured to be 14.2 kcal mol −1 , a ΔG ‡ value which is experimentally the same as that determined for the rotaxane R2 6+ . These data suggest the shuttling (Figure 2) in the BRCC of the tristable [2]rotaxane R3 6+ occurs predominantly toward having the CBPQT (•+)(2+) ring encircle the TTF unit in the BDCC. Variable-temperature, variable scan-rate CV experiments reveal (see SI) that this shuttling motion is a thermally activated mechanical process, with the rate of shuttling becoming slower with decreasing temperature. It is important to realize this fact when considering the hypothesis that the presence of the BIPY 2+ dication in the CBPQT (•+)(2+) component may act to repel the ring from the BIPY •+ of the dumbbell as a consequence of Coulombic repulsiona process that would not be reliant on thermal energy. This hypothesis can be discredited, however, since the rate of shuttling is indeed temperature dependent. From the variable-temperature, variable scan-rate CV experiments, we were able to determine that the enthalpic ΔH ‡ and entropic ΔS ‡ contributions to the transition-state energy for this shuttling process are +17.6 kcal mol −1 and +11.2 cal mol −1 K −1 , respectively. These activation parameters stand in contrast 11c to those (ΔH ‡ = +8.4 kcal mol −1 , ΔS ‡ = −31 cal mol −1 K −1 ) associated with the shuttling Figure 2. Thermodynamic landscapes constructed from variable scanrate CV data of the three rotaxanes R1 6+ , R2 6+ , and R3 6+ depicting the mechanical switching motion of the CBPQT (•+)(2+) ring in the case when all three of these rotaxanes are in their bisradical tetracationic redox states. The CBPQT (•+)(2+) ring starts out encircling the BIPY •+ unit of the dumbbell component as the BRCC. The ring then undergoes translational motion in order to encircle the π-electron-rich units (DNP or TTF) in the BDCC at a rate governed by the free energy barriers ΔG ‡ as shown. The rate of shuttling is faster in the case of R2 6+ than in the case of R1 6+ by over an order of magnitude. In the case of R3 6+ , the rate of shuttling is experimentally the same as the rate measured for R2 6+ , pointing strongly to the fact that shuttling of the CBPQT 4+ ring occurs in a mechanostereochemically selective fashion toward the TTF unit. of the CBPQT 4+ ring from DNP to a bispyrrol-TTF unit obtained for a previously reported 11c bistable [2]rotaxane employing these components. We hypothesize that the comparatively large enthalpic contribution to the barrier associated with the shuttling of the CBPQT (•+)(2+) ring is a consequence of the energy required to unpair 25 the radical electrons of two interacting BIPY •+ radical cations before any mechanical motion can occur. We also propose that the transition state is further stabilized by folding 10h of the flexible oligoethylene glycol chains that result when intramolecular donor−acceptor interactions 14e occur between the BIPY 2+ unit of the ring and the TTF unit of the dumbbell. These intramolecular donor−acceptor interactions also explain the differences in the ΔG ‡ values between R1 6+ and R2 6+ as well as between R1 6+ and the catenane C1 6+ . The fact that the difference in the energy barriers to shuttling for R1 6+ and R2 6+ is 1.9 kcal mol −1 , approximately the difference in the binding energy 11c between the CBPQT 4+ ring with diethylene glycol derivatives of TTF (−7.66 kcal mol −1 ) and DNP (−6.26 kcal mol −1 ), respectively, provides further evidence in support of the hypothesis. The greater affinity of TTF for the BIPY 2+ unit of the CBPQT (•+)(2+) ring results in increased stabilization of the transition state than does the DNP unit, and so the barrier to shuttling is larger in the case of the rotaxane R1 6+ . The fact that the barrier to shuttling is slightly lower in the case of the catenane C1 6+ compared to R1 6+ is a result of the geometry of the macrocyclic polyether, which enforces 26 stabilizing donor−acceptor interactions of the DNP with the BIPY 2+ unit. All these kinetic data for C1 6+ , R1 6+ , R2 6+ , and R3 6+ are summarized in Table 1.
Journal of the American Chemical Society
In order to provide additional evidence from CV (Figure 3), that after the one-electron reoxidation of the trisradical RSCC to the bisradical BRCC of the [2]rotaxane R3 6+ , shuttling of the CBPQT (•+)(2+) ring occurs predominantly in the direction of the TTF unit, we employed TTF as a redox probe 27 for the position of the ring. In a previous communication 14c dealing with the tristable [2]rotaxane R3 6+ , we reported on extending the lifetime of the MSCC that has the CBPQT 4+ ring encircled around the DNP unit through incorporation of the BIPY 2+ unit. This BIPY 2+ unit acts as an electrostatic barrier to relaxation of the MSCC to the GSCC, which has the CBPQT 4+ ring encircled around the TTF unit. Upon oxidation of the TTF unit, the CBPQT 4+ ring is very quickly repelled along a pathway activated by Coulombic energy over the BIPY 2+ electrostatic barrier to encircle the DNP unit. Upon reduction of the TTF 2+ dication back to its neutral state, the CBPQT 4+ ring remains around the DNP unit for some time before passing over the BIPY 2+ barrier by means of a thermally activated process accessing the TSCC as the transition state to this shuttling in order to encircle the TTF unit once again, restoring the ground-state distribution of 10:1/GSCC:MSCC. This relaxation is governed 14c by a barrier of 19 kcal mol −1 in MeCN at 298 K with 0.1 M TBA·PF 6 , an increase of 3 kcal mol −1 from the previously reported 11c [2]rotaxane, omitting the central BIPY 2+ moiety.
In the CV, we scanned ( Figure 3a) at 50 mV s −1 a potential window, starting at +1 V and reversing the scan at −0.60 V. Starting at +1 V generates the TTF 2+ dication, enticing the CBPQT 4+ ring to pass over the BIPY 2+ electrostatic barrier and encircle the DNP unit. Rereduction of TTF 2+ dication occurs by means of two one-electron processes, after which the CBPQT 4+ ring remains encircled around the DNP unit, kinetically trapped before passing over the electrostatic BIPY 2+ barrier to encircle the more favored TTF unit. Scanning Figure 3. (a) The red trace CV taken at a scan rate of 50 mV s −1 of the [2]rotaxane R3 6+ reveals evidence for the formation of the MSCC through an oxidation−reduction cycle of the TTF moiety. The CV begins at +1 V, with the oxidation of the TTF unit to its TTF 2+ dication, which repels the tetracationic CBPQT 4+ ring over the BIPY 2+ electrostatic barrier to encircle the DNP unit. Scanning toward 0 V reduces the TTF 2+ dication back to its neutral form, resulting in the MSCC wherein the CBPQT 4+ remains encircled for some time around the DNP subunit before passing over the BIPY 2+ electrostatic barrier back onto TTF to form the GSCC. At this scan rate, the amount of "free" TTF still remaining, as measured from the return scan, is 50%. The black trace CV also recorded at a scan rate of 50 mV s −1 starting at +1 V, but with the scan limit of −0.60 V, which is enough to ensure the formation of the RSCC. The return scan shows that the amount of free TTF is now only 10% and corresponds to the GSCC/MSCC distribution ratio of 10:1 at equilibrium. (b) Schematic representation for the formation of the MSCC which can be reset to the GSCC through thermal relaxation or by accessing the RSCC in the pathway described above. further past 0 V toward −0.60 V invokes the three-electron reduction process, generating the RSCC. It is important to note that prior to this three-electron reduction, the CBPQT 4+ ring has not had enough time to relax fully back to the TTF unit, a conclusion which can be reached by taking a scan only to the potential of 0 V and back. In this CV experiment, approximately 50% of free TTF still exists, which is significantly far from the equilibrium distribution of 10% free TTF, i.e., 10:1/GSCC:MSCC. A scan rate of 50 mVs −1 is slow enough such that (i) nearly all of the BRCC generated after the initial one-electron oxidation is allowed to shuttle to the BDCC and (ii) becomes reoxidized after shuttling has occurred, as indicated by the observation that almost no second reoxidation peak at 0 V can be detected. Scanning further past 0 V toward +1 V reveals two oxidation peaks, the first of which corresponds to the initial oxidation of free TTF, followed by the oxidation of encircled TTF combined with the second oxidation of free TTF. Quantification of the relative amount of free TTF from an integration analysis of these two oxidation peaks reveals that the amount of free TTF detected, after proceeding through a reoxidation pathway where only the BDCC is oxidized (and not the BRCC), is approximately 10%, a percentage that is consistent with the ground-state equilibrium distribution ratio. These results provide strong evidence that, after the one-electron oxidation of the trisradical RSCC of the tristable rotaxane R3 6+ to form the BRCC, the CBPQT (•+)(2+) of the BRCC shuttles in a mechanostereochemical selective fashion onto TTF in the BDCC (Figure 3b) as a consequence of the faster kinetics associated with this process in comparison with translation toward the DNP unit. Furthermore, by taking advantage of this mechanostereochemical selective pathway forged out by the trisradical species, we can restore the rotaxane to its GSCC. Since it is possible to control the reoxidation pathway depending on the scan rate employed, we investigated further how the CBPQT 4+ ring is propelled when the reoxidation occurs, while in the BRCC, wherein CBPQT (•+)(2+) is still encircling the BIPY •+ radical cation, forming the TSCC. We performed a similar experiment (Figure 4a) by scanning a potential window starting at +1 V and reversing the scan at −0.60 V, only this time, at 1000 mV s −1 . This scan rate is fast enough to ensure that almost none of the BRCC has had time to shuttle to the BDCC, and so nearly all the oxidation will occur while the ring is encircled around the BIPY •+ radical cation corresponding to the TSCC for the rotaxane R3 6+ . Starting at a potential of +1 V generates the TTF 2+ dication. Scanning toward 0 V reduces the TTF 2+ back to its neutral state through two one-electron processes. Scanning further toward −0.60 V forms the trisradical RSCC. In this reoxidation pathway, the highly transient TSCC is momentarily attained as a result of the second two-electron oxidation at 0 V, that follows after the first one-electron oxidation of the noninteracting BIPY •+ of the CBPQT 2(•+) ring encircling the BIPY •+ of the dumbbell component. The TSCC is not stable since it possesses a large amount of Coulombic potential energy, and a priori, the CBPQT 4+ ring has two choices once again − it can either be repelled toward the TTF unit or toward the DNP unit. Scanning past 0 V toward +1 V reveals two oneelectron oxidation processes corresponding to the first oxidation of free TTF and a combination of the first oxidation of encircled TTF and the second oxidation of free TTF. Quantification of these oxidation waves reveals the existence of approximately 30% free TTF, indicating that a majority of the ring has been pushed onto the TTF unit rather than onto the DNP unit from the TSCC. It is important to note that, prior to reduction, approximately 90% of the CBPQT 4+ ring is still located on the DNP unit. The amount of free TTF can be reduced by 60 to 30% on going along this reoxidation pathway, relying only upon the TSCC, which is almost the GSCC equilibrium of 10% free TTF. Taken together, these results demonstrate (Figure 4b) that the restoration of the GSCC can be achieved through a redox-activated pathway, and the reliance on an entirely thermally activated pathway is not necessary.
X-Ray Crystallography. The solid-state structure of the fully oxidized C1·6PF 6 was previously reported 22 by us, and we have reproduced a depiction of it here for the sake of convenience. In particular, the structure reveals (Figure 5a) that the DNP unit is located inside the cavity of the CBPQT 4+ ring, while the BIPY 2+ unit is located as far as possible as a result of Coulombic repulsion. We now report our investigation of the catenane in its reduced form. Solid-state evidence of the rotaxane R3 6+ provides evidence for the formation of the MSCC as a result of an oxidation−reduction cycle implemented on the TTF unit. The CV begins at +1 V with the oxidation of the TTF unit to its TTF 2+ dication, which repels the tetracationic CBPQT 4+ ring over the BIPY 2+ electrostatic barrier to encircle the DNP unit. Scanning toward 0 V reduces the TTF 2+ dication back to its neutral state, forming the MSCC, wherein the CBPQT 4+ ring remains encircled for some time around the DNP unit before passing over the BIPY 2+ electrostatic barrier back onto the TTF unit to form the GSCC. At this scan rate, the amount of "free" TTF still remaining, as measured by the return scan, is 90%. The black trace CV also taken at a scan rate of 1000 mV s −1 also starts at +1 V but with the scan limit of −0.60 V, which is enough to ensure the formation of the RSCC. The return scan shows that the amount of free TTF is now only 30%. (b) Schematic representation for the formation of the MSCC, which can be reset to the GSCC through thermal relaxation or by going through the RSCC in the pathway discussed above. bisradical redox state has been obtained by single crystal X-ray crystallography. The [2]catenane C1·6PF 6 was dissolved in MeCN, and excess of zinc dust 17j was added to the solution in order to reduce the catenane to its trisradical form. Slow vapor diffusion of iPr 2 O into this solution after removal of the zinc dust afforded single crystals suitable for X-ray analysis. The solid-state structure 28 reveals (Figure 5b,c) that there are four PF 6 − counterions per catenane molecule, an observation which implies the catenane is in the bisradical tetracationic C1 2(•+)(2+) state, even though the initial solution comprised 22 the catenane in its trisradical form. The BIPY •+ radical cation component of the macrocyclic polyether resides inside the cavity of the CBPQT (•+)(2+) ring, while the DNP unit is located alongside. The BIPY •+ radical cation is located inside the cavity at an angle of 13°from the principal axis of the cyclophane. We hypothesize 29 that this relative orientation maximizes the amount of π-orbital overlap with the BIPY •+ radical cation of the ring. The unit cell of the crystal contains four catenane molecules located around a center of inversion. Analysis of the plane-to-plane separation between the BIPY •+/2+ and DNP units provides further evidence that the [2]catenane is present in its bisradical form, allowing us to make definitive assignments to the dicationic BIPY 2+ and radical cationic BIPY •+ units. The BIPY •+ radical cation unit inside the cavity of the CBPQT (•+)(2+) ring is located closer to one side of the ring than the other. The plane-to-plane separation of the outside BIPY •+ radical cation of the ring with the BIPY •+ radical cation of the macrocyclic polyether is 3.09 Å, while the plane-to-plane separation with respect to the BIPY 2+ dication is 3.39 Å. The plane-to-plane distance between the DNP unit and the dicationic BIPY 2+ unit is 3.33 Å, a distance which is consistent with π-stacking 30 brought about by donor−acceptor π-orbital overlap. Furthermore, the torsional twists of all BIPY •+ and BIPY 2+ units about their 4,4′-C−C bonds are less than 6°. It is known 17j,31 that this relatively small twist is observed in the case when BIPY •+ units have been reduced to their radical cationic forms or when BIPY 2+ dications are engaged 9e,10a in donor− acceptor interactions, in comparison to free BIPY 2+ . This effect, which produces a reduction in the torsional angles of the BIPY 2+/•+ units, is a direct result of electron density populating π-antibonding orbitals, which leads to increased double-bond character between the 4,4′-C−C bonds. As a result, this bond is also observed to decrease in length compared with the bond in free BIPY 2+ dications, while the 3 ( ′ ) ,4 ( ′ ) /4 ( ′ ) ,5 ( ′ ) -C−C bond lengths are all observed to increase. In the case of reduced BIPY •+ radical cations, an entire electron is populating the antibonding orbital, so these bond distortions are even more exaggerated compared to the case of the BIPY 2+ units engaged in donor−acceptor interactions, where only partial electron density is populating this orbital. Nonetheless, the end result is the same ( Table 2) for both donor−acceptor and radical− radical interactions pointing to the similarities 32 in the fundamental orbital interactions between these two seemingly different recognition motifs.
Journal of the American Chemical Society
The solid-state extended packing structure (Figure 5d) is also consistent with the bisradical state and provides insight as to why the bisradical state of the catenane is the one which is isolated in solution, even though the initial solution contained the trisradical form of the catenane. The outside BIPY •+ radical cation of the CBPQT (•+)(2+) ring π-stacks with another outside BIPY •+ radical cation from an adjacent catenane molecule, with a plane-to-plane separation of 3.18 Å. In a similar fashion, the DNP unit is observed to be π-stacked, although in an offset fashion, with another DNP unit from an adjacent catenane molecule on the opposite side, with a plane-to-plane separation of 3.28 Å. This interplanar π−π stacking motif between two BIPY •+ radical cations on one side and two DNP units on the other continues throughout the crystal in such a manner as to form one continuous stack replete with discrete domains of radical−radical and donor−acceptor interactions. We believe that this stacking arrangement brings about an element of stability which is not possible to achieve if the catenane were in its trisradical form. When in the trisradical form, the DNP unit is not able to enter into donor−acceptor interactions with the inside BIPY •+ radical cation of the ring and will find itself as a "fifth wheel", disrupting the favorable extended packing structure. Overall, the observation of the bisradical in the solid-state crystal structure supports the mechanism of switching in solution, which sees the bisradical as an intermediate state as one where the CBPQT (•+)(2+) ring can be engaged in either donor−acceptor or radical−radical interactions.
UV−vis Spectroscopy. In order to confirm the results obtained from CV, namely, the fact that the MSCC can be restored to the GSCC through accessing the RSCC, UV−vis spectroscopic experiments were performed on the tristable rotaxane R3 6+ in MeCN at 263 K. The initial spectrum reveals (Figure 6a) an intense charge-transfer (CT) band centered on 840 nm, which is characteristic 27,33 of the encirclement of the CBPQT 4+ ring around a TTF unit. This observation confirms that the ground-state distribution favors the GSCC as the major translational isomer. Aliquots of an Fe(ClO 4 ) 3 solution were added in sequence in order to oxidize 27 the TTF unit of R3 6+ .
The spectra first of all shows a decrease in the intensity of the 840 nm band with the simultaneous growth of the bands centered on 440 and 600 nm, which are characteristic 27 of the TTF •+ radical cation. When further aliquots of Fe(ClO 4 ) 3 solution were added, the 440 and 600 nm bands disappeared, and a new band, which is characteristic 27 of the TTF 2+ dication, resulted at 363 nm. Furthermore, a low-intensity CT band centered on 530 nm indicates 9e the encirclement of the CBPQT 4+ ring around the DNP unit.
In order to reduce the TTF 2+ dication back to its neutral form, small quantities of zinc dust were added to the solution sequentially, while monitoring the UV−vis spectrum. The reappearance (Figure 6b) of the TTF •+ radical cation bands, followed by their subsequent decrease in intensity (Figure 6c), confirms that the TTF 2+ dication is reduced back to its neutral form, passing through the TTF •+ radical cation as an intermediate on the way. Importantly, no CT band centered on 840 nm could be observed after the neutral TTF unit had been restored; an observation which indicates that the MSCC has been populated and trapped by the BIPY 2+ electrostatic barrier. At this point, an excess of zinc dust was added to the solution (Figure 6d) in order to reduce 17j the BIPY 2+ units to their radical cation forms and bring about formation of the RSCC, which was evidenced by the appearance 17h,j of intense bands centered on 541 and 860 nm. Reoxidation, by exposing the solution to ambient O 2 , resulted in a drastic increase of the CT band at 840 nm, indicating that the GSCC has been restored to approximately its ground-state distribution at equilibrium. These results agree well with those obtained from CV experiments which also reveal that the MSCC can be reset to the GSCC through accessing the RSCC.
1 H NMR Spectroscopy. In order to lend further support to the fact that the MSCC of R3 6+ can be trapped by means of electrostatic repulsion and then reset back to the GSCC by accessing the RSCC, we performed 1 H NMR spectroscopy in CD 3 COCD 3 at 233 K. The initial spectrum of R3·6PF 6 shows (Figure 7), in particular, six resonances between 7.2 and 7.6 ppm, which can be assigned 34 to the aromatic protons of the DNP. These downfield resonances associated with the DNP protons indicate that this unit is free from being encircled by the CBPQT 4+ ring, an observation which is consistent with the GSCC being the major translational isomer at equilibrium. Resonances associated with the cis and trans olefinic protons of the TTF unit encircled by the CBPQT 4+ ring were observed clustered around 6.4 ppm. Upon addition of excess oxidizing agent, tris(p-bromophenyl)ammoniumyl hexachloroantimonate, resonances associated with the olefinic TTF protons vanished altogether, 35 and two new resonances between 9.8 and 10.0 ppm were observed. These two resonances are associated with the aromatic protons of the dicationic TTF 2+ unit that result after oxidation. In addition, resonances located between 7.2 and 7.6 ppm, associated with the aromatic DNP protons free of the CBPQT 4+ ring, were no longer observed, and instead, a new set of peaks associated 34 with the protons of the DNP unit encircled by the CBPQT 4+ ring was observed. In particular, a new doublet which appears around 2.7 ppm that can be assigned to the 4/8 protons of the DNP unit: the doublet is shifted upfield drastically as a consequence of [C− H···π] interactions occurring 34 with the bridging paraphenylene units present in the CBPQT 4+ ring. The resonances for the 2/6 and 3/7 DNP protons are also observed in further upfield positions between 6.2 and 6.6 ppm. The assignments of these resonances were confirmed (see the SI) by 1 H− 1 H COSY spectroscopy. These results support the notion that the CBPQT 4+ ring can be made to translate over the central BIPY 2+ electrostatic barrier and onto DNP as a consequence of Coulombic repulsion associated with the TTF 2+ dication. Next, we prepared a pure solution of the MSCC of R3 6+ in CD 3 COCD 3 at 233 K. Briefly, R3·6PF 6 was dissolved in Me 2 CO, and a slight excess of Fe(ClO 4 ) 3 was added to the solution in order to oxidize the TTF unit to its dicationic form. The solution was then cooled in a dry ice acetone bath followed by the addition of an excess of ascorbic acid as a solid to serve as a reducing agent for the TTF 2+ dication. As soon as the color of the solution became red, a saturated solution of cold aqueous NH 4 PF 6 was added, and the resulting reddish purple precipitate was collected by filtration and washed with cold H 2 O. The dried solid was placed in an NMR tube, dissolved in COCD 3 , and cooled in a dry ice acetone bath, following which the 1 H NMR spectrum was recorded at 233 K. Importantly, the spectrum reveals that the resonances associated with the protons of the TTF 2+ dication between 9.8 and 10.0 ppm are no longer present in the spectrum, while the resonances for the aromatic DNP protons are still shifted upfield, an observation which indicates 34 that the CBPQT 4+ ring remains encircled around the DNP unit even when the TTF unit becomes neutral once again. The additional fact that the resonances for the DNP protons do not appear as perfect doublets and triplets provides further evidence that the TTF unit has been reduced to its neutral form wherein its cis and trans isomers are in slow exchange, resulting in an added degree of complexity in the observed spectrum. The kinetic stability of the MSCC is such that at these temperatures, recording a 1 H− 1 H COSY 2D spectrum (see SI) over the course of an hour is possible. As a consequence, all the proton assignments could be corroborated by this 2D technique. If the CD 3 COCD 3 solution is allowed to stand at room temperature, relaxation of the MSCC to ground-state distribution occurs (see SI) by a thermally activated pathway but only on the order 21 of days.
Zinc dust was added to the cooled solution of the MSCC of the [2]rotaxane R3·6PF 6 in order to reduce the BIPY 2+ units to their radical cations, yielding 17j the RSCC. The solution became dark purple after a few minutes, an observation which is indicative of the formation of the trisradical state. The zinc dust was then filtered off, and while on a dry ice acetone bath, air was bubbled through the solution to introduce oxygen in order to oxidize the BIPY •+ radical cations back to their dicationic forms. After a few minutes, the purple color retreated, and a green colored solution emerged. The 1 H NMR spectrum of this green solution confirmed that the rotaxane had indeed returned to the GSCC. The resonances associated with the aromatic protons of the DNP unit once again appeared downfield between 7.2 and 7.6 ppm, indicating 34 that the DNP unit is free of the CBPQT 4+ ring. Overall, the 1 H NMR spectrum is nearly identical (see SI) to the original spectrum of the rotaxane recorded as the GSCC, prior to any redox experimentation. These results are consistent with UV−vis spectroscopic and CV experiments which show that the GSCC can be recovered from the MSCC by reduction to the RSCC, followed by reoxidation.
Quantum Mechanical Calculations. In order to examine the hypothesis that the monoradical tricationic CBPQT (•+)(2+) ring is capable of recognizing both π-electron-rich donor molecules and radical cationic BIPY •+ units, we carried out a series of ab inito calculations on the bisradical tetracationic CBPQT (•+)(2+) ⊂ MV •+ inclusion complex using several DFT methods, including B3LYP and M06. These methods suffer, however, from self-interaction errors and do not give the correct charge distribution for this complex. Although the functionals of such DFT methods are adjusted to produce accurate energies, the actual charge distributions may not be reliable. Indeed, for these systems, the DFT charges lead to descriptions incompatible with both experiment and ab initio calculations. Therefore, we applied second-order Møller− Plesset perturbation theory (MP2) at 6-31G level to optimize the geometry in the polarizable continuum model (PCM) for acetonitrile (ε = 37.5 and R 0 = 2.18 Å).
The Loẅdin charge distribution (Figure 8a) on the three BIPY 2+/•+ units of the CBPQT (•+)(2+) ⊂ MV •+ complex are 1.9, 1.0, and 1.0, corresponding to the experimental charge assignment. The interplanar distance between the two BIPY •+ units is 3.04 Å, which is much shorter than the interplanar distance (3.61 Å) between the middle BIPY •+ and the BIPY 2+ , indicating the favored interaction between the BIPY •+ units. The magnitude of the dihedral angle between the two pyridine units of each BIPY 2+/•+ units differs according to the charge on each of these units; the dihedral angles are 26.7°for the BIPY 2+ , 5.7°for the middle BIPY •+ , and 0.9°for the remaining BIPY •+ .
In the case of the inclusion complex of CBPQT (•+)(2+) ⊂ TTF, the Loẅdin charge distribution (Figure 8b) on the two BIPY 2+/•+ units is 1.9 and 1.0. At the optimized geometry, TTF is closer to the BIPY 2+ unit. The interplanar distance to BIPY •+ is 3.65 Å, whereas it is 3.58 Å to BIPY 2+ . Similar structural parameters are also found in the case of the inclusion complex of CBPQT (•+)(2+) ⊂ DNP, where the Loẅdin charge distributions (Figure 8c) on the two BIPY 2+/•+ units are 1.9 and 1.0. At the optimized geometry, the interplanar distance from DNP to BIPY •+ is 3.46 Å, whereas to BIPY 2+ , it is 3.43 Å. Without radical−radical interactions, closed-shell molecular entities, such as DNP and TTF, slightly favor BIPY 2+ unit over BIPY •+ , most likely in part at least as a consequence of the stronger Coulombic interactions that polarize the π-electrons on DNP or TTF in addition to a lesser amount of Paulirepulsion energy. These theoretical results are consistent with In each case, the dihedral angles between pyridines are in black, interplanar distances in blue, and the charges associated with each unit are in red. All geometries were optimized at MP2/6-31G in PCM MeCN solvent. the experimental data and confirm that the CBPQT (•+)(2+) ring has a "split personality", behaving as both a donor-and radicalloving species at the same time.
Journal of the American Chemical Society
In order to understand the delocalized spin-pairing interaction, we built a model system containing only two MV •+ units and examined the singlet−triplet (S−T) gap, an indicator of noncovalent bond strength just as in the case of covalent bonding between atoms. Calculations at the level of CASSCF(2e,2o)/cc-pVDZ plus a diffuse function show that, at the distance observed (3.15 Å) in the crystal structure, the S−T energy gap is 10.5 kcal mol −1 which decreases to zero as the plane-to-plane distance increases. The exponential decay of the S−T gap means that the bonding strength is proportional to the overlap of the singly occupied orbitals which decays exponentially in space. The spin pairing of the bisradical CBPQT (•+)(2+) ⊂ MV •+ complex was investigated at the CASSCF(2e,3o)/6-31G level with PCM. One more orbital was included in the active space for the correlation from the empty orbital on BIPY 2+ unit. We found that the bisradical intermediate CBPQT (•+)(2+) ⊂ MV •+ is thermodynamically metastable at 5.8 kcal mol −1 higher (Figure 9) than the infinitely separated CBPQT (•+)(2+) and MV •+ . It is necessary, however, to break the radical bonding between two BIPY •+ units before the disassociation process occurs, making it kinetically stable. A pull-out scan of the potential energy singlet surface points to a 7.6 kcal mol −1 barrier height at a pullout distance 1.6 Å. Beyond this point, the electrostatic repulsion dominates and pushes the center MV •+ out of the cavity of the ring. The 1.3 kcal mol −1 S−T gap at 1.6 Å suggests that this repulsion plays a role to lower the barrier, enabling the disassociation process to occur without totally breaking the noncovalent bonding between the two BIPY •+ units. This calculated barrier height of 7.6 kcal mol −1 is somewhat lower than the experimental value 17j of approximately 16 kcal mol −1 , an outcome which is not unexpected. Indeed, we expect CASSCF(2e,3o) to underestimate the barrier because it includes the correlation of two pairing electrons while omitting correlation from other electrons, e.g., the van der Waals interactions, between π-electrons in Å-stacking of arometics. It is worth noting that, in the area of the pull-out distances of 2−3 Å, the S−T gap decreases to almost zero, from the almost net zero overlap between the two singly occupied orbitals. The small orbital overlap found for these geometries is not a result of the spatial separation as is the case when the middle BIPY •+ is pulled out for more than 5 Å but rather because the nodal structure of the orbitals results in positive and negative contributions to the overlap integral and cancel out.
■ CONCLUSIONS
We have characterized by variable scan-rate CV the dynamic behavior of two bistable [2]rotaxanes, a [2]catenane, and one tristable [2]rotaxane, all containing a BIPY 2+ unit which on reduction serves as a radically enhanced recognition site for the CBPQT 4+ ring. Once the trisradical RSCC has been formed in the two bistable rotaxanes, the bistable [2]catenane, and the tristable rotaxane, a one-electron oxidation of the weakly interacting BIPY •+ radical cation to its BIPY 2+ dication results in the formation of a bisradical state coconformation which undergoes shuttling to a bisradical donor state coconformation. In the case of the bistable [2]rotaxanes and [2]catenane, shuttling onto the adjacent electron-rich unit, TTF or DNP, occurs. We have shown that the shuttling of the bisradical state coconformation onto the TTF unit occurs faster than relaxation onto the DNP unit. The difference in the barriers governing these relaxation processes is 1.9 kcal mol −1 , a value which is approximately equal to the difference in the binding energy for the corresponding TTF-and DNP-containing [2]pseudorotaxanes with the CBPQT 4+ ring, respectively. We hypothesize that the height of the free energy barrier is dictated by the relative affinity of the adjacent π-electron rich unit, such that the stronger the recognition, the faster the shuttling and the smaller the barrier. In the case of the tristable [2]rotaxane, there is strong evidence that the ring relaxes preferentially onto the more electron-rich TTF unit in preference to the DNP one. These results have opened up the possibility of restoring the GSCC from the MSCC through the RSCC by using either (i) the TSCC reoxidation pathway, (ii) the BRCC reoxidation pathway, or (iii) a thermally assisted pathway, demonstrating a working design principle for the realization of molecular flash memory devices by employing a rotaxane which makes use of electrostatic barriers in combination with tristability.
* S Supporting Information
For general methods, further electrochemical and NMR spectroscopic characterization. This material is available free of charge via the Internet at http://pubs.acs.org. | v3-fos-license |
2020-11-05T09:08:27.594Z | 2020-10-30T00:00:00.000 | 228931491 | {
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} | pes2o/s2orc | Carbon Nanodots Synthesized from Dunaliella salina as Sun Protection Filters
Carbon nanodots (CNDs) are more and more being exploited for various applications including biological ones. To this end, they have been thoroughly studied for their potential as antibacterial, wound healing, and bioimaging agents. In this study, we examined the sun protection properties of CNDs. Dunaliella salina was selected as a promising precursor for the synthesis of CNDs which were compared with those produced by citric acid, a widely used precursor for such materials. The CNDs were examined spectrophotometrically, and the sun protection factors were calculated. Additionally, in vitro experiments were carried out to evaluate their UV protection properties and to obtain better insight into whether CNDs are suitable to be used as filters for the development of new sunscreens. The results were conclusive that both CNDs possess favorable properties that potentiate their use for the development of sunscreens. However, the CNDs from Dunaliella salina were found to be superior to those derived from citric acid. Therefore, they can further be exploited as sun protection filters.
Introduction
It is well known that exposure to sunlight has many benefits for human health. However, overexposure has been associated with various skin problems, such as sunburn, accelerated skin aging [1], even cancer [2]. This is the reason that the World Health Organization has classified ultraviolet light as carcinogenic [3]. Although the most damaging type of irradiation for the skin is UV-C (280-200 nm), it poses no threat, since it is filtered from the ozone layer [4]. Among the other two types, UV-B (320-280 nm) is the most dangerous, since it has almost 40 times higher energy than the UV-A (400-320nm) type and thus, its erythemal power is a thousand times as high as that of UV-A [1,5].
The use of sunscreens has been proved to protect the skin from UV damages. Many compounds are being synthesized and examined in terms of their UV absorbance properties. These substances fall between two main categories: inorganic photoactive compounds and organic UV absorbers [6]. The organic compounds are often toxic and exhibit low photostability, while the inorganic analogs have a higher agglomeration tendency at concentrations needed to achieve sufficient protection. Therefore, the demand for new compounds that can be used in sunscreens is increasing. Such compounds must C 2020, 6, 69 2 of 12 have good UV absorption properties and photostability, negligible toxicity, and must be environmentally friendly, while, ideally, they should combine more than one beneficial activity. To this end, many efforts have been made (and their number still increases) to develop new sunscreens, using compounds from natural resources, such as plant extracts [1,5,7,8]. This is due to many activities that plant extracts exhibit, including antioxidant, antibacterial, etc.
Carbon nanodots (CNDs) are a type of nanomaterial that can potentially combine all the above favorable properties and present unique optical properties, among the carbon-based nanomaterials. Many studies have been carried out in the last few years, demonstrating their fluorescent properties (emission is generated, usually, after excitation at the UV region), photostability, negligible toxicity towards eukaryotic cells, and environmental friendliness [9][10][11]. Additionally, CNDs have been found to possess other non-fluorescent properties, such as antibacterial, anticancer, wound healing etc. [12,13]. In our laboratory, CNDs from human fingernails have been used to produce CNDs that exhibit wound healing properties [14]. In another study, we proved clearly that nitrogen-doped and nitrogen and sulfur co-doped CNDs exhibit antibacterial properties [15]. Based on the above, CNDs exhibit fluorescence, by absorbing UV light, and have many other favorable properties. Altogether, that can be put to good use to produce multifunctional sunscreens.
This study aims to explore the potential in vitro sun protection properties of CNDs. To this end, CNDs from two different sources were synthesized and compared. For the one kind of CNDs, a well-established synthesis based on the hydrothermal treatment of citric acid was employed. The other kind of CNDs was produced using a carbonization procedure and Dunaliella salina as the carbon-based biomass. Strains of the green flagellate Dunaliella salina (Dunal) Teodoresco 1905 (Chlorophyta, Chlamydomonadales) are usually found in hypersaline environments [16]). This organism is very efficiently adapted to such extreme environmental conditions rendering it a valuable species for producing high added value biomolecules. It accumulates glycerol as an osmoregulator at high salinity, and β-carotene at high UV radiation to protect its photosynthetic machinery from stress [17]. These molecules not only are in abundance in Dunaliella salina but also exhibit better biological activities, compared to their synthetic analogs [18]. Moreover, the mix of components present in the microalgae results in superior activities, compared with single components, highlighting the potential of microalgae for the development of novel cosmetics for skin [19,20]. The sun protection factors of the as-synthesized CNDs were calculated and compared to obtain a better insight into whether the CNDs possess sun protection properties, or the properties are dependent on the precursor material. The suitability of the CNDs was further examined by an in vitro toxicity and a UV protection study.
Instruments
A Perkin Elmer Spectrum (Two FTIR PerkinElmer, Waltham, MA, USA) was used to record FTIR spectra (of powdered samples), using an attenuated total reflectance accessory. High resolution-transmission electron microscopy (HR-TEM) images were obtained with a JEOL JEM-2100 microscope (Jeol SAS, Croissy-Sur-Seine, France) operated at 200 kV equipped with LaB6 filament. Samples for HR-TEM measurements were prepared by depositing a drop of CNDs dispersion on carbon-coated copper grids and drying, at room temperature. The UV-Vis absorption spectra of CNDs solutions were recorded on a Lambda 35 UV-Vis spectrometer (Perkin Elmer, Rodgau, Germany). All fluorescence measurements and photostability experiments of CNDs solutions were performed on an RF 5301 PC spectrofluorometer (Shimadzu, Kyoto, Japan) in a quartz cell, with 1-cm pathlength and excitation and emission slits set at 5 nm bandpass. Lyophilization of the samples was carried out by an Alpha 1-4 LD freeze-dryer (Christ, Osterode, Germany). Digital images were captured with an Infinity 2-2 digital camera (Lumenera, Danville, Canada), mounted on an Olympus BX43 microscope.
Dunaliella Salina Culture Conditions
The strain of Dunaliella salina used was Duna 32 (Athual, strain bank, University of Athens, Athens, Greece), isolated from the Greek salt works of Megalo Embolo (Aegean Sea, Angellochori, North Greece). It was cultivated in quadruplicate batches of 5 L volume of mildly aerated (0.5 vvm) synthetic seawater (Tropical Marine™ salts dissolved in deionized water, (Wartenberg, Germany) of 120 ppt salinity at a temperature of 26 ± 1 • C, a photoperiod of 12:12 h L: D, 100 µmol photons m −2 ·s −1 . The seawater was enriched with 1mL N-replete Conway medium and 0.1 mL Conway medium vitamin stock [21]. The strain was cultivated until senescence (at least 20 days).
Synthesis of CNDs
For the synthesis of CNDs from Dunaliella salina (CNDs-DS), 30 mg dried algal biomass was placed into a crucible and heated into a pre-heated oven for 2 h at 250 • C. After cooling down to room temperature, water was added to the black powder and the mixture was ultrasonicated for 5 min. Then, the mixture was centrifuged at 3500 rpm for 5 min and the supernatant was collected. The procedure was repeated until the collected supernatant exhibited no fluorescence. The collected supernatants were filtered through 0.20 µm syringe filters and then freeze-dried. The obtained powder (~12.8 mg; synthetic yield: 38.7%) was stored in the dark, at room temperature.
The citric acid-based CNDs (CNDs-CA) were synthesized by placing 0.20 g of citric acid in a stainless-steel Teflon-lined autoclave and heating in an oven at 200 • C, for 3 h. After cooling to room temperature, double-distilled water was added, and the solution was centrifuged at 8000 rpm for 10 min. The supernatant was collected, filtered through 0.20 µm syringe filters, and then freeze-dried. The obtained powder (~114 mg; synthetic yield: 57%) was stored in the dark, at room temperature.
Sun Protection Factor (SPF) Calculation
To determine the SPF of the CNDs, the UV spectrum of the CNDs was recorded between 290 and 320 nm. SPFs were calculated using Mansur's Equation (1), based on previous studies [1,2].
where CF is the correction factor (=10), EE(λ) is the erythemogenic effect of radiation at wavelength λ, I(λ) is the solar light intensity at wavelength λ and ABS(λ) is the absorbance of the tested solution at a wavelength λ. The EE(λ) × I(λ) values are constant according to Sayre et. al. [22].
In Vitro Toxicity Study
The toxicity study was carried out, according to our previous publications [14,23,24]. In brief, HaCaT cells were seeded into 24-well plates and incubated overnight. Different concentrations of CNDs (100, 200, 400, 600, and 800 µg mL −1 ) were added to each well. Cells incubated in the absence of CNDs were taken as control. In all cases, three parallel samples were prepared for each tested concentration. After incubating for 24 h, the culture supernatant was removed, and cells were washed with PBS. Cell viability was assessed using the crystal violet assay. The optical absorbance was measured at 570 nm and the % cell viability was calculated using the formula: where % cell viability represents the number of alive cells, OD non-treated represents the absorbance of control cells and OD treated represents the absorbance of the cells treated with CNDs.
In Vitro UV Protection
HaCaT cells were seeded in 24-well plates and incubated overnight. After complete adhesion of cells in the plate surface, 200 µg mL −1 CNDs were added and cells were incubated overnight. Next, the medium was removed, and cells were washed twice with PBS. A small amount of PBS was added so that a thin layer of PBS was above cells, and cells were irradiated using a UV lamp in the range 280-340 nm (peak emission at 314 nm). The dose was 60 mj/cm 2 . Next, the PBS was replaced with fresh medium, and cells were left to incubate for 24 h. After incubation, the cells were counted following the crystal violet assay, according to our previous study [25].
Cell Cycle Analysis
HaCaT cells were treated with CNDs as described in Section 2.7. After irradiation, cells were harvested, and their DNA content was analyzed by flow cytometry according to our previous study [14].
Statistical Analysis
All assays were replicated three times and p values lower than 0.05 were considered to be statistically significant. Results were expressed as mean ± standard deviation and the levels of significance between samples were compared by one-way ANOVA using Student's t-test, as a post-hoc test for comparison of means. Statistically significant differences at p < 0.05, p < 0.01 and p <0.001 are denoted in Figures with *, ** and ***, respectively.
CNDs Characterization and Optical Properties
Detailed characterization data of the citric acid-based CNDs can be found in our previous study [15]. As regards the Dunaliella salina-based CNDs, Figure 1 depicts their FTIR spectrum. The broad absorption band around 3300 cm −1 is characteristic of O-H stretching vibrations. The broad peak at 2930 cm −1 is attributed to the stretching vibration of sp 3 C-H moieties. Peaks attributed to C=C stretching and -COO-asymmetric and symmetric stretching can be seen at 1600 and 1420 cm −1 , respectively [24,26,27]. The peaks at 1137 and the shoulder at 996 cm −1 can be ascribed to C-O, and C=O stretching vibrations [28,29]. As can be seen in Figure 2, the average size of CNDs-DS is between 3.0 and 3.5 nm, which is typical of CNDs structure. Figure 3 shows the UV-Vis spectrum of the CNDs-DS. A weak shoulder can be seen at ∼270 nm. This can be attributed to the n-π* and π-π* transitions of the -C-O bonds, from carboxyl groups, or to the π-π* transitions or aromatic -C=C bonds [23,24,30,31]. The fluorescence emission spectra of the CNDs-DS for excitation wavelengths between 280 and 480 nm are illustrated in Figure 4. Maximum fluorescence emission was recorded at 470 nm for excitation at 380 nm. The fluorescence emission was found to be excitation dependent, since emission shifts from 400 to 550 nm, as the excitation wavelength increases. The photostability of CNDs was examined by recording the UV-Vis absorbance and the fluorescence emission intensity (for excitation at 300 nm) after 2 h of irradiation with a 150W Xenon lamp. The photostability was examined in the UV-B region (290-320 nm) because as potential sun protection filters they should be stable after irradiation in this region. The CNDs were found to be stable with respect to their optical properties rendering them good candidates for use in sunscreens ( Figure 5). Figure 3 shows the UV-Vis spectrum of the CNDs-DS. A weak shoulder can be seen at ∼270 nm. This can be attributed to the n-π* and π-π* transitions of the -C-O bonds, from carboxyl groups, or to the π-π* transitions or aromatic -C=C bonds [23,24,30,31]. The fluorescence emission spectra of the CNDs-DS for excitation wavelengths between 280 and 480 nm are illustrated in Figure 4. Maximum fluorescence emission was recorded at 470 nm for excitation at 380 nm. The fluorescence emission was found to be excitation dependent, since emission shifts from 400 to 550 nm, as the excitation wavelength increases. The photostability of CNDs was examined by recording the UV-Vis absorbance and the fluorescence emission intensity (for excitation at 300 nm) after 2 h of irradiation with a 150W Xenon lamp. The photostability was examined in the UV-B region (290-320 nm) because as potential sun protection filters they should be stable after irradiation in this region. The CNDs were found to be stable with respect to their optical properties rendering them good candidates for use in sunscreens ( Figure 5). Figure 3 shows the UV-Vis spectrum of the CNDs-DS. A weak shoulder can be seen at~270 nm. This can be attributed to the n-π* and π-π* transitions of the -C-O bonds, from carboxyl groups, or to the π-π* transitions or aromatic -C=C bonds [23,24,30,31]. The fluorescence emission spectra of the CNDs-DS for excitation wavelengths between 280 and 480 nm are illustrated in Figure 4. Maximum fluorescence emission was recorded at 470 nm for excitation at 380 nm. The fluorescence emission was found to be excitation dependent, since emission shifts from 400 to 550 nm, as the excitation wavelength increases. The photostability of CNDs was examined by recording the UV-Vis absorbance and the fluorescence emission intensity (for excitation at 300 nm) after 2 h of irradiation with a 150W Xenon lamp. The photostability was examined in the UV-B region (290-320 nm) because as potential sun protection filters they should be stable after irradiation in this region. The CNDs were found to be stable with respect to their optical properties rendering them good candidates for use in sunscreens ( Figure 5).
SPF of CNDs
To quantitatively assess the effectiveness of a sunscreen formulation, the SPF factor was calculated. The absorbance values of CNDs solutions (concentrations between 0.5 and 11 mg mL −1 ) for both CNDs species were recorded between 290 and 320 nm (with an increment of 5 nm). Using the Mansur equation, the SPFs were calculated and can be seen in Figure 6. According to the results, a solution with an SPF of ∼37 can be prepared using 11 mg mL −1 (1.1% w/v) CNDs-DS. A solution containing the same concentration of CNDs-CA has an SPF of 18. As can be seen from Figure 6, in all tested concentrations the CNDs-CA exhibit nearly two times lower SPF compared to CNDs-DS. This signifies that although both CNDs have sun protection properties, their properties are dependent on other parameters, such as the precursors employed and the synthesis procedure. Since better results were obtained from the CNDs-DS, further experiments were carried out using this material. Until now, there have been only two reports dealing with the use of CNDs to enhance the UV protection properties of other materials. In the first study, the authors examined various CNDs as an additive to PVA films and found that they can enhance their UV properties [32]. The CNDs were synthesized from citric acid, ethylene glycol, and N,N'-bis(2-aminoethyl)-1,3-propanediamine, and a 0.7% w/w addition of CNDs in the PVA films resulted in an SPF of 22. In the second study, authors functionalized cotton fabrics with CNDs from citric acid, ethylenediamine, and borax [33] and the resulting fabrics exhibited an SPF of 28 (the bare fabrics exhibited an SPF of 9).
As stated in the introduction, many natural resources are also being studied to extract compounds with UV protection properties. Each of them has its pros and cons. For instance, the ink from sepia exhibits much higher SPF compared with the examined CNDs. However, the formulations containing eumelanin (deriving from melanin from sepia ink) have not been put on the market, due to aesthetic reasons (sunscreens have an unpleasant dark color) [20]. The CNDs-DS solution exhibits a pale-yellow color, as can be seen from the UV-Vis spectrum, and this could be aesthetically more pleasant, compared with a dark-colored product. Hence, CNDs are considered as a propitious material for sun protection applications. However, the use of Dunaliella salina is more favorable since it is a low-cost, renewable material. Taking into consideration that most sunscreens have a content of 10% w/v in nanoparticles, the superiority of CNDs-DS as sunscreen additives is further strengthened since a 10 times lower content of CNDs-DS can be used to produce sunscreens with high SPF. The reduction in the mass of nanoparticles used in sunscreens can have many benefits, such as reduction of the cost and avoidance of potential side effects (caused by a high concentration of nanomaterials).
SPF of CNDs
To quantitatively assess the effectiveness of a sunscreen formulation, the SPF factor was calculated.
The absorbance values of CNDs solutions (concentrations between 0.5 and 11 mg mL −1 ) for both CNDs species were recorded between 290 and 320 nm (with an increment of 5 nm). Using the Mansur equation, the SPFs were calculated and can be seen in Figure 6. According to the results, a solution with an SPF of~37 can be prepared using 11 mg mL −1 (1.1% w/v) CNDs-DS. A solution containing the same concentration of CNDs-CA has an SPF of 18. As can be seen from Figure 6, in all tested concentrations the CNDs-CA exhibit nearly two times lower SPF compared to CNDs-DS. This signifies that although both CNDs have sun protection properties, their properties are dependent on other parameters, such as the precursors employed and the synthesis procedure. Since better results were obtained from the CNDs-DS, further experiments were carried out using this material. Until now, there have been only two reports dealing with the use of CNDs to enhance the UV protection properties of other materials. In the first study, the authors examined various CNDs as an additive to PVA films and found that they can enhance their UV properties [32]. The CNDs were synthesized from citric acid, ethylene glycol, and N,N'-bis(2-aminoethyl)-1,3-propanediamine, and a 0.7% w/w addition of CNDs in the PVA films resulted in an SPF of 22. In the second study, authors functionalized cotton fabrics with CNDs from citric acid, ethylenediamine, and borax [33] and the resulting fabrics exhibited an SPF of 28 (the bare fabrics exhibited an SPF of 9).
In Vitro Assessment of CNDs Toxicity, UV Protection, and Cell Cycle
Despite their favorable UV absorption properties, CNDs will be of no use if they are toxic towards eukaryotic cells. In this context, we examined the cell viability of HaCaT keratinocyte cells, As stated in the introduction, many natural resources are also being studied to extract compounds with UV protection properties. Each of them has its pros and cons. For instance, the ink from sepia exhibits much higher SPF compared with the examined CNDs. However, the formulations containing eumelanin (deriving from melanin from sepia ink) have not been put on the market, due to aesthetic reasons (sunscreens have an unpleasant dark color) [20]. The CNDs-DS solution exhibits a pale-yellow color, as can be seen from the UV-Vis spectrum, and this could be aesthetically more pleasant, compared with a dark-colored product. Hence, CNDs are considered as a propitious material for sun protection applications. However, the use of Dunaliella salina is more favorable since it is a low-cost, renewable material. Taking into consideration that most sunscreens have a content of 10% w/v in nanoparticles, the superiority of CNDs-DS as sunscreen additives is further strengthened since a 10 times lower content of CNDs-DS can be used to produce sunscreens with high SPF. The reduction in the mass of nanoparticles used in sunscreens can have many benefits, such as reduction of the cost and avoidance of potential side effects (caused by a high concentration of nanomaterials).
In Vitro Assessment of CNDs Toxicity, UV Protection, and Cell Cycle
Despite their favorable UV absorption properties, CNDs will be of no use if they are toxic towards eukaryotic cells. In this context, we examined the cell viability of HaCaT keratinocyte cells, after treatment with different concentrations of CNDs-DS, as can be seen in Figure 7. Although the use of normal human epidermal keratinocytes over HaCaT cells (spontaneously immortalized aneuploid human keratinocytes) is still controversial, the use of the latter is highly accepted, as they offer more reproducible results, compared with the former [34,35]. It is obvious that CNDs-DS are non toxic to the cells at concentrations in the range of 100-600 µg mL −1 .
C 2020, 6, x FOR PEER REVIEW 8 of 12 Figure 6. Sun protection factors of CNDs solutions at different concentrations; data are presented as means ± standard deviation; n = 3.
In Vitro Assessment of CNDs Toxicity, UV Protection, and Cell Cycle
Despite their favorable UV absorption properties, CNDs will be of no use if they are toxic towards eukaryotic cells. In this context, we examined the cell viability of HaCaT keratinocyte cells, after treatment with different concentrations of CNDs-DS, as can be seen in Figure 7. Although the use of normal human epidermal keratinocytes over HaCaT cells (spontaneously immortalized aneuploid human keratinocytes) is still controversial, the use of the latter is highly accepted, as they offer more reproducible results, compared with the former [34,35]. It is obvious that CNDs-DS are non toxic to the cells at concentrations in the range of 100-600 μg mL −1 . Next, we evaluated the potency of CNDs-DS to protect cells from UV irradiation. Although the spectrophotometrically calculated SPF was promising, it is of high importance to evaluate the in vitro effect of the CNDs. For this reason, cells were firstly treated with the CNDs-DS, so that they could enter cell cytoplasm and then they were irradiated with UV light. The cell viability was measured and compared with that of control cells, which were irradiated without any pre-treatment with CNDs-DS. As can be seen in Figure 8, a pre-treatment with 100 µg mL −1 of CNDs-DS can increase the viability of cells by 23% (compared with control), while a pre-treatment with an amount of CNDs-DS twice as much, increases cell viability by 34%, compared with control cells. effect of the CNDs. For this reason, cells were firstly treated with the CNDs-DS, so that they could enter cell cytoplasm and then they were irradiated with UV light. The cell viability was measured and compared with that of control cells, which were irradiated without any pre-treatment with CNDs-DS. As can be seen in Figure 8, a pre-treatment with 100 μg mL −1 of CNDs-DS can increase the viability of cells by 23% (compared with control), while a pre-treatment with an amount of CNDs-DS twice as much, increases cell viability by 34%, compared with control cells. It is known that UVB radiation can cause damage to cell DNA. Therefore, obtaining information about the proliferation state of cells is also important. The DNA damage has been associated with abnormal cell cycle progression. On the other hand, the physiological cell cycle progression is of high importance to maintain normal cell function. To gain a better insight into the UV protection properties of the CNDs-DS, we examined the cell cycle progression of cells subjected to irradiation, previously treated with CNDs-DS, and non-treated. As can be seen in Figure 9, exposure of cells to UVB irradiation increased the number of sub-G1 cells by four times, compared with the nonirradiated cells (statistically significant at p < 0.001). When cells were pre-treated with CNDs-DS, the number of cells in the sub-G1 phase was 50% lower compared with the control cells (statistically significant for p < 0.01). Additionally, it can be seen that the UVB irradiation arrested cells in the G0/G1 phase (increasing the percentage of cells in this phase) (statistically significant for p < 0.05), while it decreased the number of cells in the S and the G2/M phases, compared with the negative control (statistically significant at p < 0.01). Interestingly, cells pre-treated with the CNDs-DS exhibit a cell cycle profile which is more similar to that of the negative control cells, signifying that CNDs-DS hinder the arrest of cells in the G0/G1 phase (no statistically significant differences were observed). It is known that UVB radiation can cause damage to cell DNA. Therefore, obtaining information about the proliferation state of cells is also important. The DNA damage has been associated with abnormal cell cycle progression. On the other hand, the physiological cell cycle progression is of high importance to maintain normal cell function. To gain a better insight into the UV protection properties of the CNDs-DS, we examined the cell cycle progression of cells subjected to irradiation, previously treated with CNDs-DS, and non-treated. As can be seen in Figure 9, exposure of cells to UVB irradiation increased the number of sub-G 1 cells by four times, compared with the non-irradiated cells (statistically significant at p < 0.001). When cells were pre-treated with CNDs-DS, the number of cells in the sub-G 1 phase was 50% lower compared with the control cells (statistically significant for p < 0.01). Additionally, it can be seen that the UVB irradiation arrested cells in the G 0 /G 1 phase (increasing the percentage of cells in this phase) (statistically significant for p < 0.05), while it decreased the number of cells in the S and the G 2 /M phases, compared with the negative control (statistically significant at p < 0.01). Interestingly, cells pre-treated with the CNDs-DS exhibit a cell cycle profile which is more similar to that of the negative control cells, signifying that CNDs-DS hinder the arrest of cells in the G 0 /G 1 phase (no statistically significant differences were observed). CNDs-DS; * denotes statistically significant differences at p < 0.05, ** at p < 0.01 and *** at p < 0.001
Conclusions
In this study, the in vitro UV protection properties of CNDs from Dunaliella salina and CNDs from citric acid were examined. Our results demonstrate that both CNDs species exhibit sun protection properties, however, to a different degree. The CNDs deriving from Dunaliella salina CNDs-DS; * denotes statistically significant differences at p < 0.05, ** at p < 0.01 and *** at p < 0.001.
Conclusions
In this study, the in vitro UV protection properties of CNDs from Dunaliella salina and CNDs from citric acid were examined. Our results demonstrate that both CNDs species exhibit sun protection properties, however, to a different degree. The CNDs deriving from Dunaliella salina exhibit an enhanced SPF, compared to CNDs from citric acid. The CNDs-DS exhibit negligible cytotoxicity towards eukaryotic cells, while they were found to protect cells from UV irradiation. This was further validated by the cell cycle analysis, which confirmed that the percentage of cells treated with CNDs-DS in the cell cycle phases were similar to non-irradiated cells. Despite that this is the first step trying to shed light on another potential application of CNDs, the results corroborate the real prospect of CNDs being used in sunscreen formulations to enhance their SPFs. Since CNDs can combine other beneficial properties (bestowed by heteroatom doping) they can be the basis to develop multifunctional sunscreens. More research should be carried out on examining the UV protection of CNDs in sunscreen formulations. Additionally, further studies are needed to strengthen the biocompatibility and their other biological properties. | v3-fos-license |
2019-05-16T13:03:49.592Z | 2019-05-14T00:00:00.000 | 155086348 | {
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} | pes2o/s2orc | The solution structure of the human IgG2 subclass is distinct from those for human IgG1 and IgG4 providing an explanation for their discrete functions
Human IgG2 antibody displays distinct therapeutically-useful properties compared with the IgG1, IgG3, and IgG4 antibody subclasses. IgG2 is the second most abundant IgG subclass, being able to bind human FcγRII/FcγRIII but not to FcγRI or complement C1q. Structural information on IgG2 is limited by the absence of a full-length crystal structure for this. To this end, we determined the solution structure of human myeloma IgG2 by atomistic X-ray and neutron-scattering modeling. Analytical ultracentrifugation disclosed that IgG2 is monomeric with a sedimentation coefficient (s20, w0) of 7.2 S. IgG2 dimer formation was ≤5% and independent of the buffer conditions. Small-angle X-ray scattering in a range of NaCl concentrations and in light and heavy water revealed that the X-ray radius of gyration (Rg) is 5.2–5.4 nm, after allowing for radiation damage at higher concentrations, and that the neutron Rg value of 5.0 nm remained unchanged in all conditions. The X-ray and neutron distance distribution curves (P(r)) revealed two peaks, M1 and M2, that were unchanged in different buffers. The creation of >123,000 physically-realistic atomistic models by Monte Carlo simulations for joint X-ray and neutron-scattering curve fits, constrained by the requirement of correct disulfide bridges in the hinge, resulted in the determination of symmetric Y-shaped IgG2 structures. These molecular structures were distinct from those for asymmetric IgG1 and asymmetric and symmetric IgG4 and were attributable to the four hinge disulfides. Our IgG2 structures rationalize the existence of the human IgG1, IgG2, and IgG4 subclasses and explain the receptor-binding functions of IgG2.
Up to 75% of the total antibody content in serum is composed of the IgG class. The IgG class is divided into four subclasses, IgG1, IgG2, IgG3, and IgG4. The differences between these arise due to their variable regions, upper C H 2 domains, and the hinge (Fig. 1). The hinge consists of the upper, middle, and lower hinge (Fig. 2). The core hinge (upper and middle) contains 15, 12, 62, and 12 residues for IgG1, IgG2, IgG3, and IgG4, respectively. The IgG subclasses present much variety in structure and function, even though the constant domains possess over 95% sequence homology (1). IgG2 is the second most abundant subclass with an average concentration of 3 mg/ml in adult serum (1). IgG2 has a serum half-life of 21 days as for the IgG1 and IgG4 subclasses. IgG2 has a unique role as this is the predominant IgG subclass that binds to bacterial capsular polysaccharide antigens (2,3). Also, IgG2 shows an increased resistance to microbial proteases for reasons of the unique sequence of the lower hinge (4,5). IgG2 binds complement C1q weakly, and thus IgG2 predominantly activates the complement cascade through the alternative pathway (6). Of the three classes of human Fc␥R receptors, IgG2 binds to only Fc␥RII and Fc␥RIII and not to Fc␥RI. Through its ligand binding and the formation of antigen-antibody immune complexes, IgG2 activates antibody-dependent cell-mediated cytotoxicity through macrophages and polymorphonuclear leukocytes, in order that antibody-dependent cell-mediated phagocytosis will clear any pathogens such as bacteria. In biotechnology, IgG2 is regarded as the antibody with the least overall effector function, and thus it makes a perfect scaffold for designing therapeutic antibodies with lack of function, similar to IgG4. Several Food and Drug Administration-approved drugs are based on IgG2, including evolocumab, denosumab, panitumumab, brodalumab, and erenumab (Antibody Society).
IgG2 is ϳ150 kDa and has the typical IgG structure consisting of two heavy chains (H) 3 and two light chains (L) that are divided into variable (V) and constant (C) domains (Fig. 1). The heavy chains are connected via four disulfide bonds that form between cysteine residues in the hinge. Structurally, human IgG2 from myeloma forms covalent dimers through inter-protein disulfide bonds arising from the hinge cysteine residues (7). IgG2 possesses three different isoforms termed IgG2A, IgG2A/B, and IgG2B with different hinge disulfide bonds (8 -10). IgG2A is regarded as the "classical" or "canonical" IgG2 structure with four intact disulfide bonds in the hinge (Fig. 1). The IgG2A/B isoform contains one Fab region disulfide-linked to the hinge, and the IgG2B isoform contains both Fab regions disulfide-linked to the hinge. The significance of these isoforms on the structure and function is currently unknown.
Structural information on the IgG subclasses is lacking because only two crystal structures for full-length human antibodies are available, namely IgG1 b12 and IgG4 (PDB codes 1HZH and 5DK3) (11,12). This is attributed to the inherent flexibility in the antibody hinge in the order of IgG3 Ͼ IgG1 Ͼ IgG4 Ͼ IgG2 that makes crystallizations difficult (13). The crystal structures only offer a single "snapshot" of a potential broad range of IgG structures in physiological conditions (11). Electron micrographs of human-mouse chimeric IgG2 in vacuo show the existence of different shapes (14). Although no fulllength IgG2 crystal structure is yet available, crystal structures for the Fab and Fc regions of human IgG2 are available (PBD codes 3KYM, 4HAF, 4HAG, and 4L4J) (15)(16)(17). Myeloma IgG2 has been studied by EM, differential scanning microcalorimetry, and fluorescence to reveal an asymmetric structure with one Fab region closer to the Fc region than the other Fab region, similar to that seen for IgG1 and IgG4 (18). Human monoclonal and polyclonal IgG2, human myeloma IgG2, human-mouse chimeric human IgG2, and humanized IgG2 have been previously studied using X-ray or neutron solution scattering or analytical ultracentrifugation (8, 14, 15, 19 -27). The recent studies of human monoclonal IgG1 and IgG4 utilized modeling to fit the scattering curves in terms of molecular structures (28,29). More accurate modeling for human IgG1 and IgG4 based on joint X-ray and neutron-scattering data sets with Monte Carlo simulations has been performed using a newly developed workflow termed SASSIE (30). 4 The outputted structures are atomistic in their nature, because they are physically-realistic models with correctly-joined amino acid and glycan residues. These outputs revealed asymmetric solution structures that resembled the IgG1 and (in part) the IgG4 crystal structures. Here, we used joint small-angle X-ray and neutron-scattering (SAXS and SANS), analytical ultracentrifugation (AUC), and Monte Carlo modeling to analyze 123,371 physically-realistic IgG2 structures. The resulting best-fit atomistic models revealed that classical IgG2 possesses a Y-shaped symmetric conformation in solution. This outcome explained in structural terms for the first time the different IgG2 isoforms and the ligand-binding functions of IgG2 to C1q and the three human Fc␥R receptors.
Purification and characterization of IgG2
Human IgG2 from myeloma plasma was subjected to Superose 6 gel filtration to ensure that this was monodisperse immediately prior to AUC, SAXS, and SANS experiments. It was eluted as a large main peak at ϳ16 ml, with a minor peak at 14.5 ml that was discarded (Fig. 3). Nonreducing and reducing SDS-polyacrylamide gels were run for IgG2, IgG1 6a, IgG1 19a, and IgG4 B72. Fig. 3, lane 3, the heavy chains were observed at an apparent molecular mass of 55.4 kDa, and the light chains were observed between 21.5 and 31 kDa, both as expected (Fig. 3). The corresponding nonreducing and reducing samples for the other antibodies IgG1 6a, IgG1 19a and IgG4 B72.3 were also consistent with previous studies, noting that IgG4 forms trace amount of a half-molecule (28,29).
Native and deglycosylated myeloma IgG2 was subjected to native MS to determine its molecular mass size range. The mass spectra show that native and deglycosylated IgG2 existed as three main populations. For native IgG2 (Fig. 4A), the masses for the three populations were 154,527 Ϯ 52, 156,392 Ϯ 139, and 157,988 Ϯ 62 Da. The observed molecular masses were higher than the calculated molecular mass of 147.4 kDa from the sequence of IgG2 anti-LINGO1 Li33 (Fig. 2), suggesting polydispersity arising from variable protein and glycan contents, but as this spanned 3,461 Da (2.2%) in mass, this polydispersity was comparatively low. The amounts of the three species were 8.9% for 154.5 kDa, 48% for 156.4 kDa, and 43.1% for 158.0 kDa. The high mass error range was attributed to the different glycoforms present in native IgG2. For deglycosylated IgG2 (Fig. 4B), the signals were clearer with reduced error ranges. The masses and corresponding distributions for the three populations were decreased by 3,199 The two heavy chains each possess V H , C H 1, C H 2, and C H 3 domains, and the two light chains each possess V L and C L domains. The heavy chains are connected by four Cys-Cys disulfide bridges at Cys-223, Cys-224, Cys-227, and Cys-230. There is one N-linked oligosaccharide site at Asn-297 on each of the C H 2 domains. The hinge region between the Fab and Fc fragments is composed of 19 residues (ERKCCVECP-PCPAPPVAGP) between Val-219 and Ser-239 (EU numbering). Below the black diagram, the distance between the centers of mass of the two Fab regions (blue, yellow) was denoted as d1. Those between the two Fab and Fc regions were denoted as d2 and d3. The antibody is shown as a 2-fold symmetric structure with d2 ϭ d3. In general, d2 and d3 are unequal. In the text, the smaller of the two values is denoted as min(d2,d3), and the larger of the two is denoted as max(d2,d3).
Solution structure of IgG2
Figure 2. Sequence alignment of IgG2 with human IgG1 6a and IgG1 19a and IgG4. The IgG2 sequence was taken from IgG2 Li33 (13). The IgG1 6a and 19a sequences were taken from Ref. 27. A and B, V L , and C L domains. C-E, V H and C H 1 domains and the hinge. F and G, C H 2 and C H 3 domains. H, comparison of hinge sequences from human IgG1, IgG2, and IgG4 subclasses. E and F, yellow indicates the contact residues involved in the IgG1-Fc complex with the C1q globular head, and blue indicates the contact residues required for interacting with Fc␥RI, and green indicates the contact residues that interact with both C1q and Fc␥RI. Figure 3. Purification of IgG2. The IgG2 elution peak from a Superose 6 10/300 gel-filtration column is shown on the left (mAU, milli-absorbance units). The nonreduced and reduced SDS-PAGE analysis of the IgG subclasses is shown on the right with H 2 L 2 representing the intact antibody molecule, H the heavy chain, and L the light chain. Lanes 1 and 10 contain Mark 12 molecular mass markers labeled in kDa. Lanes 2-3, 4 -5, 6 -7, and 8 -9 contain nonreduced and reduced IgG2, nonreduced and reduced IgG1 6a, nonreduced and reduced IgG1 19a, and nonreduced and reduced IgG4 B72.3, respectively.
Solution structure of IgG2
again spanning 3,400 Da in mass. The reduction was attributed to the removal of two biantennary glycan chains at Asn-297 (each with an approximate mass of 2,200 Da) through PNGase digests. Compared with IgG2 anti-LINGO1 Li33 as a baseline, the protein molecular masses of the three species were increased by up to 4.0 -10.6 kDa (2.7-7.2%).
Analytical ultracentrifugation of IgG2
The size and shape of IgG2 were examined using sedimentation velocity runs in AUC experiments. The SEDFIT analyses involved fits of up to 300 scans. Excellent agreement between the experimental boundary scans and fitted lines was seen (left panels, Fig. 5). The size distribution analyses c(s) for IgG2 showed a major monomeric species in solution and accompanied by a negligible dimer peak (right panels, Fig. 5). The monomer peak was observed at mean s 20, w 0 values of 7.33 Ϯ 0.07 S for IgG2 in H 2 O and 7.07 Ϯ 0.20 S in 2 H 2 O, within error of each other (Fig. 6A). These s 20, w 0 values were comparable with values of 6.4 -7.0 S previously reported for IgG2 (8,14,15,19,20) but not with the value of 5.4 S reported in one study (21). These previous studies did not state the protein partial specific volume, and the buffer density and viscosity in use, thus explaining small differences with earlier s 20, w 0 values. Some variation is attributed to instrumental effects; the s 20, w 0 values from 67 different laboratories showed a Ϯ4.4% deviation in an AUC study of reproducibility (30). Given that the IgG2A/B and IgG2B isoforms possess different hinge disulfide bonds (8 -10), the appearance of a single c(s) peak indicated that, if present, these two other isoforms showed similar shapes to the classic IgG2A structure.
Given the scatter in s 20, w 0 values (Fig. 6A), the sedimentation rates of IgG2 did not display a clear dependence on sample concentration or buffer, indicating that the overall shape remained unchanged (Fig. 6A)
X-ray and neutron-scattering data for IgG2
The IgG2 solution structure was jointly analyzed by both SAXS and SANS as complementary approaches (Table 1). SAXS monitored the shape of the hydration shell surrounding IgG2 as well as its overall antibody structure, whereas SANS using heavy water buffer monitored the overall shape of the unhydrated structure because the hydration shell was largely invisible in this buffer (32).
SAXS was used to examine IgG2 at 0.5-4.0 mg/ml at 20°C in PBS-50, PBS-137, and PBS-250, using time-frame analyses to ensure the absence of radiation damage effects. Overall, crosssectional Guinier analyses resulted in high-quality linear plots in three distinct regions of the I(Q) curves, as expected for antibodies, from which the R g , R xs-1 , and R xs-2 values were obtained within satisfactory Q.R g and Q.R xs limits (Fig. 7, A-C), as in our previous studies (28,29). The lowest Q values were not used in the Guinier R g fits to minimize any potential effect of trace aggregates in the samples. The X-ray R g values showed an apparent concentration dependence. These increased with concentration from 5.24 to 5.71 nm for PBS-50, 5.02 to 5.41 nm for PBS-137, and 5.16 to 5.38 nm for PBS-250 (Fig. 8A). This increase was attributed to X-ray radiation-induced damage of IgG2, because a small increase in intensities was seen in I(Q) at low Q values, and because this concentration effect was not seen in the AUC and neutron data (Figs. 6A and 8B). SAXS data above 1.5 mg/ml were thus discarded for reasons of radiation damage. The R xs-1 and R xs-2 values were unchanged (Fig. 8A). The average R xs-1 values were 2.64 Ϯ 0.03, 2.59 Ϯ 0.04, and 2.61 Ϯ 0.03 nm, and the average R xs-2 values were 1.41 Ϯ 0.06, 1.34 Ϯ 0.08, and 1.34 Ϯ 0.04 nm for PBS-50, PBS-137, and PBS-250 buffers, respectively. The R g , R xs-1, and R xs-2 values in the three buffers were within error of each other. The R g values of 5.0 -5.2 nm here agreed with the earlier R g values of 5.0 to 5.8 nm for humanized IgG2 (26), and they were slightly larger than those for humanized IgG2 of 4.76 Ϯ 0.048 nm (20,25), panitumumab of 5.1 nm (22), and polyclonal human IgG2 of 4.8 nm (27).
SANS was also used to examine IgG2 at 0.3-4.0 mg/ml at 20°C in PBS-50, PBS-137, and PBS-250 in 2 H 2 O. Likewise, the neutron Guinier analyses also revealed high-quality linear plots in three distinct regions of the I(Q) curves from which the R g , R xs-1 , and R xs-2 values were obtained within satisfactory Q.R g and Q.R xs limits (Fig. 7, D-F). The R g , R xs-1 , and R xs-2 values were consistent within error (Fig. 8B) The first experimental value was from the Guinier R g analysis (Fig. 8), and the second one was from the GNOM P(r) analysis ( Fig. 9). b The sedimentation coefficients s 20, w 0 were for IgG2 at 0.54, 1.55, 0.90, and 0.64 mg/ml, respectively, and not as in column 1.
Solution structure of IgG2
These neutron R g , R xs-1 , and R xs-2 values were lower than those for X-rays, with this being attributed to the near invisibility of the surface hydration shell in heavy water, as well as the highnegative solute-solvent contrast difference compared with that of IgG2 (34). The R g values reported here were slightly larger than that of 4.76 Ϯ 0.06 nm for human anti-streptavidin IgG2 in 10 mM sodium acetate (pH 5.2) in 2 H 2 O (23).
The distance distribution function P(r) provided structural information on full-length human IgG2 in real space, with this being equivalent to a histogram of all the inter-atom distances within IgG2. The X-ray P(r) analyses gave R g values similar to those from the X-ray Guinier analyses, showing that the two analyses were self-consistent (filled and open symbols in The filled circles between the arrowed data points represent the Q.R g and Q.R xs ranges used to determine the R g and R xs values. The Q-ranges used for the R g , R xs-1 , and R xs-2 values were 0.15-0.28, 0.31-0.47, and 0.65-1.04 nm Ϫ1 , respectively. Two neutron curves (4 mg/ml in PBS-137 and 0.45 mg/ml in PBS-50) were omitted for clarity.
Solution structure of IgG2
most frequently occurring inter-atomic distances within the structure. Two peaks, M1 and M2, were identified in all the P(r) curves at r values of 4.8 Ϯ 0.3 and 7.6 Ϯ 0.3 nm, respectively. The M1 peak corresponds to distances within each Fab and Fc region, and the M2 peak corresponds to distances between the Fab-Fab and Fab-Fc regions. No concentration dependence in the M1 and M2 positions was observed (Fig. 9C).
The neutron P(r) analyses of IgG2 in heavy water revealed similar R g values compared with the Guinier analyses (filled and open symbols in Fig. 8B). The majority of L values was 17 nm except for 0.59 and 2.38 mg/ml IgG2 in PBS-50 (average L of 16.9 Ϯ 0.4 nm) (Fig. 9B). The M1 and M2 peaks were observed for most of the P(r) curves, except for 0.33 mg/ml IgG2 in PBS-250, and showed r values of 4.8 Ϯ 0.5 and 7.6 Ϯ 0.3 nm, respectively. The neutron and X-ray M1 and M2 values were in excellent agreement, and the reduced neutron L values compared with the X-ray L values were attributed to the hydration shell being not visible in neutron scattering.
Scattering models for IgG2
The IgG2 starting model was generated using the crystal structures of the human IgG2 Fab and Fc regions (see "Experimental procedures"). Residues missing in the Fc region were replaced with the corresponding residues from the other heavy chain (see "Experimental procedures"). The starting structure was Y-shaped with the Fab arms crossed over one another. This starting structure was energy-minimized using NAMD (see "Experimental procedures").
In the Monte Carlo simulations based on backbone dihedral angles, the 19 IgG2 residues (Fig. 2E) represented the fulllength hinge, which was assigned to be flexible ( Fig. 1) and was varied in four different simulations ("Experimental procedures"). The first search involved 200,000 simulations to yield 106,799 sterically-accepted models that included asymmetric as well as symmetric IgG2 models. The second, third, and fourth searches reduced the maximum rotation angle per step from 30 to 15°to include smaller movements of the IgG2 hinge. These involved distance constraints of 1 or 0.75 nm between the ␣-carbons of each of the four cysteine residue pairs that form hinge disulfide bonds (Fig. 1). A total of 100,000 simulations were carried out for each of these two distance constraints using five different starting structures (20,000 simulations for each structure). This resulted in 12,597 and 3,975 accepted models for distance constraints of 1 and 0.75 nm, respectively. Different asymmetric and symmetric IgG2 starting structures with or without crossover Fab regions explored four types of Fab arrangements, limited any biased structures that favor certain conformations, and allowed the sampling of the maximum conformational space. Overall, 123,371 models were accepted for evaluation from a total of 400,000 simulated ones.
X-ray scattering modeling fits for IgG2
The 123,371 models were converted to their hydrated smallsphere representations for comparison with the SAXS curves. Their R g values ranged between 3.85 and 6.21 nm (Fig. 10, A and B; Table 2). The R xs-1 and R xs-2 ranges were 1.34 -3.36 and 0.02-
Solution structure of IgG2
2.30 nm, respectively. The models thus covered a broad range of conformational space as desired (gray, Fig. 11A). Following an examination of the R-factors, the experimental X-ray curves used for the fits were taken to be those for 0.5, 1.0, and 1.5 mg/ml IgG2 in PBS-50, PBS-137, and PBS-250, respectively, for which radiation damage was seen to be minimal. The scattering curve fits gave rise to a wide range of R-factors from 3.5 to 22.9% in a V-shaped distribution with its minimum close to the experimental R g value (gray, Fig. 10, A and B). The lowest R-factors for the three X-ray scattering curves in PBS-50, PBS-137, and PBS-250 were 4.0, 3.5, and 3.6%, respectively, showing that these structures were improved compared with the starting IgG2 structure R-factors of 5.1, 4.8, and 5.1%. The use of R-factor cutoffs of 5.5, 5.0, and 5.7% as filters resulted in the selection of 35,141, 30,088, and 42,292 good-fit models for the three scattering curves in PBS-50, PBS-137, and PBS-250 buffers, respectively (orange, Fig. 10, A and B; Table 2), and reduced the number of accepted models by two-thirds. Views of the 30,088 models for 1 mg/ml IgG2 in PBS-137 showed a broad conformational distribution (gold, Fig. 11B).
The ␣-carbon disulfide distance constraints of 0.75 nm greatly limited the possible R g values of the structures. When the 123,371 models were filtered for distances of Յ0.75 nm between the four cysteine pairs (blue, Fig. 10, A and B), only 5,242 models remained (cyan and blue, Fig. 11E). After the R-factor filters were applied to the 5,242 models for each of the PBS-50, PBS-137, and PBS-250 curves, this left 1,474, 1,247, and 1,100 models, respectively ( Table 2). The fit of the best-fit IgG2 model with the lowest R-factor for each X-ray experimental curve showed good visual agreements out to a Q value of 1.1 nm Ϫ1 (Fig. 12, A-C); note that the same best-fit model was identified for the X-ray fits in PBS-50 and PBS-250, in agreement with the observed lack of conformational change in these buffers. The M1 and M2 values of the X-ray best-fit structures in Fig. 12, A-C, were 4.0 and 7.7 nm, in good agreement with the observed values of 4.8 Ϯ 0.3 and 7.6 Ϯ 0.3 nm (Fig. 9C). Also, the P(r) curves for the best-fit models showed a smaller L value of 15-16 nm compared with the experimental L value of 18 nm.
The d1 value represented the separation between the centers of the two Fab regions (Fig. 1). The min(d2,d3) and max(d2,d3) values represented the minimum and maximum separation The 123,371 goodness-of-fit R-factors were compared with the X-ray and neutron R g values calculated for the IgG2 models. All 123,371 models are shown in gray. The 5,242 models filtered using an ␣-carbon separation of 0.75 nm for each of the four pairs of cysteine residues in the hinge (Fig. 1) are shown as blue circles. The 13 best-fit models that were accepted for each X-ray and neutron pair according to three filters (X-ray and neutron R-factor cutoffs and disulfide separations) are shown as yellow circles and arrowed. The experimentally observed Guinier R g values are shown by vertical solid lines with error ranges of Ϯ 5% shown by dashed lines. A, hydrated X-ray models were compared with the experimental X-ray curve of 0.5 mg/ml IgG2 in PBS-50 where the orange circles show 35,141 models with the R-factor below 5.5%. B, hydrated X-ray models were compared with experimental X-ray curve of 1 mg/ml IgG2 in PBS-137, where the orange circles show 30,088 models with the R-factor below 5%. C, unhydrated neutron models were compared with the experimental neutron curve of 0.45 mg/ml IgG2 in PBS-50 in 100% 2 H 2 O, where the red circles show 44,835 models with the R-factor below 6%. D, unhydrated neutron models were compared with the experimental neutron curve of 1 mg/ml IgG2 in PBS-137 in 100% 2 H 2 O, where the red circles show 10,731 models with the R-factor below 3.75%.
Table 2
Modelling of the X-ray (upper) and neutron (lower) scattering data for human myeloma IgG2 a Models that satisfy the R-factor and disulfide filters and the R g , R xs-1 , and R xs-2 parameters for both X-rays (X) and neutrons (N) are displayed. Because the R-factor depended on which scattering curve comparison was used, this was therefore denoted as NA for not available.
Solution structure of IgG2
between the centers of each Fab-Fc pair (d2 and d3). The 123,371 models covered a large range of d1, min(d2,d3), and max(d2,d3) values, in reflection of the asymmetric and symmetric nature of the IgG2 models. After the R-factor and ␣-carbon disulfide distance filters were applied, the ranges of d1, min(d2,d3), and max(d2,d3) distances were much reduced to similar values of 5.6 -7.5, 6.8 -8.4, and 7.6 -8.9 nm in the three fits ( Table 2). The reductions were explained by a convergence to a single best-fit conformational ensemble in the PBS-50, PBS-137, and PBS-250 buffers. The d1 distances were also smaller than the min(d2,d3) distances, indicating that the distance and angle between the two Fab regions are smaller than the Fab-Fc angles ( Table 2). The min(d2,d3) and max(d2,d3) ranges overlapped. These considerations indicated that the best-fit IgG2 models adopted a largely symmetric Y-shape structure according to the X-ray modeling fits.
Neutron-scattering modeling fits for IgG2
The 123,371 models were converted to their unhydrated small-sphere representations for comparison with the SANS curves. Their R g values ranged between 3.77 and 5.70 nm (Fig. 10, C and D; Table 2). The R xs-1 and R xs-2 ranges were 1.55-2.96 and 0.05-2.14 nm, respectively. The modeled R g , R xs-1 , and R xs-2 values were smaller than those for the corresponding X-ray R g , R xs-1 , and R xs-2 values because of the invisibility of the hydration shell using neutrons in heavy water. Following an R-factor examination of the available experimental scattering curves with three to five different concentrations in PBS-50, PBS-137, and PBS-250 in heavy water, the best experimental neutron curves were taken to be 0.45, 1.0, and 1.99 mg/ml IgG2 in PBS-50, PBS-137, and PBS-250, respectively. The R-factor cutoffs were 3.75% for 1 mg/ml IgG2 in PBS-137 and 6% for 0.45 mg/ml IgG2 in PBS-50 when these two curves were compared ( Table 2). When the curve for 1.99 mg/ml IgG2 in PBS-250 was compared with 0.45 mg/ml IgG2 in PBS-50 (100% 2 H 2 O), the R-factor cutoff was 8.6%. This R-factor cutoff was too lenient, giving 57,566 models, and thus this was reduced to 8.2%, giving 35,215 models ( Table 2). The lowest R-factors for the three scattering curves in PBS-50, PBS-137, and PBS-250 buffers were 4.15, 2.85, and 6.13%, respectively, which were again improved compared with the starting IgG2 structure values of 6.1, 4.0, and 8.6%. The R-factor cutoff filters resulted in 44,835, 10,731, and 35,213 models, respectively (red, Fig. 10, C and D, and Table 2). The 10,731 models for 1 mg/ml IgG2 in PBS-137 showed a broad conformational distribution (Fig. 11C). The modeled R g minimum was centered on the experimental R g value, thus showing good agreement (red, Fig. 10, C and D).
The ␣-carbon disulfide distance constraints of 0.75 nm resulted in only 5,242 models remaining out of 123,371 (see above) (Fig. 11E). After filtering for R-factors, 13, 13, and 19 models remained for the three curves in PBS-50, PBS-137, and PBS-250, respectively (yellow, Fig. 10, C and D; Table 2). The best-fit IgG2 models with the lowest R-factor for the three buffers agreed with the experimental neutron curves up to a Q-value of 1.0 nm Ϫ1 (Fig. 12, D-F). The P(r) curves were in good agreement when overlaid, although a smaller L value of 15 nm was seen compared with the experimental L value of 17 nm. The M1 and M2 values of the neutron best-fit structures in Fig. 12, D-F, were 4.0 and 7.5 nm, in good agreement with the observed values of 4.8 Ϯ 0.5 and 7.6 Ϯ 0.3 nm (Fig. 9D). Surprisingly, this turned out to be the same IgG2 model in all three fits. The R-factor of 3.5% for PBS-137 was the lowest of the three.
The application of the joint R-factor cutoff filter and the 0.75-nm ␣-carbon disulfide distance constraints to the SANS modeling restricted the range of d1, min(d2,d3), and max(d2,d3) distances in a similar fashion to the SAXS modeling ( Table 2). After the R-factor and disulfide distance filters were applied, far fewer neutron models were acceptable (13-19 models) compared with the X-ray models (1,100 -1,474 models). The ranges of d1, min(d2,d3), and max(d2,d3) distances were altered to similar values of 6.4 -7.4, 7.5-8.1, and 7.9 -8.7 nm, respectively, in the three fits compared with X-rays ( Table 2). The neutron d1 values of 6.4 -7.4 nm were higher than those for X-rays of 5.6 -7.5 nm, although these ranges overlapped, suggesting that the IgG2 structures with wider Fab regions were favored in the neutron fits. The range of neutron d1 values was narrower than for the X-ray d1 values. Interestingly, the min(d2,d3) and max(d2,d3) distances of the three sets of filtered neutron models were consistent with each other, and the The graphics were rendered using Tachyon in VMD. A, density plot for all 123,371 models is shown as a mesh with the Fc region shown as a gray solid surface. This is the reference for B-F. B, models that satisfied an X-ray R-factor cutoff below 5% for the curve at 1 mg/ml in PBS-137 in 100% light water. The two Fab regions are shown in gold and orange (30,088 models). C, models that satisfied a neutron R-factor cutoff of 3.75% for the curve at 1 mg/ml in PBS-137 in 100% heavy water. The two Fab regions are shown in red and purple (10,731 models). D, models that satisfied both the X-ray and neutron R-factors. The two Fab regions are shown in brown and tan (4,866 models). E, models that satisfied using an ␣-carbon separation of 0.75 nm between each of the four pairs of cysteine residues in the IgG2 hinge. The two Fab regions are shown in cyan and blue (5,242 models). F, 13 final best-fit models for IgG2 in PBS-137 that meet the X-ray and neutron R-factor cutoff and disulfide filters. The two Fab regions are shown in purple and black (13 models).
Solution structure of IgG2
ranges overlapped. It was concluded from the neutron modeling that IgG2 adopted a symmetric Y-shape structure, in agreement with the X-ray modeling.
Joint X-ray and neutron best-fit IgG2 models
The final best-fit models were identified by using both the SAXS and SANS R-factor cutoffs and the disulfide ␣-carbon distance constraints of Յ0.75 nm as filters. Compared with the distributions of the 30,088 and 10,731 best-fit models for the X-ray and neutron R-factor cutoff filters, respectively (Fig. 11, B and C), the application of both R-factor cutoff filters reduced the best-fit models to 4,866 (brown, Fig. 11D). These 4,866 IgG2 models showed Fab regions that encompassed the majority of conformational space around the Fc region. The ␣-carbon disulfide distance constraint of Յ0.75 nm had severely restricted the allowed positions of the Fab regions around the Fc region (Fig. 11E). When the SAXS and SANS R-factors and disulfide distance constraints were jointly applied, the number of IgG2 best-fit models were reduced to 13, 13, and 19 models for PBS-50, PBS-137, and PBS-250, respectively ( Table 2). The 13 best-fit models for PBS-137 adopted a symmetrical Y-shape (Fig. 11F). Overall, nine best-fit IgG2 models fitted all six SAXS and SANS experimental I(Q) and P(r) curves in PBS-50, PBS-137, and PBS-250. This outcome indicated little or no differences in the IgG2 solution structures in three salt concentrations or between light and heavy water. This agreed with the AUC analyses (Fig. 6A).
Dimensionless Kratky plots of (Q.R g ) 2 .I(Q)/I(0) vs Q.R g provided information on the folded state and flexibility of IgG2 (Fig. 12, G-L). They showed a characteristic two-peak curve similar to that shown previously (24). The X-ray data offered better signal-to-noise ratios than the neutron data. Comparison between the best-fit modeled and experimental Kratky plots showed good agreement up to a Q.R g of 6 for X-rays and 4 for neutrons. The increased X-ray intensities beyond Q.R g of 6 was attributed to potential flexibility in the IgG2 structure that had not been considered in the modeling. A similar intensity increase beyond Q.R g of 4 for neutrons may also indicate flexibility, but it may also include a flat background due to an incoherent scattering contribution that had not been discounted.
The s 20, w 0 values of the nine scattering best-fit models were calculated using HYDROPRO ("Experimental procedures") to compare these with the experimental values (Fig. 6A). Using the density (1.00529 g/ml) and viscosity (0.01002 poise) parameters for PBS-137 buffer at 20°C and a partial specific volume of 0.7 ml/g, the mean s 20, w 0 value was 7.04 Ϯ 0.05 S, which was less than that of 7.32 Ϯ 0.02 S seen experimentally. The energyminimized IgG2 starting structure prior to the Monte Carlo simulations gave an s 20, w 0 value of 7.10 S. This difference in s 20, w 0 values suggested that the best-fit IgG2 scattering model was slightly more elongated in its solution structure than the starting IgG2 structure.
Discussion
The X-ray and neutron-scattering data for human myeloma IgG2, coupled with atomistic Monte Carlo simulations of the dihedral angles in the main-chain backbone, have revealed novel molecular details of its solution structure. Importantly, this provided the first molecular explanation of the different
Solution structure of IgG2
functional IgG2 interactions with its protein ligands. Comprehensive data sets were obtained on human myeloma IgG2 for reason of its availability. Mass spectrometry and AUC showed only a 2% range in mass and a single c(s) peak, respectively, and thus the polydispersity in these samples was low and did not preclude molecular structure analyses. The IgG2 structure was unaffected in scattering experiments in concentration series in three salt concentrations and in light and heavy water. The IgG2 modeling was based on separate crystal structures for the Fab and Fc regions to generate a starting model that was refined by energy minimization and subjected to dihedral angle Monte Carlo modeling. Three filters based on the X-ray data, neutron data, and disulfide distances in the hinge region identified nine best-fit structures. The resulting classical human IgG2 revealed a symmetric Y-shaped conformation in solution that was able to account for its different functional interactions with complement C1q and the Fc␥R receptors.
Together, solution scattering and Monte Carlo modeling have offered molecular structural information on the IgG1, IgG2, and IgG4 subclasses. The IgG2 starting structure included the full IgG2 hinge 220 ERKCCVECPPCPAPPVAGP 238 . Of the 123,371 physically-realistic IgG2 models derived from this hinge, the joint X-ray and neutron best-fit strategy brought down the number of filtered structures from 30,088 and 10,731 models, respectively, to a joint total of 4,866 models (brown, Fig. 13A). Thus the comparison of hydrated and unhydrated scattering structures proved to be effective. The further filter of Յ0.75 nm for the disulfide bridges between the four ␣-carbon cysteine pairs in the hinge gave 5,242 permitted structures (blue, Fig. 13A). The 13-19 best-fit models with the lowest R-factors that passed the double-scattering and disulfide filters revealed that human IgG2 adopts a Y-shaped symmetric conformation in solution. The 13 best-fit models for PBS-137 in light and heavy water were shown (black, Fig. 13A), of which nine models at d1 of ϳ7 nm fitted all the X-ray and neutronscattering curves from six different buffers. These nine structures are available in supporting Material, alongside their computed scattering curves and the experimental data.
Similar methods were used to determine the solution structures of human monoclonal IgG1 and IgG4 by joint X-ray and neutron Monte Carlo scattering fits. 4 That work identified two ␣ and  clusters of symmetric and asymmetric structures, respectively. The clusters were defined by d1 distances of Յ7 or Ն7 nm for the ␣ and  clusters, respectively (Fig. 13B). The best-fit models of IgG1 corresponded to the  cluster of asymmetric structures, and this agreed with the crystal structure of intact human IgG1 b12 (red, Fig. 13B). The best-fit models of IgG4 B72.3 corresponded to both the ␣ cluster of symmetric structures and  cluster of asymmetric structures (blue, Fig. 13B). Interestingly, the IgG2 best-fit models did not correspond to either of the best-fit clusters for IgG1 or IgG4, and instead they were located between these at d1 ϭ 7 nm (black, Fig. 13B). The three analyses indicated that these three IgG subclasses show different conformations. This outcome explains the evolution of the human IgG subclasses such that human IgG1, IgG2, and IgG4 exhibit distinct structural and functional properties. IgG1 and IgG4 have two Cys-Cys bridges in their hinges, whereas IgG2 has four Cys-Cys bridges; in addition, IgG2 lacks a second Gly residue in its hinge that is present in IgG1 and IgG4 (35,36). Furthermore, IgG2 has a shorter hinge than IgG1 and IgG4 (Fig. 2H). These three features are expected to make the IgG2 hinge more rigid, compared with the IgG1 and IgG4 hinges, and alter its function. For example, it can now be seen why IgG2 is able to perform a unique structural role as the only IgG subclass that binds predominantly to bacterial capsular polysaccharide antigens (2,3).
The outcome of scattering modeling and the number of bestfit models depends on the inputs, i.e. the quality of the experimental scattering curves, the starting model, and the number of Monte Carlo models and their filtering. Each are discussed in turn. (i) For example, the lowest X-ray R-factors for IgG2 of 4.2-4.7% were higher than those for IgG1 of 2.6 -2.9% and IgG4 of 2.5-2.6%. 4 This difference is attributable to the larger Q-range of 0.13-2 nm Ϫ1 used for IgG2 here (with higher noise at larger Q values), whereas these Q-ranges were lower at 0.09 -1.1 and 0.15-1.1 nm Ϫ1 for IgG1 and IgG4, respectively. Nonetheless, all three studies resulted in R-factor versus R g graphs with clear minima that identified an ensemble of best-fit structures. The final R g values of the IgG2 models of 4.8 nm (Table 2) Figure 13. Distribution of the Fab-Fab and Fab-Fc distances in human IgG2. The analyses are shown for 1 mg/ml IgG2 in PBS-137. The inter-Fab distance, d1, between the center-of-mass of the two Fab arms and the absolute difference in Fab to Fc distances, d2-d3, are shown (Fig. 1). A, all 123,731 models from the Monte Carlo simulations are shown in gray. The 30,088 models with an X-ray R-factor below 5% are shown in orange. The 10,731 models with a neutron R-factor below 3.75% are shown in red. The 4,866 models filtered by both the X-ray and neutron R-factor filters are shown in brown. The 5,242 models that have less than 0.75 nm ␣-carbon separations for each of the four pairs of cysteine residues in the hinge are shown in blue. The 13 best-fit models that satisfy the X-ray and neutron and disulfide filters are shown in black. B, IgG2 models (black) denote those that meet the X-ray and neutron and disulfide filters from A and are compared with those for IgG1 (red) and IgG4 (blue) that were calculated in the same way.
Solution structure of IgG2
were similar to those of 4.9 nm for IgG4, but less than that of 5.2 nm for IgG1. 4 (ii) The assumptions used for generating the initial models can be important. For example, the earlier neutronscattering fits for human monoclonal anti-streptavidin IgG2 employed an IgG2 starting model based on the crystal structure of full-length mouse IgG2A with three Cys residues in the hinge and not four (PDB code 1IGT) and two Gly residues in its hinge, and only varied three amino acids in the IgG2A upper hinge to generate 56,511 acceptable models (23). Unsurprisingly, these authors determined an asymmetric IgG2 structure. In this study, Fab and Fc crystal structures for human IgG2 were used alongside variation of the full-length human IgG2 hinge with all 19 residues, including four Cys residues in the hinge and only a single hinge Gly residue (Fig. 2E). This study resulted in symmetric IgG2 structures that well-explained the biological function of IgG2 (see below). Further structural analyses with monoclonal IgG2 will clarify these differences further. (iii) A large number of starting models facilitated the identification of best-fit structures. Starting from 704,000, 700,000, and 400,000 trial models for IgG1, IgG4, and IgG2, respectively, the numbers of evaluated physically realistic models with no steric overlap were 231,492 (IgG1), 190,437 (IgG4), and 123,371 (IgG2). These resulted in final totals of 28, 2,748, and 13 best-fit structures, respectively (Fig. 13B). The joint X-ray and neutron fits were the key filter in reaching the final 28 models for IgG1, whereas the joint X-ray and neutron fits together with the disulfide separation filters were key in reaching the final nine best-fit models for IgG2. The rather larger number of final best-fit IgG4 models resulted from the relatively unrestricted shorter IgG4 hinge conformation that gave many more compatible models.
There are three different isoforms of IgG2, namely IgG2A (classical), IgG2A/B, and IgG2B, which are found in both human monoclonal IgG2 and myeloma-derived IgG2 (8 -10). The isoforms vary in the disulfide bond connectivity in the IgG2 hinge, where these studies suggested that the two Cys-223-Cys-223 and Cys-224 -Cys-224 disulfide bonds between the two heavy chains (Fig. 1) can be broken with the formation of new disulfide bonds with the Fab regions. The light and heavy chains in the Fab region are connected by a Cys-135-Cys-214 bridge (Fig. 1). In the IgG2B isoform, Cys-223 from one heavy chain can form an inter-chain disulfide bond with the C-terminal Cys-214 in the light chain. Cys-224 from one heavy chain can form an inter-chain disulfide bond with Cys-135 in the other heavy chain (10,(37)(38)(39)(40) or an intra-chain disulfide bond with Cys-135 in the same heavy chain (8,41). Also, Cys-223 can form an intra-chain disulfide bond with Cys-135 in the same heavy chain where Cys-224 forms an inter-chain disulfide bond with Cys-214 in the light chain (9). The disulfide bond variations in IgG2A/B and IgG2B are still not fully understood, and the impact of these different disulfide bond variants upon antigen binding as well as effector functions is currently unknown.
Although no SAXS and SANS data were collected on the individual IgG2A/B and IgG2B isoforms, our IgG2 best-fit models for human IgG2 provided new insight into these two other isoforms formed by potential Cys-223-Cys-214 and Cys-224 -Cys-135 bridges. These alternative disulfide arrangements may result in more compact global structures than the classic IgG2A isoform (8,40). Interestingly, both these disulfide bond variants were indeed found in our library of 123,371 IgG2 models. Thus, 53 models showed ␣-carbon separations below 0.75 nm for Cys-223-Cys-214, and another 126 showed separations below 0.75 nm for Cys-224 -Cys-135. However, none of these models satisfied the joint X-ray/neutron R-factor cutoff filter and the inter-chain disulfide separation of below 0.75 nm for the best-fit IgG2 models, showing that their structures were distinct from that of IgG2 in its IgG2A isoform as studied. The above 53 models gave R-factors of 5.1-16.2% for X-rays and 4.6 -15.9% for neutrons, both in PBS-137. The above 126 models gave R-factors of 4.9 -8.9% for X-rays and 4.3-11.0% for neutrons. In comparison, the best overall R-factor was lower at 3.5% for IgG2 ( Table 2). The s 20, w 0 values of the 53 and 126 models were 7.2 Ϯ 0.2 and 7.2 Ϯ 0.1 S, respectively, which were not much different from the experimental value of 7.32 Ϯ 0.02 S in PBS-137 and the best-fit modeled value of 7.04 S above. Although a small difference of about 0.3 S was seen between IgG2A and IgG2B, this difference was considered to be low. Overall, even though the IgG2A/B and IgG2B isoforms showed different solution structures, they were not more compact than the classic IgG2A isoform.
A Y-shaped symmetric structure of IgG2 (or IgG2A) had been determined by our atomistic modeling. This outcome differs from the postulated moderately asymmetric structures for the IgG2A and IgG2B isoforms based on comparison with the asymmetric IgG1 crystal structure (8,11) and the asymmetric structures reported elsewhere from EM and neutron-scattering (18,23). By EM, the conclusion of asymmetric IgG2 structures was attributed to the study of an assumed IgG2A/B structure with an asymmetric disulfide arrangement at its hinge (18). The differences from the previous neutron-scattering modeling that gave an asymmetric solution structure could arise from the use of the mouse IgG2a crystal structure to fit the neutron data instead of a human IgG2 Fab and Fc structure (23). In that study, the neutron data on human anti-streptavidin IgG2 were measured at a high concentration in nonphysiological buffers containing 10 mM sodium acetate (pH 5.2), which may have caused conformational changes, whereas here we have used more dilute concentrations for our AUC runs as well as our X-ray and neutron data collection, all at pH 7.4.
The atomistic best-fit models for human IgG2 provided new molecular insight into its binding to the Fc␥RII and Fc␥RIII receptors, but not to C1q of complement nor to the Fc␥RI receptor. This key assessment was performed using recently available crystal structures of the Fc region of human IgG1 complexed with these ligands (Fig. 14). The C1q globular head in complex with the IgG1-Fc region (PDB code 6FCZ) (42) was aligned with the nine best-fit models of IgG2 through their Fc regions, giving a satisfactory r.m.s.d. of 0.149 nm in ␣-carbon positions. Clear steric clashes between the C1q domains and the Fab2 region of IgG2 were visible, explaining why C1q cannot bind to IgG2 (Fig. 14A). The Fc␥RI (CD64) receptor in complex with the IgG1-Fc region (PDB code 4X4M) (43) was also aligned with the nine IgG2 best-fit models through their Fc regions, resulting in an r.m.s. of 0.168 nm (Fig. 14B). Here, clear steric clashes between the D1 and D3 domains of the threedomain "sea-horse" Fc␥RI structure were visible with the Fab1 and Fab2 regions of IgG2, explaining why IgG2 cannot bind to
Solution structure of IgG2
Fc␥RI. The views of Fig. 14, A and B, show that the Fab regions were too close to the Fc region to permit C1q and Fc␥RI binding. In addition, the IgG2 sequence does not possess the key amino acid contact residues required for the complexes between the IgG2-Fc region and each of C1q and Fc␥RI as revealed by their recent co-crystal structures (Fig. 2, E and F). In distinction with these first two cases, the dissociation constant K D of IgG2 with the Fc␥RIIIA Val-158 receptor is 14 M (44), showing that the IgG2-Fc␥RIIIA complex is formed, albeit weakly. To examine this, the Fc region of the nine best-fit IgG2 models were aligned with three crystal structures for the Fc-Fc␥RIII complex (PDB codes 3SGJ, 5VU0, and 5YC5) to give satisfactory low r.m.s. values of 0.100, 0.102, and 0.102 nm, respectively (Fig. 14C) (45)(46)(47). The two orthogonal views of the superimposed IgG2 complexes with Fc␥RIIIA Val-158 show that these IgG2 complexes with the two-domain receptor were sterically compatible with the position of the Fab regions and were therefore allowed. The ability of scattering modeling to generate atomistic structures for the full-length IgG2 mole-cules has provided important clarifications of the ability of IgG2 to bind to C1q and the Fc receptors.
Purification and composition of IgG2
Purified myeloma IgG2 from human plasma (Athens Research, Athens, GA) was further purified by gel filtration using a Superose 6 10/300 column (GE Healthcare) to remove aggregates, then concentrated using Amicon Ultra spin concentrators (50-kDa molecular mass cutoff), and dialyzed at 4°C against its ultracentrifugation and scattering buffers (see below). The N-linked oligosaccharides at Asn-297 on the C H 2 domains (Fig. 1) were represented as a complex-type biantennary oligosaccharide with a Man 3 -GlcNAc 2 core and two NeuNAc⅐Gal⅐GlcNAc antennae (48). Using SLUV2 in the SCT software package, the IgG2 molecular mass was calculated to be 147.4 kDa from the IgG2 sequence (Fig. 2) based on the Fab and Fc crystal structures (PDB codes 3KYM and 4HAF, respectively); its unhydrated volume was 189.9 nm 3 ; its hydrated volume was 231.0 nm 3 (based on a hydration of 0.3 g of water per g of glycoprotein and an electrostricted volume of 0.0245 nm 3 per bound water molecule); its partial specific volume (v) was 0.7294 ml/g, and its absorption coefficient at 280 nm was 15.33 (1%, 1 cm pathlength) (49).
All data were recorded in PBS with different NaCl concentrations. The buffer with 137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.7 mM KCl, and 1.5 mM KH 2 PO 4 (pH 7.4) was termed PBS-137. When 50 mM NaCl or 250 mM NaCl were used, these were termed PBS-50 and PBS-250, respectively. Buffer densities were measured using an Anton Paar DMA 5000 density meter, for comparison with the theoretical values calculated by SEDNTERP (50). This resulted in densities of 1.00529 g/ml for PBS-137 at 20°C (theoretical, 1.00534 g/ml), 1.00145 g/ml for PBS-50 at 20°C (theoretical, 1.00175 g/ml), and 1.0098 g/ml for PBS-250 at 20°C (theoretical, 1.00998 g/ml), all in 100% light water. A buffer viscosity of 0.01002 poise was used for the light water buffers. The densities were increased to 1.11183 g/ml for PBS-137 at 20°C (theoretical, 1.11247 g/ml), 1.10839 g/ml for PBS-50 at 20°C (theoretical, 1.10889 g/ml), and 1.116752 g/ml for PBS-250 at 20°C (theoretical, 1.11711 g/ml), all in 100% 2 H 2 O. A viscosity of 0.01200 poise was used for the heavy water buffers.
Native MS of IgG2
IgG2 was deglycosylated with PNGase F (New England Biolabs, Herts., UK) according to the manufacturer's protocol. The native and deglycosylated IgG2 samples were placed into spin concentrators (Amicon Ultra 500, MWCO
Solution structure of IgG2
collision energy of 10 eV. The mass range was 1,000 -18,000 m/z. Proteins were sprayed using nano-electrospray ionization from gold-coated capillaries prepared in-house using a Flaming Brown P97 needle puller and a Quorum Q150R S sputter coater.
Sedimentation velocity data for IgG2
Sedimentation velocity data were obtained on two Beckman XL-I analytical ultracentrifuges equipped with AnTi50 rotors for IgG2 samples in PBS-50, PBS-137, and PBS-250 at 20°C in each of 100% H 2 O and 100% 2 H 2 O. Data were collected at rotor speeds of 40,000 rpm. in two-sector cells with column heights of 12 mm. Sedimentation analysis was performed using direct boundary Lamm fits of up to 300 scans using SEDFIT (version 14.6) (52,53). SEDFIT resulted in size-distribution analyses c(s) that assumed all species to have the same frictional ratio f/f 0 . The final SEDFIT analyses used a fixed resolution of 200 and optimized the c(s) fit by floating f/f 0 , the meniscus, and bottom of the sedimentation boundaries until the overall r.m.s. deviations (Ͻ 0.02) and visual appearance of the fits were satisfactory. The percentage of oligomers in the total loading concentration was derived using the c(s) integration function. Partial specific volumes of 0.73 and 0.70 ml/g were used for samples in 100% H 2 O and 100% 2 H 2 O, respectively. HYDROPRO version 10 was used to calculate the sedimentation coefficients based on the molecular structure of human IgG2 (54), using an atomic level shell calculation and a hydrodynamic radius of 0.29 nm of each element in the model.
X-ray and neutron-scattering data for IgG2
X-ray scattering data were obtained on Instrument BM29 at the European Synchrotron Radiation Facility, Grenoble, France (55). Data were acquired using a Dectris Pilatus 1M detector with a resolution of 981 ϫ 1043 pixels (pixel size of 172 ϫ 172 m). Sample volumes of 50 l were loaded into PCR strip tubes for the BioSAXS automatic sample changer (56). Each sample in the quartz capillary was moved continuously during beam exposure to reduce radiation damage. Sets of 10 time frames, with a frame exposure time of 1 s each, were acquired, alongside real-time checks that confirmed the absence of radiation damage during data acquisition. After this, any frames containing radiation damage were removed, and the remaining frames were averaged. EDNA software provided automatic data processing in which the intensities I(Q) were automatically scaled by concentration (57). The Biosaxs Customized Beamline Environment (BsxCUBE) software was used for control of the automatic sample changer, and the sample settings were loaded from the Information System for Protein Crystallography Beamlines database (ISPyB) (55,58). IgG2 samples were studied in each of PBS-50, PBS-137, and PBS-250 at 20°C at eight concentrations between 0.5 and 4.0 mg/ml in a dilution series. Data for samples at above 1.5 mg/ml were not used due to radiationinduced damage.
Neutron-scattering data were obtained on Instrument D22 at the Institut Laue-Langevin, Grenoble, France. The data were acquired using a two-dimensional 3 He detector with 128 ϫ 128 pixels of 7.5 ϫ 7.5 mm 2 in size. The sample-to-detector and collimation distances were both 5.6 m. The wavelength was 0.60 nm. Sample volumes of 400 l were used. Samples were measured in rectangular Hellma cells of 2 mm thickness in a thermostatted sample rack set at 20°C. IgG2 was studied in PBS-50, PBS-137, and PBS-250 in 100% 2 H 2 O buffers at 20°C. The dialyses were performed on site immediately prior to D22 experiments to reduce the risk of aggregate formation. IgG2 concentrations were 0.30, 0.45, 0.59, 1.19 and 2.38 mg/ml for PBS-50, 0.5, 1.0, 2.0, 3.0 and 4.0 mg/ml for PBS-137, and 0.33, 1.99 and 2.66 mg/ml for PBS-250.
In a given solute-solvent contrast, the R g is a measure of structural elongation if the internal inhomogeneity of scattering densities within the glycoprotein has no effect. Guinier analyses at low Q (where Q ϭ 4 sin /; 2 is the scattering angle, and is the wavelength) give the R g and the forward scattering at zero-angle I(0) (59) as shown in Equation 1, this expression is valid in a Q.R g range up to 1.5. If the structure is elongated, the mean radius of gyration of cross-sectional structure R xs and the mean cross-sectional intensity at zeroangle (I(Q)Q) Q 3 0 are obtained from Equation 2, the cross-sectional plot for Igs exhibits two distinct regions, a steeper innermost one and a flatter outermost one (60), and the two analyses correspond to R xs-1 and R xs-2 , respectively. The R g and R xs analyses were performed using the SCT software package (45 P(r) corresponds to the distribution of distances r between volume elements. This provides the maximum dimension of the antibody L and its most commonly occurring distance M in real space. For this, the X-ray I(Q) curve utilized up to 1,043 data points in the Q range between 0.03 and 4.92 nm Ϫ1 . The neutron I(Q) curve utilized up to 108 data points in the Q range between 0.1 and 1.7 nm Ϫ1 . Dimensionless Kratky plots of (Q.R g ) 2 ⅐I(Q)/ I(0) vs Q.R g were calculated using the Guinier R g values to provide information on the folded state and flexibility of IgG2 (62)(63)(64)(65).
Generation of starting structure of IgG2
A full-sequence starting model was created for human IgG2 using two crystal structures of the separate Fab and Fc regions and that for human mAb IgG2 anti-LINGO1 Li33 represented Solution structure of IgG2 the IgG2 Fab region (PDB code 3KYM) (15). The human IgG2 Fc region was used directly (PDB code 4HAF) (16). The EU numbering was used here where Asn-297 ( Fig. 1) is equivalent to Asn-297 in IgG1 (29,66,67). 4 In the Fab region, the hinge residues 223 CCVECPPCPAPPVAGP 238 and the last Cys residue on the light chain (C terminus) were unresolved. In the Fc region, most of the unresolved residues ( 235 VAGP 238 , 265 DVSHEDPE 272 , 294 EQF 296 , and 325 NKGLP 329 ) were on one of the two heavy chains, but they were resolved in the other heavy chain. 445 PGK 447 on both heavy chains at the C terminus was also unresolved. The missing Fc residues were reconstructed by replacing the entire heavy chain with a duplicate of the complete heavy chain using superimposition using PyMOL version 1.3 (Schrödinger, LCC). The root mean square difference of the superimposition of the newly built and original heavy chains of 209 and 190 residues was low at 0.0852 nm, showing excellent agreement between the two structures. The missing hinge 223 CCVECPPCPAPPVAG 237 and the C-terminal residues for both the light and heavy chains were modeled with backbone and angles of 10°using the PyMOL script build-_seq (PyMOL Script Repository, Queen's University, Ontario, Canada). All disulfide bonds were retained. Force field parameterizations were generated, and the hydrogen atoms were added to the starting IgG2 structure using the glycan reader component of CHARMM-GUI (68,69) and the CHARMM36 force field (70 -74). This includes the disulfide bond between the light chain and heavy chain. The starting structure was then energy-minimized for 2,000 steps in NAMD (version 2.9) as the simulation engine (https://sassie-web.chem.utk.edu/sassie2/). 5
Dihedral Monte Carlo simulations
Dihedral Monte Carlo simulations in SASSIE used the Complex Monte Carlo module whereby 400,000 models were sampled rapidly (75). This module varied backbone dihedral angles for the IgG2 hinge residues 220 ERKCCVECPPCPAPPVAGP 238 . A Metropolis sampling methodology was used to sample the energetically-allowed dihedral angles, using only the dihedral component of the CHARMM potential to determine the energy of each configuration (70). Sterically-overlapping IgG structures were removed during sampling. Overall, from the total of 400,000 generated IgG2 models, 123,371 models were accepted for the scattering fits.
Different conformational searches of IgG2 were as follows.
(i) First, 200,000 simulations were performed without any disulfide bond constraints in which the entire hinge 220 ERKC-CVECPPCPAPPVAGP 238 was varied in the sampling. Maximum rotation angle steps of 30°were used in this simulation. From this search, 106,799 models (53%) were sterically acceptable with no overlaps.
(ii) Using the models from search i, four were selected as new starting structures. These were selected by measuring the ␣-carbon distances between the hinge residue pairs Cys-223-Cys-223, Cys-224 -Cys-224, Cys-227-Cys-227, and Cys-230 -Cys-230 to be under 1 nm, and these were the only four models that met this criterion. A simulation of 20,000 structures for each of these four starting structures was performed with the constraint that the four cysteine pairs involved in inter-heavy chain disulfide bonding remained within 1 nm of one another (i.e. "disulfide distance" constraints). The entire hinge 220 ERKCCVECPPCPAPPVAGP 238 was varied in the simulation with maximum rotation angle steps of 15°. This produced 9,560 accepted structures that retained their 1 nm separation (12%) out of the total of 80,000 simulations.
(iii) Subsequently, a further five starting structures were defined by setting the ␣-carbon distances between the four pairs of hinge cysteines Cys-223-Cys-223, Cys-224 -Cys-224, Cys-227-Cys-227, and Cys-230 -Cys-230 to be under 0.75 nm in the best-fit models from the 9,560 structures (as above). Four of the structures showed no crossover at their hinges, meaning that each Fab region remained on the same side of the IgG structure as their C H 2 and C H 3 domains (Fig. 1). One of the five structures showed a slight crossover of its hinges. The first four starting structures were subjected to disulfide distance constraints of 0.75 nm between each of the four hinge cysteine pairs in a simulation of 80,000 structures, from which 3,108 (4%) were accepted. The fifth structure was subjected to constraints of 1 nm of ␣-carbon separations between each of the four hinge cysteine pairs in a simulation of 20,000 structures, of which 3,037 models were accepted (15%). The entire hinge 220 ERKCCVECPPCPAPPVAGP 238 was varied in the sampling with maximum rotation angle steps of 15°u sed in all five simulations.
(iv) As a control, the same starting structures used for ii and iv were subjected to two different filters of either 1 or 0.75 nm ␣-carbon separations in the Cys pairs to confirm that the filters in SASSIE were not biased in producing accepted models during the simulation and that the SASSIE simulation produced randomized trial models. One of the starting structures from ii was subjected to a further simulation of 20,000 structures using a constraint of Յ0.75 nm in the ␣-carbon separation for each of the four cysteine pairs in the hinge. The entire hinge 220 ERKC-CVECPPCPAPPVAGP 238 was varied in the sampling with maximum rotation angle steps of 15°, and 867 models from 20,000 were accepted (4%).
Scattering curve calculations and analyses
The scattering curves for the 123,371 accepted models were calculated using the SCT software package (49). This is a coarse-grained method that converts the atomistic models into small sphere models for use with the Debye equation adapted to spheres to calculate the theoretical scattering curves I(Q) (76). For comparison with neutron data, the sphere models were left unhydrated; however, smearing corrections were applied (wavelength 0.60 nm; wavelength spread 10%; beam divergence of 0.016 radians). For comparison with X-ray data, hydration spheres were added to create a hydration shell corresponding to 0.3 g of water/g of protein (33,34). The atomic coordinates were converted into small sphere models using a grid with a cubeside length of 0.54298 nm and a cutoff of four atoms, and these parameters were optimized using SCT to reproduce the unhydrated protein volume. The target dry volume was 189.9 nm 3 (the modeled dry volume was 189.9 nm 3 ) and the target wet volume was 250.1 nm 3 (the modeled hydrated volume was 5 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. where is a scaling factor used to match the theoretical curve to the experimental I(0) value. An iterative search to minimize the R-factor was used to determine . The theoretical scattering curves that matched the experimental scattering curves were accepted as valid models of the antibody solution structure. The experimental curves were fitted using a Q-range of 0.13-2 nm Ϫ1 for the X-ray and neutron curves. A cutoff R-factor, below which models were assigned as bestfit, depended on the experimental scattering curve, its signalto-noise ratio, and its Q range. To determine this cutoff, two experimental curves were used to calculate two R-factors for each of the 123,371 curves. The correlation between the two R-factors was assessed using both the Pearson r and Spearman r s coefficients (31). By gradually excluding the models with higher R-factors, this identified the point at which the ranking of the fits was no longer consistently determined for the two curves. The cutoff was chosen as the point where both the r and r s coefficients dropped below 0.5. 4 If there is a correlation between the two compared curves where r and r s are not equal to 0.5, then the cutoff R-factor filter selected is the minimum R-factor for that experimental curve plus 1-2%. Here, the number of accepted models was reduced by approximately twothirds using the R-factor cutoff filter to select for the better models with a lower R-factor. To analyze the models, the distances between the centers of mass of the two Fab regions (d1) and the distances between the centers of mass of each Fab region to the Fc region (d2 and d3) were measured (Fig. 1). Note that because IgG2 is symmetric, the differentiation between Fab1 and Fab2 and the corresponding d2 and d3 values is for clarity. The Fab1 and Fab2 structures in the models were distinguished according to their chain names. The asymmetry of an antibody structure was measured by the absolute difference between the two Fab-Fc distances, abs(d2-d3). | v3-fos-license |
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} | pes2o/s2orc | Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay
This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.
Introduction
Infection by SARS-CoV-2 virus (SARS-2) occurs mainly through inhalation of virusladen respiratory droplets [1]. When the virus reaches the lower lung airways, ACE2expressing pneumocytes-II epithelial cells take up SARS-2 through interaction between host cell ACE2 receptors and the viral spike-protein (S-protein) [2][3][4]. Thus, neutralization is mainly achieved by antibodies (Abs) that interrupt the binding of ACE2 receptors and this does not prove inhibition of ACE2 binding, as critical ACE2-binding residues within the shared epitope may not be challenged by the tested Ab or serum. A neutralizing epitope was recently shown to overlap with an infection-enhancing epitope in a SARS-CoV pre-clinical study on macaques [22], and thus, steric clashing of antibodies against these epitopes does not guarantee ACE2-RBD binding neutralization. This strategy involves coating ELISA plates with SARS-2-RBD, employing neutralizing sera or unknown Abs, applying known competing biotinylated neutralizing Abs, then adding chromogen-streptavidin followed by substrate for color generation and detection via plate common readers. (B) This strategy involves coating ELISA plates with ACE2, then employing a mixture of neutralizing sera and human IgG-Fc-RBD conjugate (hFc-RBD), followed by anti-human IgG-Fc-HRP-conjugated secondary Abs (Anti-hFc-2 ry Ab-HRP) and substrate. (C) This strategy utilizes the capture ELISA principle, where anti-histidine rabbit Abs are coated onto the plates, followed by capture of the applied His-tagged RBD. Neutralizing sera or purified antibodies are applied, followed by human IgG-Fc-conjugated ACE2 (hFc-Ace2), then anti-hFc-2 ry Ab-HRP and substrate. (D) Our strategy, which is reported herein, involves coating the ELISA plates with RBD, applying neutralizing sera and hFc-ACE2 (or biotin-tagged ACE2), then adding anti-hFc-2 ry Ab-HRP (or chromogen-streptavidin), respectively, with substrate for color generation and detection.
Eventually, ACE2-coated plates were employed, and a mixture of RBD and neutralizing serum was added to the plates [17,18] ( Figure 1B). The advantage of this configuration is that it requires fewer processing steps. However, it was difficult to use controls, as RBDneutralizing Ab binding inhibited ACE2-RBD binding. Thus, bound, or excess unbound RBDs, were washed away with the serum before 2 ry Abs were added. Therefore, serum interaction with RBD, whether as non-specifically bound or excess unbound molecules, could not be detected or confirmed by controls. This assay strategy also requires manipulation of serum/neutralizing Ab concentration, serum to RBD ratio, and mixing with RBD before it is added to the ACE2-coated plates. Thus, neutralization (%) can be easily evaluated only at very few high serum concentration values, as RBD and sera have to be mixed first before addition to the wells. In addition, nAb/RBD mixing time is critical, as binding kinetics influence the extent/percentage of RBD-nAb binding over time [23,24]; as such, the mixing/incubation time requires further investigation in assay strategy B. In contrast, standard ELISA procedures guarantee antigen-specific binding to reach true equilibrium, even for viscous sera, after 1-2 h of incubation. Lastly, in He Y. et al., the concentration of purified Abs that were employed in this assay strategy were high (50 µg/mL). This would have affected binding affinity and might even have allowed for the binding of weakly neutralizing Abs [23].
Recently, a more complicated RBD/S-protein coating was employed. Rabbit antihistidine Abs (1 µg/mL) was first coated on microtiter plates, then His 6 -RBD or His 6 -S-protein (10 µg/mL) solution was added to the wells in a similar manner to sandwich ELISA ( Figure 1C) [25]. Sera or purified Abs were subsequently added to neutralize indirectly-coated RBD or S-protein. Human IgG-Fc-conjugated ACE2 (hFc-ACE2) was applied, followed by 2 ry Abs and substrate. This strategy is similar to our approach ( Figure 1D) [8,25], although the initial anti-His coating step is not included in our strategy since RBD or S-protein binds well to ACE2 when coated directly onto the plate [8]; however, no additional controls were employed, in strategy (C), to determine the amount of indirectly bound S-protein. Assays that use anti-His coating require extra time and effort due to addition of competitor ACE2 protein, which is generally longer than standard ELISA. This protocol used an unknown quantity of S-protein, which was indirectly coated on the plates, and suboptimal amounts of ACE2 (0.1 µg/mL). Low quantities of ACE2 (below saturation) could result in the presence of unbound S-protein, i.e., neither bound by ACE2 nor by neutralizing Abs. This would influence total optical density (OD) readings, because neutralization and ACE2 binding could occur at the same time. Therefore, in our assay strategy, we used enough ACE2 to reach saturation to ensure the assay's sensitivity for neutralization detection.
Currently, the most common cELISA strategy [6,7], which has also been reported by our group [8], involves allowing RBD-or S-protein-coated plates to interact with immune sera, i.e., become neutralized. Thereafter, hFc-ACE2 (or biotinylated-ACE2, for testing human sera) is added, followed by anti-hFc 2 ry Ab-HRP (or streptavidin-HRP for human serum) and substrate. This strategy involves sequential addition of binding proteins (serum antibodies, 2 ry antibody, and ACE2), and thus, facilitates full use of controls. This enables the detection of non-specific interactions, e.g., between RBD and serum proteins, such as complement proteins and detects cross-reactivity of 2 ry Abs with primary/murine Abs or residual serum proteins, e.g., by including blank ACE2 control. In the reported tests, 100 ng of RBD (50 µL of 1 µg/mL solution) was used for plate coating; however, only 50 ng of ACE2 (100 µL of 1 µg/mL solution) was added. Despite that, a four-to five-fold increase in ACE2 quantity was required to achieve equimolar saturation of coated RBD ( Figure 1D). Through development of our assay, we found that 50 ng of directly coated RBD provided a sufficient OD 450 signal, even when it was partially bound to ACE2. The resulting signal was within the linear range of Beer-Lambert's Law, and protein sparing as well, because 200-220 ng of ACE2 is needed, per well, to saturate coated-RBD and result in an OD 450 signal (plateau) (Figure 2). The binding affinity of ACE2 added to immobilized RBD. RBD (50 ng/100 µL per well) was immobilized on ELISA plates, then ACE2 solutions with various concentrations were added to the immobilized RBD. Bound ACE2 and saturation were determined from the OD 450 signal and fitted to a four-parameter logistic function, providing an R 2 = 0.99 and a binding affinity dissociation constant of K D = 10 ng/100 µL, as determined at 50% of the maximum signal reading. The highest reading that resulted in significant OD increase corresponded to 250 ng/100 µL of hFc-ACE2. Therefore, a 5:1 concentration ratio of hFc-ACE2 to RBD (or 200 ng/100 µL, 4:1 ratio of ACE2/biotinylated-ACE2) is required for saturation with a statistically significant maximum OD signal (p < 0.05). This corresponds to an equimolar ratio between the two proteins that achieves a 1:1 binding ratio. Asterisks represent the statistical significance level of each data point (compared to the preceding data point) using the student t-test, * p ≤ 0.05; ** p < 0.01; *** p < 0.001; ns is non-significant, p > 0.05.
All reported assay strategies have assumed linear calculations, as the signal OD value is divided by a blank and then subtracted from 100% to yield percent neutralization. This assumes linear RBD-ACE2 binding or competition, as well as full ACE2 saturation at low serum concentration [6] or in the absence of serum [7]. However, all of these assumptions are incorrect ( Figure 2). While Abe et al. [6] employed a different calculation approach, they used area under the curve (AUC) displacement as an independent neutralization assessment parameter in their cELISA as an alternative relative neutralization measurement.
While potently neutralizing sera can maintain neutralization at dilutions of up to 1/200-1/1000, we found significant positive interference (>10% OD 450 of non-neutralizing control) in assays with high serum concentrations (>2%) if the serum was not heat-treated (not shown). The correlation between neutralization results of cELISA and pseudovirion cell entry assay was R 2 = 0.76, but this dropped to 0.6 between cELISA and PRNT assays (VNT equivalent), potentially due to serum-associated interference [6]. Ultimately, none of the reported assay methods or protocols employed heat inactivation of the sera, even at high concertation's, nor did they employ calibration curves to evaluate diluted immune serum signals. We found that these two steps were essential for accurate determination of the neutralization efficacy of immune sera.
Development of cELISA Assay
Several critical aspects of cELISA assay development were not sufficiently considered in previous assay designs. These include (a) the ACE2 solution concentration (>K D ) employed, (b) the total amount of ACE2 applied (to saturate bound RBD), (c) the use of a non-linear calibration curve for calculations (considering the inaccuracies introduced from a linear assumption), (d) maximizing the sensitivity of the OD signal through adjustment of the RBD quantity, while maintaining the optimum RBD/ACE2 ratio, (e) ensuring assay specificity by eliminating non-specific binding interference of high serum concentrations to added ACE2 or 2 ry Abs, and (f) ensuring that the conventional validation parameters conformed to standard assay specifications by evaluating accuracy, precision, quality of non-linear fit, specificity, and sensitivity (i.e., detection and quantification limits). In order to ensure that the binding equilibrium is shifted towards ACE2 binding of RBD, unless nAbs are present, the concentration of added ACE2 solution must exceed the K D of ACE2-RBD binding for a given coated amount of RBD. Otherwise, even in presence of ACE2, minimal binding would be expected, or the equilibrium could shift towards RBD binding to a weakly neutralizing competitor Ab, instead of ACE2. The minimum ACE2 concentration required for favorable binding equilibrium to coated RBD at 50 ng/100 µL per well was K D = 10 ng/100 µL (0.083 nM) for Fc-ACE2. However, this still does not ensure the saturation of all coated RBD with ACE2. Therefore, the concentration should exceed the K D and the binding extent should be near saturation, i.e., all coated and accessible RBDs (26.6 kDa) should bind to Fc-ACE2 (120 kDa). Thus, an equimolar ratio per well of both proteins may be employed (RBD/ACE2 ratio of 0.23). Ultimately, Fc-ACE2 total mass per well should be four-to five-fold the amount of coated RBD to ensure maximum coverage/saturation. We employed this ratio in our binding curve (Figure 2), as this was the maximum amount of ACE2 that resulted in a significant increase in OD 450 signal.
When using lower amounts of RBD, which has commonly been done, accessible, unbound RBDs are available in the presence of a suboptimal amount of ACE2, resulting in simultaneous, non-competitive Ab-neutralization and ACE2/RBD binding. However, due to the non-linearity of binding approaching saturation, changes in OD readings are very minor (at the plateau), while the amount of ACE2 still increases. Therefore, this may significantly influence readings near the saturation point. To solve this issue, standard neutralizing Abs with known neutralization efficacies (N 50 or IC 50 ) must be employed, or a saturating amount of ACE2 must be used. Otherwise, significant differences between the OD readings (signal replicates) from different amounts of bound ACE2 may be observed, reducing the accuracy of the assay. Binding equilibria are non-linear and follow a sigmoidal pattern (Figure 2), even in the absence of a competitor probe, e.g., neutralizing Abs. Therefore, accounting for neutralization by the missing or unbound ACE2 using only the difference between OD readings and the blank (absent serum) plate wells assumes a linear relationship between bound ACE2 (%) and OD readings (Figure 2), which is grossly inaccurate. This linear relationship assumption in the calculation is, unfortunately, common across all reported forms of the cELISA assay, and this is likely one of the main reasons for the technique's limited correlation with cell-based assays.
The OD 450 readings are relative for every measurement, as they depend on substrateenzyme type, properties, concentration, and reaction time. Thus, a calibration curve or external standard is required for each measurement. This has also, unfortunately, not been employed in any of the reported cELISA assays, as they falsely assumed that the highest reading corresponded to 100% bound ACE2, and that values less than this maximum represented unbound ACE2, i.e., full neutralization of immobilized RBD in a linear fashion. This is incorrect due to a number of factors, including positive interference from nonspecific binding. Therefore, a calibration curve must be established to quantify the amount of bound or free ACE2 as an external standard for the determination of neutralization (%).
The total amount of coated RBD should be high enough to obtain a clear OD readout signal, thus increasing test sensitivity. However, it should not be high enough to exceed the Beer-Lambert cut-off of 0.9 absorbance unit in order to avoid convoluting the calculations further with an additional non-linear component. The immune serum must not interact with any of the ingredients, so as not to impart significant interference. This last point is very important, as we found that the serum non-specifically binds to ACE2 and/or 2 ry Abs at concentrations exceeding 2-5%, even with extensive plate washing. We found in early development of the assay that OD readings from sera wells at various serum concentrations (RBD + 5-20% serum + ACE2 + 2 ry Abs) could be twice as high as maximum OD readings from blank serum wells in the calibration curve (100% RBD + ACE2 + 2 ry Ab) that should have maximum theoretical OD signal. This shows that serum complement activation at high concentrations non-specifically traps ACE2. Similarly, irrelevant serum from a PBS-immunized group of mice (n = 5) at a high concentration (2-20%) was also found to result in a low but significantly high OD reading interference in blank ACE2 control (RBD + 2-20% serum + 2 ry Ab) compared to background OD readings, despite the absence of antigen-specific Abs and ACE2. This shows that complement activation could also trap 2 ry Abs. Therefore, as commonly used in biotechnology techniques, heat inactivation of the serum was conducted to eliminate this non-specific binding/trapping effect. Following heat inactivation, the background interference was minimal (<5% of total OD), even at the highest employed serum concentrations (1/10 dilution), thus increasing assay specificity and sensitivity to neutralization detection and accurate quantification. Another alternative technique involves purifying and isolating RBD-specific IgGs to evaluate them directly in the assay. While purified Abs are easier to evaluate, serum testing requires these precautions and a number of controls to ensure that no significant interference or non-specific interactions are present. Moreover, conventional validation parameters and compendial limits are also important to investigate, even if their boundaries are often less strict for immunological and biological techniques because of their inherent variability. Important validation parameters include accuracy, specificity, precision, sensitivity, and non-linear calibration curve fit quality. Finally, serum assay results should correlate and conform to gold standard cell-based neutralization assays to prove validity and usefulness as an alternative high-throughput screening technique for neutralization efficacy determination.
The relationship between binding affinity/extent and added total substrate/binder protein concentration kinetics follows a non-linear sigmoidal pattern "exponential rise to plateau". Thus, the simplest mathematical function to represent binding is the Michaelis-Menten adsorption equation. Alternatively, the more flexible four-parameter logistic sigmoidal function, which results in a better fit with more complex binding affinity curves, could also be used (Equation (1)): where A 1 and A 2 are the minimum and maximum measured/found OD signals that corresponds to the amount of bound ACE2, p is the power exponent representing the growth rate of the signal as the binder protein concentration increases, and x o is the x-axis ACE2 concentration value at 50% binding, i.e., half-height of the maximum OD signal ( Figure 2). The amount of bound ACE2 can only be determined via constructing and interpolating an ACE2-RBD binding calibration curve. Employing ACE2 concentrations below saturation will reduce test sensitivity to detect neutralization. Applying excess ACE2 beyond saturation will not change the signal at the plateau (Figure 2), thus wasting expensive ACE2 protein, which could be disrupting to the binding equilibria. Therefore, the ideal amount of ACE2 is at the beginning of the RBD/ACE2 binding plateau, where the differences in OD-readings beyond which becomes non-significant statistically, e.g., 250 ng/100 µL concentration point in Figure 2. This can be calculated experimentally or provided from a supplier's RBD certificate of analysis effective 50% binding concentration/molar ratio to RBD value, if available. Finally, the OD-reading should be adjusted to lie within a linear absorbance range of 0.1 to 0.9, in accordance with Beer-Lambert's Law, as greater values will impart non-linearity to the response and unnecessarily complicate the calculations.
In our procedure, we took into account all eight of the issues mentioned above. We conducted our cELISA assays using the configuration presented in Figures 1D and 3 on several immune murine sera to evaluate neutralization efficacy. We interrogated the procedure using conventional assay method development validation parameters, such as non-linear calibration curve fit and significance, precision, accuracy, specificity, and sensitivity in terms of the limit of quantification and detection of neutralization. Lastly, we conducted a standard cell-based neutralization efficacy assay (VNT assay) to compare and correlate the neutralization efficacies of both tests.
Evaluation of Conventional Validation Parameters
We adopted rigorous compendial analytical validation parameters to qualify the neutralization detection and quantification capabilities of this assay. Non-linear calibration curve fit quality is important to confidently determine and interpolate the neutralization values of each well at each serial serum dilution, as demonstrated in the procedure. Goodness of fit was evaluated by correlation coefficient value (R 2 > 0.95). Furthermore, standard deviations between each two successive mean-bound ACE2 amounts in the calibration curves (n = 5) was statistically significant (p < 0.05, paired student t-test). This ensures that there is no overlap between two successive neutralization values. Our procedural set-up confirms significant differences and excellent fitting of logistic function. Our 4P logistic fit of the calibration curve gave correlation coefficient (R 2 ) values that were often in the range of 0.98 to 0.99. Precision or repeatability evaluation was conducted on four different serum samples with various neutralization efficiencies through determination of the 50% neutralization (N 50 ) and percent neutralization extent (N%) values. N% is the percent reduction/neutralization of ACE2-RBD binding at a given serum dilution, and N 50 is the serum dilution (or antibody concentration) responsible for reducing/neutralizing ACE2-RBD binding to 50%. The N 50 and N% for each serum (at different dilutions) had coefficients of variance below <5%. Since the calibration curve should simulate neutralization conditions to different extents (N%), we immobilized different amounts of RBD on ELISA plates. This is because when neutralization occurs, the RBDs become inaccessible to ACE2, i.e., less RBDs are available, so we deliberately reduced the amount of coated RBDs.
Accuracy was determined by adding irrelevant serum (which does not bind to RBD) to the calibration curve with various coated-RBD concentrations, ranging from 2.5 to 50 ng/100 µL/well. These corresponded to 95% to 0% of neutralization, respectively. The blank (subtracted) calibration curve OD 450 readings (in the presence of irrelevant murine serum) remained within ±5% of the labeled bound ACE2 (%) standard value (in absence of the serum), without statistically significant differences (student t-test, p > 0.05, n = 4). Specificity was evaluated by testing the neutralization potential of a non-neutralizing irrelevant serum sample and determining whether it had a significant neutralizing response at any dilution. The non-neutralizing serum had no detectable neutralization at the highest concentration and the level of interference was below 2%, which was attributed to acceptable random error. The sensitivity of the assay was evaluated by its capacity for quantitative detection of neutralizing sera. The neutralization detection limit was calculated from the mean background OD value plus three standard deviations (OD 450 = 0.056), which corresponded to 0.3% bound ACE2. The quantitation limit was the mean background OD value plus 10 standard deviations (OD 450 = 0.083), which corresponded to 7% bound ACE2. These represent very sensitive detection and quantification limits ( Figure 1D), as the lowest OD signals result from the lowest amount of bound hFc-ACE2 (high neutralization extent), and thus, in turn, they have the lowest amount of bound 2 ry Abs, which are responsible for the color change.
Virus Neutralization Assay: Orthogonal Validation
Twenty-five individual murine immune sera were tested (n = 25) for neutralization using both VNT and cELISA assays. Mice were immunized with various SARS-CoV-2 vaccines or a PBS negative control. Group mean neutralization extent (%) was calculated using both assays by averaging the neutralization efficacy (%) for each serum dilution (1/20, 1/40, or 1/80) ( Figure 4A). cELISA assays were additionally conducted with and without heat treatment ( Figure 4B,C). Finally, the serum neutralization profile and dilution value corresponding to N 50 were determined by both assays and compared ( Figure 4D). The calculation protocol for the cELISA technique is provided in the detailed protocol.
VNT assays were conducted as described previously (Figures S1 and S2) [26,27]. Briefly, overlay medium, block buffer, and polysorbate/phosphate saline (wash buffer) were prepared. The primary probes (mouse sera) were heat inactivated. Vero E6 cells were cultured in DMEM medium. SARS-2 virus isolate (QLD02, GISAID accession EPI_ISL_407896, provided by Queensland Health Forensic and Scientific Services, Queensland Department of Health, Australia) was used for this assay. Vero E6 (5 × 10 4 ) cells were seeded in 96-well plates with DMEM medium, and incubated overnight at 37 • C and 5% CO 2 . After incubation, the medium was removed, heat inactivated mouse serum was serially diluted five-fold, and viral inoculum (~260 FFU/well) was incubated with serially diluted sera for 1 h at 37 • C. In a similar process to the mouse serum, a final concentration of 10 µg/mL of the nAb, S309 [28], was serially diluted five-fold, then incubated with similar amount of SARS-CoV-2 virus. The mixture (50 µL) was then added to each well of the cell plates to infect the cells. The overlay medium was added onto the cells, and the plates were re-incubated for 14 h at 37 • C and 5% CO 2 prior to fixing the cells with 80% acetone. The plates were dried, blocked using blocking buffer for 1 h at room temperature. The block buffer contains milk diluent sera (KPL, Seracare) and 0.1% Tween in PBS. Plates were then probed with anti-spike antibody (CR3022) [29] and followed by IR dye ® -conjugated 2 ry Abs (LI-COR Biosciences, Lincoln, NE, USA), both diluted in blocking buffer, were added to each of the seeded cell wells. The plates were read using an Odyssey Infrared Imaging System infrared high-resolution scanner LI-COR CLX (LI-COR Biosciences, Lincoln, NE, USA). Spots denoting the number of infected Vero E6 cells were counted using the procedure below. The correlated data belong to six groups of mice, comprising 24 different weakly-to-moderately neutralizing murine sera at different dilutions: (A) the correlation between combined group mean neutralization (%) values from the VNT assay and heat-inactivated sera cELISA assay was R 2 = 0.98, n = 10; (B) the correlation between individual mouse immune serum neutralization (%) values from the VNT assay and heat treated serum cELISA assay was R 2 = 0.975, n = 25; (C) the correlation between individual mouse immune serum neutralization (%) values from the VNT assay and untreated serum cELISA assay was R 2 = 0.54, n = 25; (D) the correlation between reciprocal serum dilutions that corresponded to 50% neutralization efficacy (N 50 ) between both assays (VNT and heat-treated serum cELISA) using heat-inactivated weak-to-moderately neutralizing murine sera profiles that were within the detectable range of both assays.
VNT assay plate spots (signaling viral cell entry; Figure S2) were counted using ImageJ Fiji software version 1.53e (a free, open-source application, https://imagej.net/ software/fiji/, accessed on 15 May 2021). The plate images were cropped, and the color threshold was adjusted to the default settings: black and white (B&W), in RGB color space with the parameters: Red = 33, Green = 74, and Blue = 49, against a dark background. The diluted serum wells across all plates were processed using the same parameters, including immune serum plates and the naïve serum plate. After threshold adjustment, we conducted particle counts for every well. The counts were divided by virus only (blank serum) positive control numbers to yield N% at a given dilution. For individual sera that exceeded 50% neutralization values at 1/20 or 1/40 dilutions, the reciprocal serum dilution that corresponded to 50% neutralization was interpolated to yield N 50 values.
Assay Limitations
Our novel assay strategy is not limited to the detection of RBD-bound nAbs. Nterminus domain-bound nAbs can be determined by coating the plates with 100 µL of 3.32 µg/mL proline-substituted S-protein instead of RBD (21.23 kDa, on amino acid Mwt basis). This is an equimolar concentration to our assay RBD concentration, as S-protein monomer (141 kDa, on amino acid Mwt basis) contains a single RBD, while the trimer (423 kDa, on amino acid Mwt basis) contains three RBDs. An initial quick binding affinity titration may be required to establish the optimum S-protein coating concentration that would yield a suitable OD reading range for detection and quantification. A similar 6.6fold increase in coating solution concentration using full-length S-protein, as substitute for RBD, was suggested in a recently published cELISA assay to yield comparable results to RBD-coated plates [6]. However, the limitation in all currently employed cELISA assays, including ours, is its lack of detection of priming process inhibitory Abs compared to cell-based assays. These Abs prevent furin and cathepsin-L enzymes from priming spike protein; however, they do not prevent RBD/ACE2 binding, so a different assay is required to evaluate this neutralization mechanism.
Reagent Setup
Caution: Personal protective equipment (lab coat, goggles and acid-resistant gloves) should be worn, especially while handling sulfuric acid or OPD substrate. The reagent quantities required are as follows: • hFc-ACE2 ( Weigh out 193 mg sodium carbonate and 380 mg sodium hydrogen carbonate, then dissolve both in 100 mL of deionized water. Adjust the pH to 9.6, if necessary.
2.
Prepare PBST wash buffer (800 mL/plate): Add eight PBS tablets to 4 L of deionized water. Stir with a magnetic bar and stirrer, until the tablets are completely dissolved, then add 1 mL Tween 20 using a needleless 2 mL syringe.
3.
Prepare 2% w/v BSA block solution (25 mL/plate): Weigh out 5 g of BSA and dissolve this in 250 mL of PBST wash buffer.
4.
Prepare 0.5% w/v BSA solution (35 mL/plate): Weigh out 2.5 g of BSA and dissolve this in 500 mL of PBST wash buffer. Alternatively, transfer 125 mL of 2% w/v block solution and dilute to a final volume of 500 mL in a suitable glass bottle using PBST wash buffer.
6.
Prepare 2 ry Ab solution (10 mL/plate): Warm the 2 ry Ab stock solution to room temperature, transfer 16.65 µL into 50 mL of 0.5% BSA solution and mix well.
7.
Prepare OPD substrate (10 mL/plate) (prepare fresh before addition): Dissolve three substrate buffer tablets in 60 mL of deionized water in a glass bottle using a magnetic bar and stirrer, until completely dissolved. Cover the bottle with foil to protect from light, then add three OPD substrate tablets and stir, until completely dissolved.
8.
Prepare 1N sulfuric acid (10 mL/plate): Transfer 1.35 mL of sulfuric acid (98%) to 20 mL of deionized water in a suitable glass bottle and complete volume to 50 mL with deionized water.
Assay Protocol
Experimental procedure takes about 4.5 + 8.5 h to complete and can be conducted over 2 days.
1.
Dissolve RBD in a suitable volume of distilled water to prepare 1 mg/mL stock solution in a 1 mL Eppendorf tube.
2.
Dilute RBD to a final concentration of 0.5 µg/mL RBD in carbonate buffer in a 50 mL Falcon tube, or other suitable container, by transferring the necessary volume from the stock solution prepared in Step 1.
3.
Add a volume of 100 µL of RBD (0.5 µg/mL) in carbonate buffer to coat each well of the ELISA sample plates (S-plates), giving a total RBD amount of 50 ng/well. 4.
In parallel, prepare a calibration curve plate (CC-plate) by adding 100 µL of, proteinfree, carbonate buffer to five rows (D-H), starting from column 3, down to column 9. Add 25 µL of carbonate buffer to column 2 for five rows (D-H) ( Figure 5).
5.
Coat the wells of the CC-plate by adding 200 µL of the RBD solution prepared in Step 2 to column 1 for five rows (D-H), then conduct two-fold serial dilutions using a multichannel pipette by taking 100 µL from column 1 and transferring it to column 3. Mix the solution by pipetting 50 µL up and down five times, then transfer 100 µL to column 4, and repeat until you reach column 9. Discard the last 100 µL you withdraw from column 9. This should leave three blank rows (A-C) across all columns, as well as rows (D-H) in column 2 ( Figure 5). Figure 5. Schematic depicting cELISA plates: calibration curve (CC)-plate and sample (S)-plate arrangement, orientation, and associated dilutions. The lowest calibration curve value (0.78%) corresponds to our assay's detection limit. Accurate quantitation starts from ≥6.25%. S-plate sample rows have RBD, BSA block, serum, ACE2, and 2 ry Ab. S-plate blank ACE2 rows have RBD, BSA block, the same serum, and 2 ry Ab. CC-plate calibration curve rows have RBD (different amounts), BSA block, ACE2, and 2 ry Ab. CC-plate background rows have BSA block, ACE2, and 2 ry Ab. To check non-specific serum interactions (with ACE2 and/or 2 ry Abs) and efficient washing (of ACE2 and 2 ry Abs), background and blank ACE2 rows are employed.
Critical: The volumes and concentration used in this step must be exact, as this will provide the calibration curve from which binding inhibition will be calculated.
6.
Add 75 µL of the stock RBD solution (from Step 2) to the CC-plate column 2, rows D-H, using a multichannel pipette, then mix the solutions by pipetting 50 µL up and down five times. 7.
Place the plates in an airtight storage container and incubate the S-plates and CC-plate for 90 min at 37 • C. 8.
After incubation, dispose of the solution from the wells of the S-plates and CC-plate and wash the plates three times with deionized water, followed by three more washes with PBST wash buffer. Dry the plates by tapping upside down on paper towels.
The RBD amounts in the CC-plate will range from 50 ng to 0.097 ng per well. This covers neutralized-RBD from 0% (for 50 ng/well, column 1) to 99.3% (for 0.78 ng/well, column 9), including a 75% point. This provides a very practical and thorough range ( Figure 5).
9.
Block the CC-plate and S-plates by adding 250 µL of 2% bovine serum albumin (BSA) solution in PBST wash buffer to all wells, including the three empty rows (A-C) of the CC-plate, which provide background OD readings for substrate control (Figure 4). 10. Place the plates in an airtight storage container and incubate all plates overnight at 4 • C or for 90 min at 37 • C. 11. Repeat washing Step 8 (Pause Point).
Addition of Immune Murine Sera (3.5 h)
12. Add 180 µL of 0.5% BSA solution in PBST to the wells in column 1 of each S-plate, and 100 µL of the same solution to all other wells of the S-plates. 13. Conduct heat treatment on a 45 µL aliquot of undiluted/neat, vortexed mouse serum in an Eppendorf tube at 57 • C for 30 min using a temperature-controlled water bath.
Critical: This timing must be adhered to. Heating the serum for too long will inactivate the nAbs, while not heating for long enough will leave serum complement proteins active, resulting in non-specific binding interactions and obscured assay results.
14. Transfer 20 µL of each heat-treated serum sample to a designated well in column 1, e.g., well 1H, of the S-plate ( Figure 5). Transfer another 20 µL of serum from the same sample to another well of column 1, e.g., well 1D, of the same S-plate ( Figure 5). Thus, each serum sample is added twice, to two different wells, of column 1; the second well will serve as a blank ACE2 control. 15. After serum addition to the S-plates, mix the contents of the column 1 wells by pipetting up and down a 50 µL volume five times. Then, conduct a two-fold serial dilution using a multichannel pipette by transferring 100 µL from column 1, rows A-H, to column 2, rows A-H, and repeat until you reach column 12. Discard the last 100 µL you withdraw from column 12. Repeat Steps 14 and 15 for all S-plates. 16. Add 100 µL of 0.5% BSA solution to all wells of the CC-plate without further treatment. 17. Repeat incubation Step 7. 19. Repeat washing Step 8.
Addition of hFC-ACE2, 2 ry Ab and Substrate (5 h)
19. Add 100 µL of 2.5 µg/mL hFc-ACE2 solution to all wells of the CC-plate. 20. Add 100 µL of 2.5 µg/mL hFc-ACE2 solution to the E-H row wells of the S-plates only using a multichannel pipette, i.e., avoid the blank ACE2 control wells in rows A-D of the S-plates ( Figure 5).
Critical: Be careful not to add ACE2 to the blank ACE2 controls: they are intended as a blank to subtract any background and minor cross-reactivity of the 2 ry Ab with murine sera.
Critical: Check the manufacturer's instructions for 2 ry Ab dilution.
24. Add 100 µL of diluted 2 ry Ab solution to all wells of the S-plates and CC-plate. 25. Repeat incubation Step 7. 26. Repeat washing Step 8, and wipe underneath the plates dry using Kimwipes.
Critical: Washing must be thorough, as any residual 2 ry Ab, especially in the CC-plate, will obscure measurements. 27. Finally, add 100 µL of OPD substrate solution to all wells of the CC-plate and S-plates.
Incubate the plates in the dark and allow them to react for 25 min at room temperature, then add a volume of 100 µL of stop solution, 1N sulfuric acid. Determine the absorbance at 450 nm OD 450 using a common plate reader for all plates.
Critical: Be careful while using OPD substrate: it must be prepared fresh and kept in the dark once prepared and during reaction time. Otherwise, oxidation will result in a high background. Once established, proceed with full measurements (Pause Point).
Calculation Procedure (2 h)
The calculation sheet (GraphPad Prism project) is included in the Supplementary Materials to facilitate the calculation procedure of both CC-and S-plates. 28. Subtract the background OD 450 (mean of the first three rows (A-C)) from individual calibration curve OD 450 for each of the five rows (D-H) of each dilution (columns 1-9) to yield blank-subtracted calibration curve individual values (five values, rows D-H, for each dilution step, columns 1-9) ( Figure 5). 29. Calculate the mean value (±standard deviation) of blank-subtracted calibration curve data and plot these against their associated concentration expressed in terms of bound ACE2 (%) (Figure 6), i.e., the OD reading average of the second and third columns correspond to the percent of available RBDs (37.5 and 25 ng, respectively) compared to the total RBD amount in column 1 (50 ng), 75% and 50% respectively. Critical: Check whether each point on the calibration curve (mean ± standard deviation) fits the following criteria: • Each point is significantly different from its neighboring points-this means a tight standard deviation is required. The mean and standard deviation OD 450 values for 25%, 50%, 75%, and 100% bound ACE2 should all be significantly different (p < 0.05).
•
The lowest mean OD 450 value is significantly higher than the background. • The highest mean OD 450 value should be in the range of 0.5 to 1.0.
33. Finally, plot the neutralization (%) value for each serum sample in each well against its corresponding reciprocal serum dilution, as in Figure 5. The reciprocal serum dilutions of columns 1, 2, 3, 4, and 5 are 10, 20, 40, 80, 160, etc. Each curve represents the serum neutralization profile of different dilutions (Figure 7), and fit neutralization (%) versus corresponding reciprocal serum data (i.e., 1/serum dilution value, so 1/20 dilution becomes 20), again using the four-parameter logistic function (Figure 7). 34. Interpolate each serum curve at 50% neutralization value (Figure 7) to determine the neutralization 50% reciprocal serum dilution (N 50 or IC 50 ), other common determinant/endpoint of neutralization efficacy. The common endpoint/determinant of neutralization parameters (nAb titer, N 95 ) can also be determined from the plot by interpolation at 95% neutralization; it is the maximum serum dilution value that results in 95% neutralization of ACE2 binding to coated RBD (Figure 7).
• Standard nAbs can also be included and treated as serum, but without heat inactivation (Step 13). For comparison purposes, nAbs can be serially diluted from their stock solution until N 50 /IC 50 is achieved. This also provides an additional external standard, as cell-based assay procedures often employ standard nAbs.
•
Interpolation at 80% or 90% neutralization yields N 80 and N 90 values, which are also commonly used neutralization efficacy endpoints.
•
If the results are not as expected, refer to troubleshooting section (Table 1).
Issue Potential Reason(s) Solution
Neutralizing extent exceeded the plate dilution down the rows Very highly neutralizing serum, N 50 exceeding 2 × 10 4 .
Repeat the test starting with a lower serum dilution, e.g., 1/100, or conduct three-fold serum serial dilutions instead of two-fold.
Neutralization unapparent at the highest serum concentration Irrelevant or non-neutralizing serum sample. Excessive heat treatment.
Repeat the assay with careful monitoring of heat treatment conditions (time and water bath temperature) and include a known standard murine neutralizing antibody (nAb) as an external standard to check assay integrity.
OD 450 of blank ACE2 wells consistently exceeds 10% of the calibration curve's 100% bound OD 450 signal Cross-reactivity of secondary antibody (2 ry Ab) with murine sera.
Employ a more species-specific 2 ry Ab with lower cross-reactivity or switch to the biotinylated ACE2 and streptavidin-HRP system Conduct a quick experiment using only the CC-plate to adjust the 2 ry Ab and substrate.
(1) Concentrate 2 ry Ab following the manufacturer's specifications at a higher value than employed, but within the recommended stated range. (2) Adjust the substrate concentration and reaction time.
Highest mean OD 450 value is 1.0.
A different, more sensitive substrate type, e.g., TMB, was employed. High 2 ry Ab concentration. OD 450 > 1.0 will further convolute the calibration curve non-linearity.
Conduct a quick experiment using only the CC-plate to adjust 2 ry Ab and the substrate.
(1) Dilute the 2 ry Ab following the manufacturer's specifications at a lower value than employed, but within the recommended stated range. (2) Adjust the substrate concentration and reaction time.
Anticipated Results
The calibration curve should be a gradient in intensity of substrate color, starting from intense color at high RBD concentration (column 1) to faded color (columns 8-10) (Figure 8). Visually, the background wells should be nearly transparent. The neutralizing sample sera should have the reverse intensity gradient of the CC-plates, starting from a fainter substrate color, changing to a more intense color, i.e., showing complete ACE2-RBD binding and absent neutralization in low serum dilution wells with consistent intense substrate color. The fitted calibration curve (OD-readings at various bound ACE2 concentrations) should match those in Figure 2. Example of using the auto-calculation sheet, in the Supplementary Materials, can also be found in Figure 9. A calculation Sheet (GraphPad Prism v8.3 project: CELISA Calculation Sheet.pzfx) is included in the Supplementary Materials; the sheet has four data tables and three figures, which automatically calculates the neutralizing titers at N 50 . The first data table has editable yellow-colored cells for blank-corrected calibration curve OD 450 readings (five replicates). The second data table has one S-plate editable yellow-colored cells for background-corrected four serially diluted sera, i.e., one S-plate. The rest of the calculation are conducted automatically by the calculation sheet, and the results include data and plots of neutralization N% for each individual serum at different dilutions, individual serum nAb titer at N 50 , and group-averaged nAb titer column graph (Figure 9).
Conclusions
This protocol offers a standardized and validated cELISA assay for the highly sensitive determination of neutralizing capacity of murine immune sera to support vaccine development against COVID-19. This protocol is of special interest as it presented the background of previously reported cELISA assays and pinpoints their shortcomings, thus ensuring standardization and optimization of key aspects of the assay. Moreover, we discussed future strategies for the development of similar assays to determine the human sera neutralization, against original or mutant variants of SARS-CoV-2. Furthermore, we have also demonstrated that the protocol method efficacy is equivalent to gold standard assays, such as VNT assay, while surpassing method validation criteria. This renders the protocol method suitable as a high-throughput in vitro efficacy assay, without physical containment requirements, which supports the progress of vaccine development. | v3-fos-license |
2019-01-22T22:25:55.631Z | 2018-12-21T00:00:00.000 | 58644886 | {
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} | pes2o/s2orc | ROS and the DNA damage response in cancer
Reactive oxygen species (ROS) are a group of short-lived, highly reactive, oxygen-containing molecules that can induce DNA damage and affect the DNA damage response (DDR). There is unequivocal pre-clinical and clinical evidence that ROS influence the genotoxic stress caused by chemotherapeutics agents and ionizing radiation. Recent studies have provided mechanistic insight into how ROS can also influence the cellular response to DNA damage caused by genotoxic therapy, especially in the context of Double Strand Breaks (DSBs). This has led to the clinical evaluation of agents modulating ROS in combination with genotoxic therapy for cancer, with mixed success so far. These studies point to context dependent outcomes with ROS modulator combinations with Chemotherapy and radiotherapy, indicating a need for additional pre-clinical research in the field. In this review, we discuss the current knowledge on the effect of ROS in the DNA damage response, and its clinical relevance.
Introduction to the DNA damage response
DNA damage refers to physical or chemical changes to DNA in cells, which can affect the interpretation and transmission of genetic information. DNA can be damaged by a variety of exogenous and endogenous insults including chemicals, radiation, free radicals, and topological changes, each causing distinct forms of damage [1]. Cells have evolved complex processes for dealing with damage to the genome. Depending on the nature of the lesion in DNA, specific pathways are activated to facilitate identification of the damaged regions and their repair [2,3]. A particularly dangerous lesion is the DNA double strand break (DSB) which can be mutagenic due to chromosomal rearrangements or loss of genetic information due to erroneous DNA repair. In response to DNA damage a network of events collectively termed as the DNA damage response (DDR) is activated. This response includes DNA damage recognition, activation of checkpoints, cell cycle arrest, and eventually final outcomes of repair, apoptosis and immune clearance [4,5]. The molecular components of the DSB induced DDR have been studied in detail, and are typically classified into three major groups -"sensors" which recognize damage, "transducers" which coordinate signaling, and "effectors" which mediate eventual outcomes ( Fig. 1) [6]. Other DNA damage response/ repair pathways include Mismatch Repair (MMR) for mismatched bases, Base Excision Repair (BER) for base modifications, Nucleotide Excision Repair (NER) for intra-strand cross links and thymidine dimers, Single Strand Annealing (SSA) for single strand DNA (ssDNA) damage and Transcription coupled repair (TCR) for transcription associated damage [3]. The DDR leading from DSBs on the other hand activate a network of related pathways including Homologous Recombination (HR), Non-Homologous End Joining (NHEJ), Microhomology Mediated End
Introduction to ROS
Reactive oxygen species (ROS) comprise of a family of short-lived molecules like O 2 -, H 2 O 2 and •OH, first described in skeletal muscle as free radicals [9]. Though initially thought to be a hazardous byproduct of mitochondrial respiration, discoveries in the last four decades have illuminated functional roles for ROS in cells-from aiding immunity (e.g. oxidative bursts in phagocytes to eliminate pathogens) [10] to acting as signaling molecules (e.g·H 2 O 2 regulating NFκB, MAPK pathways) [11]. ROS are produced endogenously by (i) mitochondria (where O 2 acts as a terminal electron acceptor for electron transport chain) [12], (ii) NADPH oxidase, a cell membrane bound enzyme [13], (iii) Peroxisomes (which contain enzymes that produce H 2 O 2 e.g. polyamine oxidase) [14], (iv) Endoplasmic reticulum (produce H 2 O 2 as a byproduct during protein folding); or upon exposure to exogenous stress like ionizing radiation (IR), chemotherapeutic drugs and environmental insults, which affect the organelles and enzymes listed above [15]. ROS production has been implicated in mediating chemotherapy or radiotherapy responses via its effects on downstream cell survival or death signaling cascades [16][17][18]. This has led to suggestions that ROS modulators could be used for cancer primary prevention, or to enhance therapeutic responses [18,19]. However, there has been little progress in translating ROS knowledge from labs to the clinic. For example, despite promising in-vitro data, most antioxidant trials in cancer prevention have yielded negative results [20,21], highlighting the need for additional basic understanding of this process in cells. This review aims to examine mechanisms by which ROS mediates the DNA damage response, and provide insights for clinical exploitation of ROS in cancer. ROS are well recognized as mediators of DNA damage. For example, Ionizing Radiation (IR) induces DSBs through direct high-energy damage to the sugar backbone of DNA, but also through free radicals generated in cells-mostly •OH from water [22]. Chemotherapeutics like doxorubicin and cisplatin increase ROS levels, which contributes to their genotoxicity [23,24]. ROS have also been reported to directly induce other forms of DNA damage through oxidizing nucleoside bases (e.g. formation of 8-oxo guanine) [25], which can lead to G-T or G-A transversions if unrepaired. Oxidized bases are typically recognized and repaired by the BER pathway, but when they occur simultaneously on opposing strands, attempted BER can lead to the generation of DSBs [26]. ROS accumulation also induces mitochondrial DNA lesions, strand breaks and degradation of mitochondrial DNA [27].
Role of ROS in DNA damage by oncogenic replication stress
An important source of endogenous DNA damage and DSB generation in cancer is oncogene induced replication stress [28]. Proto-oncogenes aid in cell growth and proliferation, but mutations or overexpression can transform them into oncogenes that drive continuous cell growth and carcinogenesis. Oncogenic cell cycles are typically associated with replication stress, which is defined as aberrant replication fork progression and DNA synthesis [29]. Replication stress ultimately results in genomic instability and paves the way for tumor development through the accumulation of additional pro-carcinogenic changes [28,30]. The DDR acts as a barrier which limits the expansion of abnormally replicating cells, and this leads to a selective pressure for DDR defects in carcinogenesis [31].
Replication stress arises from a variety of sources including aberrant origin firing, decoupling of DNA polymerase-helicase activity, and physical obstacles to the replication fork [29]. Oncogene activation leads to an increase in ROS, which in turn influences the occurrence of replication stress [32,33]. ROS oxidize dNTPs to affect polymerase activity and thereby reduce replication fork velocity in vitro [34,35]. ROS can also affect replication fork progression through dissociation of peroxiredoxin2 oligomers (PRDX2). PRDX2 forms a replisome associated ROS sensor that binds to TIMELESS, a fork accelerator. Elevated ROS lead to dissociation of PRDX2 and TIMELESS, thus slowing replication fork speed [36]. Oxidized bases occurring from ROS activity also present a physical obstacle to replication forks [37], resulting in the breakdown of replication forks at fragile sites across the genome. Fork breakdown leads to DSBs and ultimately under-replicated or overreplicated DNA [28], with concomitant genomic instability in the tumor. Modulation of replication stress by ROS has clinical implications, with the development of several agents-notably ATR and WEE1 inhibitors, which target replication stress in tumours [28].
Sensor kinases
The initial sensing of DSBs is performed by the kinases ATM/ ATR and DNA-PK, along with a network of sensor proteins [38,39]. ATM loss, which is common in cancer, leads to an increase in ROS. This elevation in ROS appears unrelated to the canonical role of ATM in the DNA damage response. ATM loss modulates mitochondrial turnover, with an increase in aberrant mitochondria and therefore ROS [40,41]. ATM-deficient cells also have increased ROS due to defects in NRF2 activity, a transcriptional factor which promotes the expression of antioxidant proteins under conditions of cellular stress [42,43]. Accordingly, inhibition of the ATM-G6PD axis exacerbates mitochondrial oxidative stress and confers synthetic lethality with FLT3 tyrosine kinase inhibitors in AML [44]. Similarly, DNA-PK deficient cells accumulate higher ROS upon oxidative stress [45,46].
The sensor kinases however can also directly be modulated by ROS levels, with distinctions between members of the family. ATM can be directly activated by oxidative stress, for example by H 2 O 2, leading to its autophosphorylation and subsequent downstream activation of the DDR pathway [47]. On the other hand ROS accumulation inhibits DNA-PKcs activity by altering its interaction with KU70/80 [45]. Oxidative stress by H 2 O 2 requires ATR for γ-H2AX accumulation and activation of the DDR [48], as well as ATR dependent phosphorylation of Chk1 [49]. Further studies are needed to explore the effect of ROS on the activity of ATR, as well as the effect of clinical-grade ATR inhibitors on cellular ROS levels. Overall, the DDR sensor kinases appear to act to prevent ROS accumulation and protect the genomic integrity, although there are likely to be context specific variations depending on cell type and nature of insult.
Chromatin remodelers
Brahma-related gene 1 (BRG-1) associated factor complex (BAF) are chromatin remodelers commonly mutated in cancer [50], and have a recently described role in the initial activation of the DNA damage response by modulating ATR activation [51,52]. Two main components of BAF complex are AT-rich interacting domain 1 A (ARID1A) and BRG1, ATPase of the BAF complex. ROS lowers ARID1A expression by promoter methylation in ovarian cancers [53,54], and ARID1A loss sensitizes ovarian cancer cells to ROS inducing agent elesclomol [55]. Importantly, ARID1A/BRG-1 loss increases reliance on OXPHOS, causing increased ROS, and synergizes with inhibitors of OXPHOS [56], offering a possible redox based therapeutic strategy for cancers harboring SWI/SNF mutations.
Histone H2AX is another chromatin factor that has been extensively studied in the DNA damage response [57]. Phosphorylated H2AX (γH2AX) helps to recruit multiple components of the DDR to the site of DNA DSBs to initiate DNA DSB repair [58,59]. Deficiency of H2AX invivo is characterized by genomic instability and radiosensitivity [60,61] arising from an impaired DDR. Interestingly, chronically elevated ROS mediates H2AX protein degradation, which is associated with decreased γH2AX and therefore improved sensitivity to platinum therapy in triple negative breast cancer [62]. Conversely, acute oxidative stress increases γH2AX activation and DDR signaling [63]. This has been suggested to blunt the treatment response to chemotherapy and radiation, and is associated with worse outcomes for colorectal [64], breast [65], and lung cancer [66]. The link between H2AX and ROS is bidirectional. γH2AX mediated activation of the Nox1-Rac1 complex [67,68] regulates ROS production [69]. However, the pathophysiological relevance of γH2AX-mediated ROS production remains unclear.
Effect of ROS on signal transduction within the DDR
Downstream of the sensor kinases are the transducer kinases Chk2 (activated by ATM) and Chk1 (activated by ATR), which phosphorylate and regulate proteins involved in DDR, DNA repair and cell cycle arrest. Menadione and camphorquinone induced ROS accumulation increases phosphorylated Chk2 [70,71]. N-acetylcysteine, an antioxidant reverses the synergistic effect between Chk2 inhibition and gemcitabine in pancreatic cancer cells highlighting the importance of ROS in activation of Chk2 [72].
Elevated levels of ROS also activate the ATR-Chk1 axis [34]. This is associated with poorer outcomes in breast cancer independent of hormonal status [73], and can mediate chemotherapy resistance in bladder cancer cells [74]. Accordingly, attenuation of ROS or ATR-Chk1 signaling confers chemosensitivity in platinum-resistant ovarian cancer cell lines with elevated levels of ROS [34]. Chk1 inhibition potentiates the cytotoxic effects of DNA-damage therapeutics in preclinical studies [75][76][77], although the relevance of ROS in this context has not been clearly defined. The ATR-Chk1 axis is a promising therapeutic target in cancer, and ROS dependent mechanisms that lead to ATR-Chk1 inhibitor resistance are worthy of further investigation.
Effect on cell cycle progression
Cell cycle arrest is an important aspect of the DDR, preventing cells with DNA damage from proceeding with cell division. In Hela cells, asperlin induced ROS leads to an ATM-Chk2 mediated G2/M arrest [78]. Similarly, ROS induced Chk1 activation leads to a p53 independent G2/M arrest in colorectal cancer cells [79]. Apart from their effects on the activation of cell cycle checkpoint proteins, ROS also promote cell cycle arrest by direct actions on the Cdc25 family of protein phosphatases (Cdc25A, B and C). The Cdc25 phosphatases promote cell cycle progression by removing inhibitory phosphates on cyclin dependent kinases (CDK) [80], and their levels/ activity are influenced by ROS. For example, ROS decreases Cdc25C protein levels to induce G2/M arrest [81]. Caulibugulone A (a family of isoquinoline quinones) induces ROS and reduces total Cdc25A levels [82]. Similarly, 17β-Oestradiol-induced ROS increases Cdc25A oxidation and reduces its phosphatase activity [83].
Mitotic entry and recovery from the G2/M arrest upon completion of DNA repair is mediated by the mitotic kinases Polo-like kinase 1 (PLK1) and AURORA-A. These kinases are frequently overexpressed in cancer and are also of interest in the context of ROS. PLK1 phosphorylates glucose-6-phosphate dehydrogenase, causing an increased PPP flux and production of NADPH, thereby increasing the antioxidant capacity of a cell. Interestingly, oxidative stress with H 2 O 2 increases PLK1 expression in a p53 dependent manner [84,85], but maintains a G2/M arrest. In contrast, ROS accumulation inhibits Aurora kinase A [86], even though PLK1 and Aurora-A are epistatic in the pathway. PLK1 and Aurora-A kinase inhibitors are currently in clinical trials, and understanding the dichotomous relation between ROS and these proteins may have clinical applications.
p53 transcriptional response, and apoptosis
p53 is a well-studied tumor suppressor that is mutated in over 50% of all cancers [87,88], and affects multiple cellular responses to DNA damage. Upon cellular stress and DNA damage; p53 is stabilized and aids in transcription of genes to determine cell fate [89]. p53 is a redox protein with clusters of cysteine residues that can be targets of ROS [90], but it can also regulate ROS in turn [91]. Furthermore, ROS accumulation has different effects on cell fate depending on p53 status; with more apoptosis in cells with functional WT p53 [92]. p53 has an important role in regulating pro and antioxidant genes depending on ROS intensity [91]. With lower ROS intensity, p53 activates antioxidant genes, while with higher ROS intensity it switches on pro-oxidant genes [93]. In response to ROS production under basal cellular conditions, p53 upregulates transcription of several antioxidant genes including manganese superoxide dismutase (MnSOD), glutathione peroxidase 1 (Gpx1), Sestrins, Glutaminase 2 (GLS2), and TIGAR, which increase PPP and NADPH production [94,95]. However, drastic increase of cellular ROS, for example by inhibition of thioredoxin reductase, an essential component of the thioredoxin antioxidant system, leads to JNK-mediated p53 activation and its downstream upregulation of pro-oxidant genes PUMA and PIGs [96]. Furthermore, under conditions of high ROS, p53 has been demonstrated to downregulate antioxidant proteins including SOD2 [97] and the anti-oxidant transcriptional factor Nrf2 [98]. This duality in p53 function with ROS intensity may decide the cell fate, with the protective arm of p53 activating processes to reduce cell stress with lower ROS intensity, while higher ROS intensity tips the balance towards cell death.
DNA repair
DNA repair is one of the effector outcomes of the DDR, but ROS so far has not been shown to affect DSB repair protein function directly. Rloops are DNA-RNA hybrids formed during replication-transcription conflicts in cells, and are a major source of genomic instability, requiring HR for resolution [29]. ROS induced R-loops are shown to require transcription coupled homologous recombination repair to protect actively transcribed genes in a Rad52 dependent manner [99]. ROS is typically implicated in regulating other DNA repair pathways such as BER, where the DNA glycosylase OGG1 is inhibited by ROS [100]. As 8-Oxo-dG can be potentially converted to DSBs, further work will be required to understand the contribution of ROS to DSB generation through this route. With clinical implications of interfering with DNA repair pathways becoming apparent, the direct effect of ROS on DNA repair proteins and its consequence in tumor development and chemoresistance warrant more studies.
Cell death/ resistance in response to chemotherapy and radiation
Resistance to chemotherapy is a commonly encountered problem in clinical oncology, leading to disease recurrence and poor outcomes. Chemotherapeutic agents such as platinum derivatives and gemcitabine upregulate ROS in vitro [101][102][103], adding to their genotoxic effects. In addition to generating nuclear DNA adducts, platinum drugs increase mtROS via formation of mitochondrial DNA adducts [104][105][106], the extent of which correlates with cytotoxicity [24,107]. Pro-oxidant strategies could therefore serve as adjuncts to improve the efficacy of chemotherapy and reduce the development of resistance [108]. For example, depletion of intracellular glutathione (GSH) using RNAi against the anti-oxidant transcription factor Nrf2 leads to increased ROS and increased sensitivity to chemotherapy in preclinical studies [103].
Radiotherapy using ionizing radiation (mega-voltage X-ray beams) is a widely used modality in cancer treatment. DNA damage can occur directly as the beam interacts with DNA strands in the nucleus, or indirectly via generation of free-radicals within the cell. The indirect method, accounting for about 80% of DNA damage, occurs when hydroxyl free radicals (•OH) are produced from the radiolysis of water molecules [109]. These molecules are able to diffuse a short distance into the nucleus to cause DNA damage. Antioxidant molecules within cells therefore can reduce the ability of ionizing radiation to cause DNA damage. Early studies observed that depletion of GSH could enhance radiosensitivity of squamous cell carcinoma cell lines [110]. More recent work has described the role in radio-resistance for Nrf2. Nrf2 is normally degraded via its interaction with a repressor protein Keap1. Decreased Keap1-Nrf2 interaction [111,112] and loss-of-function mutations of Keap1 [112,113] lead to aberrant Nrf2 activation, and therefore resistance to radiotherapy. Other mechanisms conferring radio-resistance include regulation of antioxidants by the synergistic effects of thioredoxin and GSH [114]. Cancer stem cells have active ROS-scavenging mechanisms and consequently show lower ROS levels and, less DNA damage from radiation, and therefore more radio-resistance [115].
Immunogenic cell death (ICD) after chemotherapy and radiation
ICD is increasingly appreciated as an important mechanism of chemotherapy mediated tumor cell-kill, where chemotherapy induced antigen release, immune priming and activation triggers an immune response against the tumor. The initial stages of immunogenic cell death are mediated by release of factors such as High-mobility group box 1 (HMGB1) protein and Calreticulin, and subsequent activation of the adaptive immune system through antigen presenting cells, leading to eventual T-cell mediated killing [116].
HMGB1 is a non-histone chromatin protein, which is released by dying cells into the micro-environment, where it plays a vital role in dendritic cell licensing and maturation. HMGB1 is a redox sensor, with cysteine 106 (Cys106) particularly important for the regulation of proinflammatory cytokine release [117,118]. Reduction of three cysteine residues (Cys23, Cys45, and Cys106) induces chemotaxis of inflammatory cells [119], while oxidation of all three cysteines abolishes its pro-inflammatory and chemotactic properties [120]. As with other components of the DDR, the relationship between ROS and HMGB1 release is bi-directional. The antioxidant N-acetylcysteine attenuates HMGB1 release [121], and HMGB1 release in itself increases ROS production [122], which can lead to further oxidation of HMGB1. Oxidized HMGB1 enhances apoptosis and chemosensitivity in pancreatic and colorectal cancer cell lines [123], whereas reduced HMGB1 promotes autophagy-mediated chemoresistance towards melphalan, oxaliplatin and paclitaxel [123,124]. Oxidized HMGB1 in apoptotic cells has however been reported to also mediate immunological tolerance [125], although the relevance of this finding to ICD after chemotherapy is unclear. Further studies are required to clarify the role and mechanisms underlying ROS-regulation of HMGB1 and its effects on in vivo tumor responses to chemotherapy [126].
ROS plays an active role in the pathways involved in immunogenic cell death including the induction of autophagy [127], and antigen presentation by immune cells [128,129]. Induction of autophagy via increased levels of ROS results in biochemical hallmarks of ICD evasion [130]. Similarly, irradiation of necrotic high-grade gliomas increases the anti-tumor efficacy of dendritic cell (DC) vaccines, presumably via elevated levels of carbonylated proteins [131]. This suggests that ROS modulators could potentially play an important role in DC vaccine development and as adjuncts with other forms of immunotherapy, further highlighting its clinical relevance.
Combination studies with genotoxic agents in cancer
Modulators of ROS and redox pathways have been tested in combination with chemotherapy in clinical trials with mixed efficacy (summarized in Table 1). For instance, a single arm trial of NOV-002 (a formulation of disodium glutathione disulfide) in combination with standard neoadjuvant chemotherapy (AC-T) for stage II-IIIc HER2- Table 1 Summary of clinical studies on ROS modulators in malignancies. negative breast cancer showed promising pCR rates [132], whereas phase 3 trial data on NOV-002 in non-small cell lung cancer has been disappointing [133]. While the pro-oxidant molecule Imexon demonstrated initial promising results in advanced pancreatic cancer in combination with gemcitabine [134], a larger phase II trial demonstrated no significant survival benefit or responses (ClinicalTrials.gov; NCT00637247). However, it showed single-agent clinical activity in refractory non-Hodgkin B-cell lymphoma [135], and will need to be further evaluated with chemotherapy in this setting. On the other end of the spectrum, due to the diverse effects described above for ROS in activating various DNA damage responses, high ROS is also associated with resistance to chemotherapy [18,136]. Antioxidants such as ascorbate have been tested in this setting. However most of the clinical data on ascorbate-chemotherapy combinations are not randomized [137], and further RCTs are required to determine the efficacy of these strategies.
Compound
Further clinical research on ROS needs to take certain concepts into account. Firstly, in the context of a defined genotoxic agent in a particular cancer, identifying the specific ROS species involved in a) generation of DNA damage and b) in modulating the downstream DDR, would help in identifying specific therapeutic targets. Indeed, perturbation of redox status with a pan-antioxidant or pro-oxidant would have profound effects on both pro-survival and pro-death pathways [138], and may result in attenuation of specific chemotherapeutic responses [139]. Secondly, ROS has a dose dependent effect on activity of proteins leading to differential downstream outcomes [140], which are distinct in the setting of exogenous and endogenous ROS, and need to be evaluated in phase 1 dose finding studies with appropriate pharmacodynamic/ pharmacokinetic readouts. There is a clear need for further research outlining how chemotherapy and radiotherapy related DNA damage responses are influenced by ROS and ROS modulating drugs, using established and validated pre-clinical models.
Concluding remarks
The role of ROS in DNA damage response is multifaceted and pleomorphic. A distinction is required between oxidative stress leading to DNA damage/ downstream activation of DDR, and the role of ROS in modulating components of the DDR (signaling and effectors). There is compelling evidence that dysregulation of ROS contributes towards cancer pathogenesis as well as chemoresistance and radio-resistance, in a context specific manner. However, the modest responses of existing pan-antioxidant or pro-oxidants in advanced cancers could suggest that approaches aimed to reduce or increase ROS may not suffice. Future research on the specific mechanisms in chemo/radioresistance that are mediated by distinct reactive oxygen species, in distinct cellular contexts, will be valuable towards the development of drugs targeting these mechanisms. | v3-fos-license |
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} | pes2o/s2orc | Catalytic and antimicrobial properties of α-amylase immobilised on the surface of metal oxide nanoparticles
New methods of obtaining products containing enzymes reduce the costs associated with obtaining them, increase the efficiency of processes and stabilize the created biocatalytic systems. In the study a catalytic system containing the enzyme α-amylase immobilized on ZnO nanoparticle and Fe3O4 nanoparticles was created. The efficiency of the processes was obtained with variables: concentrations of enzymes, temperatures and times, to define the best conditions for running the process, for which were determined equilibrium and kinetics of adsorption. The most effective parameters of α-amylase immobilization on metal oxides were determined, obtaining 100.8 mg/g sorption capacity for ZnO and 102.9 mg/g for Fe3O4 nanoparticles. Base on the best parameters, ZnO-α-amylase was investigated as an antimicrobial agent and Fe3O4-α-amylase was tested as a catalyst in the process of starch hydrolysis. As a result of the conducted experiments, it was found that α-amylase immobilized on Fe3O4 nanoparticles maintained high catalytic activity (the reaction rate constant KM = 0.7799 [g/dm3] and the maximum reaction rate Vmax = 8.660 [g/(dm3min)]).
Introduction
Enzymes in industry, especially in biocatalysis, become increasingly popular. Enzymes are macromolecules that act as specific (bio)catalysts, accelerating or enabling chemical reactions by reducing the activation energy of the process [1,2]. New methods of enzyme processing are aimed at reducing the costs associated with obtaining them, increasing the efficiency of processes and stabilizing the created biocatalytic systems [3]. Nanotechnological methods are becoming widely used to overcome these problems. The increased surface area of the nanoparticles makes the immobilization of enzymes effective, which ultimately promotes the catalytic activity of proteins [4,5].
An a-amylase classified as EC 3.2.1.1 belongs to glycoside hydrolases and acts on glycosidic bonds, exactly on a-1,4-glycosidic bonds [6]. The enzyme decomposes starch to two or three saccharides. In the human body it occurs in saliva and is secreted by the pancreas. The amylase group is used in industries related to fermentation processes or starch decomposition [7,8].
An immobilisation of enzymes is intended to improve the resistance of biocatalysts to changing reaction conditions that adversely affect the catalytic performance of the free enzyme. The sensitivity of enzymes to changes in process conditions results from their composition and a change in the structure of enzymes may cause their complete inactivation [9]. Immobilization has a positive effect on the structure of the catalytic protein by stabilizing, resulting in increased tolerance to pH, temperature or denaturants. Modification of the protein by connecting it to the carrier facilitates its separation from the reaction mixture after the process. The resulting products are not contaminated with the enzyme and the enzyme deposited on the carrier can be reused. The immobilization of the biocatalyst also extends its catalytic activity so that it can be used repeatedly [10]. However, the immobilisation process may be associated with the deterioration of the catalytic properties of the entire biocatalyst. This is due to the stiffening of the protein structure and the restriction of the transport of substrates and products to and from the place of the active enzyme [11].
Enzymes are characterized by additional catalytic functions. In addition to their catalytic functions, some enzymes present additional properties, e.g. antimicrobial. Depending on the future use of the enzymes, as carriers of enzymes ZnO, Fe 3 O 4 , SiO 2 , TiO 2 , Ag or Au nanoparticles can be commonly used [12,13]. By using enzymes combined with selected nanomaterials that show biocidal activity (CuO, ZnO, Ag, Cu), it is possible to obtain products with synergistic effect [14,15]. The antimicrobial properties are distinguished by enzymes from the hydrolysis group (e.g. glycosidase and peptidase) due to the possibility of decomposition of peptidoglycans contained in cell walls, as well as oxidoreductase (e.g. oxidase), which antimicrobial activity is indirect and results from products catalysed by them [16,17].
The immobilisation by an adsorption on the surface of insoluble carrier is a method often appropriate. The method is based on the formation of bonds between the enzyme and the carrier resulting from hydrogen, hydrophobic, electrostatic, ionic and van der Waals forces [18]. The weak interactions do not influence the tertiary structure of the enzyme, which allows maintaining high catalytic activity. At the other side, the bonds can easily be broken off, so that the protein can be washed away, reducing the activity of the preparation. Through its ease of application of immobilization via adsorption and the low cost of the method, it is a widely used technique. The adsorption enables the use of many types of media that are selected for a sufficiently high protein affinity to the matrix. Depending on the choice of carrier, as well as the enzyme used and the parameters of the process environment, the amount of immobilized enzyme changes [19]. The covalent immobilisation is a method that involves the formation of stable and permanent chemical bonds between the enzyme and the carrier, which limits the leaching of the enzyme from the matrix, extending the activity of the product [20].
The aim of the study was to carry out the process of aamylase immobilization on the surface of metal oxide nanoparticles and to obtain materials with antimicrobial and biocatalytic properties. The scope of the study included the determination of the influence of the parameters of the process of obtaining materials on the efficiency of this process, analysis of equilibrium and kinetic parameters of the process and determination of the activity of a-amylase immobilized on Fe 3 O 4 in the process of hydrolysis of starch and antimicrobial properties of a-amylase immobilized on ZnO nanoparticles. The Fe 3 O 4 nanoparticles allow to separate the biocatalyst from the reaction system, while ZnO nanoparticles allow to obtain material with the increased antimicrobial activity.
Synthesis of Fe 3 O 4 and ZnO nanoparticles
The solutions of FeCl 3 Á 6H 2 O and FeSO 4 Á 7H 2 O were added to the Teflon vessel in the molar ratio of iron ions 1:1. In the presence of ultrasounds (Hielscher UP400St sonicator), an aqueous solution of NaOH acting as a precipitating agent of iron hydroxide was injected and the mixture was homogenised for 3 min. Then, in the MAG-NUM II microwave reactor at 180°C for 15 min, a dehydration process was carried out to obtain Fe 3 O 4 nanoparticles. The zinc oxide nanoparticles were obtained in the presence of ultrasound by dropping into a solution of ZnSO 4 Á 7H 2 O a solution of Na 2 CO 3 (for complete precipitation of zinc hydroxide) and after 3 min the suspension was in the microwave reactor at 180°C for 10 min. The materials were filtered, washed and dried at 105°C for 24 h. The schematic diagram for the process was presented in Fig. 1.
Preparation of a-amylase immobilised on metal oxide nanoparticles
To the solution of a-amylase from Aspergillus oryzae (Sigma Aldrich) with variable initial enzyme concentration 100 mg of Fe 3 O 4 or ZnO nanoparticles were added. The process was carried out at 20-30°C in the time from 30 to 120 min. At the end of the process, the solution was filtered and the filtrate was examined spectrophotometrically to analyse the enzyme concentration and assess the efficiency of the process. The process efficiency (E) was determined from the equation: where: C enzyme,0 -spectrophotometrically measured concentration of the a-amylase solution [mg/dm 3 ], C enzyme,t -spectrophotometrically measured concentration of the a-amylase after sorption process [mg/dm 3 ]. The amount of material that was adsorbed in the ratio of the amount of oxide (q) was determined from the equation: where: m MeOx -mass of metal oxide nanoparticles [mg], V-volume of the enzyme solution. Table 1 contains the variables tested in the process of the immobilisation of a-amylase on the metal oxides nanoparticles with ranges of variability of input quantities.
On the basis of statistical analysis, the variables and the way to which they affected the efficiency of the sorption process were determined. The analysis was carried out using the program STATISTICA 10 these parameters, the most favourable conditions for the immobilisation process were selected. Then, the sorption was carried out at selected parameters in order to obtain the most suitable system of the metal oxide nanoparticles with the enzyme for further research.
Kinetic and equilibrium parameters of the aamylase immobilisation process The equilibrium and the kinetics of the processes were examined. Analogously to the study of immobilization efficiency, 100 mg of the metal oxides were prepared and then 10 cm 3 of enzyme solution of proper concentration was added. After the process, the solution was examined spectrophotometrically. The equilibrium parameters and process kinetics were determined using selected models ( Table 2).
Material characteristics
The UV-Vis spectroscopy was used to quantify the aamylase content in the solutions. The determination method was based on modified Lowry's method [23]. The measurement was performed at 670 nm with the Rayleigh UV-1800 spectrophotometer. The prepared mixtures: A: 2% solution of Na 2 CO 3 in 0.1 M NaOH, B: 1% solution of CuSO 4 . C: 2% sodium tartrate solution, D: 100 cm 3 of solution A ? 1 cm 3 B ? 1 cm 3 C, prepared 30 min before the measurement, E: Folin-Ciocalteu's solution diluted with distilled water in the ratio Table 2 Examined equilibrium models and sorption kinetics [21,22] Equilibrium models sorption processes Isotherms Non-linear form Liner form Descriptions ,
Kinetics models sorption processes Model Non-linear form Linear form Descriptions
Pseudo-first order 1:1. The samples for spectrophotometric tests were prepared by adding 1 cm 3 of the test solution, 5 cm 3 of solution D, after 10 min 0.5 cm 3 of reagent E was added and the sample remained in the dark. After 30 min, the absorbance of the samples was measured at 670 nm. The reference curves were made using a-amylase.
Studies on crystalline structure of metal oxides were carried out using Philips X'Pert Camera diffractometer with PW 1752/00 CuKa monochromator in the range of angles 2h, from 10 to 60°. The size of nanoparticles crystallites based on the metal oxides was determined on the basis of Scherrer's equation: where: d-average size of crystallites, FWHM-peak width at half of its height, which is proportional to the size of the crystallite, K-Scherrer constant, depending on the shape of the particles (for cuboid-shaped particles it takes the value of 0.89; 1.10 for spherical particles), k-X-ray wavelength, h-angle which is formed by the incident radiation with the atomic plane. The constant K was selected on the basis of the shape of particles determined by SEM and TEM microphotography.
The morphology of the obtained metal oxide nanoparticles was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The investigations were carried out using Vegall-TescanCompany and Tecnai Transmission Electron Microscope, F20 X-Twin. The Fourier Transform Infrared Spectroscopy (FT-IR) was used to determine the presence of specific functional groups in the systems studied. The FT-IR measurements were carried out using Nicolet 380 apparatus in which spectra were recorded in the range from 3900 to 400 cm -1 . The sorbent surface and diameter of pores were performed (Micromeritics ASAP2010). In order to determine the energy gap width of metal oxide nanoparticles, diffuse reflectance spectroscopy was measured in the range 200-850 nm. Reflective spectra were recorded with a UV-2600 spectrophotometer (Shimadzu) equipped with an integrating sphere of 10 cm diameter. The nature of the surface of the metal oxide nanoparticles was determined on the basis of the zeta electrokinetic potential (Malvern Instruments, ZS-90 and Brookhaven, zetaPALS).
Studies on the biocatalytic properties of aamylase for Fe 3 O 4
The aim of the study was to check the activity of the immobilized enzyme and its applicability as a bionanocatalytic material. The starch hydrolysis process was carried out using the obtained immobilized enzyme on the iron oxide nanoparticles. The starch solution from 1 to 10 g/dm 3 was added to the iron oxide with a-amylase and then starch distribution during time was studied. Using the magnetic properties of the carrier, the enzyme was separated from the reaction mixture. At the next stage, 10 ml of post-reaction mixture solution was measured into a beaker, water and 0.1 ml of 0.01 M I 2 solution in 1% KI were added. The solution was mixed on a magnetic stirrer. After 10 min the absorbance of the solution was measured on a spectrophotometer.
The kinetic parameters of this process were determined i.e. Michaelis' constant and the maximum reaction velocity was calculated from the Lineweaver-Burk equation [18]:
Studies on antimicrobial properties of a-amylase for ZnO
The antimicrobial properties were studied by observing the growth of selected microorganisms from the bacterial group. The examined microorganisms were Escherichia coli, Staphylococcus aureus and Candida albicans. The test substances were: zinc oxide, zinc oxide with a-amylase and a-amylase. The weights containing the oxide constituted a specific percentage of the nutrient solution weight (about 15 g)-0.1%, 0.5% and 1%. The size of the weights containing the enzymes themselves was calculated taking into account the efficiency of the sorption process, so that the content of the enzyme is equal to the content of the enzyme immobilized on zinc oxide. On the dishes with prepared appropriate mediums, the weights of the tested substances were applied and then the cultures were made by surface method. Growth was observed after 24 h, 48 h and 72 h after the cultures. The tested samples with microorganisms were incubated at 30°C. The effect of materials on microorganisms was evaluated according to the given scale (Table 3).
Physicochemical characteristics of a-amylases on the metal oxide nanoparticles
The phase composition of the obtained ZnO and Fe 3 O 4 oxides was examined using the XRD method (Fig. 2). Based on the obtained diffractogram and the Scherrer equation the average size of nanoparticle crystallites was determined using. The average size of crystallites in the obtained zinc oxide was 81,18 nm and for the iron oxide was 23.24 nm.
The morphology of the pure metal oxide nanoparticles was studied using scanning electron microscopy and transmission electron microscopy. In Fig. 3a-f it can be observed that the zinc oxide obtained is mostly needleshaped particles. The zinc oxide nanoparticles form elongated agglomerates up to about 10 lm in size. Single needles of zinc oxide are about 100 nm long and 5 nm thick. The nanoparticles of the iron oxide are spherical ( Fig. 3g-l). The particle diameter is about 20 nm. Nanoparticles do not occur in the form of agglomerates; single particles are clearly visible. The dimensions of metal oxide nanoparticles are consistent with the size of crystallites determined by XRD method.
The BET surface area of metal oxide nanoparticles was measured as 10.92 m 2 /g and 16.67 m 2 /g, respectively, for Fe 3 O 4 and ZnO nanoparticles ( Table 4). The study revealed that the adsorption of enzyme molecules took place mainly on the surface of oxides, not in their pores. The small size of nanoparticles increased the effectiveness of contact with a-amylase, without increasing the porosity of the material, so that the availability of a-amylase was maintained. According to Pandya et al. if the enzyme is immobilized on the surface of mesoporous silica, it is immobilized only on the external surface, which results in increased activity with limited stability of the whole system. For materials with a larger pore diameter, the enzyme is immobilized inside the pores and a high increase in enzyme stability is observed [24]. An alternative method of improving system stability was given by Patel et al., who modified the surface of Fe 3 O 4 nanoparticles with reduced graphene oxide. Additionally, the use of graphene oxide improved the catalytic properties of the enzyme, the activity of which increased by 192% compared to the free enzyme [25].
The ZnO and Fe 3 O 4 nanoparticles were characterized by a positively charged surface with an electrokinetic potential of approximately 20 mV. Go et al. confirmed that the hydrodynamic radiation and zeta potentials of NPs were highly affected by sorbed compounds. The mean zeta potential of ZnO NPs in their study was determined to be 22.9 ± 1.1 mV. A positively charged surface increases the binding efficiency of -COO-and -NH 2 groups derived from a-amylase [26].
The samples of zinc oxide, zinc oxide with immobilized a-amylase, iron oxide and iron oxide with immobilized aamylase were examined by infrared spectroscopy (Fig. 4). The band associated with the bond Zn-O is found at a wavelength number of approximately 465 cm -1 . The band associated with the bond O-H present in the material has been confirmed at a wavelength number of approximately 3382 cm -1 [27,28]. On the spectrum obtained for zinc oxide with immobilized a-amylase, a peak with a wavelength of 1652 cm -1 is observed, it is one of the characteristic peaks appearing in the diagrams of a-amylase and is associated with oscillations of N-H bonds [29,30]. For pure iron oxide, in the area 500-600 cm -1 , bands derived from the Fe-O bond in nanoparticles can be observed, while in the area 3400-3500 cm -1 the band is associated with the presence of hydroxyl groups derived from O-H bonds. The FT-IR analysis showed the presence of aamylase immobilized on iron oxide. After adsorption of aamylase a new band within 1400-1500 cm -1 appeared, which may indicate amide bond. Catalytic and antimicrobial properties of a-amylase immobilised on the surface of metal oxide… 1615 Immobilisation of a-amylase on metal oxide nanoparticles Table 5 presents averaged results of the a-amylase immobilization process. Figure 5 a,b presents an analysis of the effect of the examined parameters on the efficiency of the process (E) of a-amylase immobilization on the zinc oxide. The initial concentration of the enzyme and temperature had the greatest influence on the efficiency of enzyme deposition. The efficiency of enzyme immobilization was significantly influenced by time (linearly) and interactions between temperature and time and between enzyme concentration and temperature. In the processes of obtaining a-amylase for Fe 3 O 4 the significance of the effect was confirmed for the initial enzyme concentration. It was found that the most optimal process parameters are: enzyme concentration equal to 1 g/dm 3 , temperature 30°C and time of 120 min. The conditions ensure optimal immobilization efficiency, with the maximum amount of immobilized enzyme. The amount of absorbed protein increases significantly with the concentration, despite the reduced efficiency of sorption. A higher process temperature increases the efficiency of the process without significantly affecting the amount of absorbed enzyme. On the basis of the results, sorption of the enzyme to Fe 3 O 4 was carried out in the most favourable conditions, i.e. temperature at 20°C for 120 min in the enzyme solution of 1 g/ dm 3 concentration.
Kinetic and equilibrium parameters of the aamylase immobilisation process
Based on the results, the parameters of sorption equilibrium of the enzyme on the surface of the metal oxides were determined (Table 6). For both oxides, Langmuir isotherm has the highest coefficients of determination. The Langmuir isotherm describes the chemical adsorption which results in a single-molecular layer of the sorbent [22]. The high determination factor for Freundlich's isotherm, which concerns multilayer physical adsorption on microporous materials has also been confirmed for a-amylase deposition on ZnO [21]. It can be concluded that the sorption process of a-amylase on zinc oxide is a complex process, following the assumptions of these two models (Fig. 6). The analysis of the kinetics of enzyme immobilization processes confirmed the chemical nature of the processes. The sorption p=0.05 The functioning of the carrier surface with amine groups caused prolongation of the enzyme activity. Higher thermostability compared to free enzyme particles was also confirmed [33]. Pandya et al. observed the significance of the a-amylase deposition site for its activity [24].
The catalytic properties a-amylases on Fe 3 O 4
The catalytic properties of a-amylases were tested by hydrolysis of starch. The plot of starch decomposition over time is presented in Fig. 7a. The kinetic parameters of the reaction were determined after the hydrolysis of starch. The concentration of hydrolysis product (S) was calculated, based on the concentration of starch remaining in the system. Next, a diagram 1/S from 1/t was plotted, which is presented in Fig. 7b. This graph represents the Lineweaver-Burk equation. On the basis of the function equation the constant of K M reaction rate = 0.7799 [g/dm 3 ] and the maximum reaction rate V max = 8.660 [g/(dm 3 min)] were calculated. Despite the limited mobility of the Table 6 The equilibrium and the kinetics parameters of the a-amylase immobilisation process on the metal oxides nanoparticles The effectiveness of a-amylase combined with the carrier was confirmed by Salaonkar et al. comparing the effectiveness of hydrolysis distribution. The constant reaction rate for the bound enzyme was 0.5889 lmol/dm 3 which is consistent with the results obtained in the presented study. Compared to the reaction carried out in the presence of a free enzyme, the value of both the constant and the maximum reaction rate was comparable, which confirmed the effectiveness of deposition of the enzyme on a carrier without loss of its activity [34].
Antimicrobial properties
The antimicrobial properties of ZnO-a-amylase, pure zinc oxide and pure enzyme as reference samples were tested. Table 7 summarises the data obtained in the study. The antimicrobial activity of zinc oxide nanoparticles and aamylase on ZnO nanoparticles surfaces was confirmed. The zinc oxide showed antimicrobial properties against bacteria both gram positive and gram negative. The fungicidal properties of nanoparticles were limited. Azizi-Lalabadi et al. proved that the antimicrobial activity of ZnO nanoparticles is dependent on the species of microorganism (gram positive or gram negative). The sensitive microorganisms were gram negative bacteria, i.e. P. fluorescens, and E. coli [35].
Both Zn 2? particles and ZnO nanoparticles have an antibacterial effect. The nanoparticles reveal biocidal activity for two mechanisms: as a result of free metal ion toxicity resulting from dissolution of metals from the surface of the nanoparticles and generation of oxidative stress through generation of reactive oxygen species (ROS) using hydrogen peroxide (H 2 O 2 ) on the surface of the nanoparticles. Additionally, nanoparticles can affect the viability of microorganisms by agglomeration on the surface of bacteria and change the structure of lipids, peptidoglycans, proteins and their DNA [36].
In the presented studies, a-amylase itself demonstrated absence of antimicrobial activity. However, the addition of a-amylase improved the bioactivity of ZnO NPs. The enzyme catalyses the hydrolysis of the internal a-1,4-glycosidic bonds in starch, transforming starch into low molecular weight products such as glucose, maltose and maltotriose compounds. The basic components of the cell membrane are lipids and proteins which together make up 60 to 80%. The remaining part is made up of sugars, which are bound to lipids and proteins forming glycolipids and glycoproteins. The a-amylase affects the decomposition of sugars; however, it is only when combined with zinc oxide nanoparticles that the cell membrane of microorganisms is destroyed, thus deactivating them. Therefore, it is beneficial to obtain a complex compound with synergistic properties. Date described the process of glycoprotein breakdown using a-amylase confirming its effectiveness [37].
The activity of the composite results from the effectiveness of the connection between the components. Excessive release of a-amylase would reduce contact between ZnO and the enzyme and the activity of such a system would be equal to the pure ZnO. Anthony et al. confirmed the synergistic effects of amylase with nanoparticles on the example of silver nanoparticles. Despite the confirmed antimicrobial properties of Ag NPs, the connection of the material with the enzyme additionally increases the activity of silver nanoparticles [38]. Cui et al. by modifying the surface of Fe 3 O 4 with silica received a material with an inactive surface, which protected the catalase from biological, thermal and chemical degradation. However, the inactive surface inhibited activity of the carrier [39]. Homaei and Saberi successfully immobilized a-amylase on gold nanoparticles while maintaining temperature stability compared to free a-amylase. The enzyme also showed higher activity than the free form. Due to the improved stability of the enzyme on the surface of gold
Conclusion
In the processes of a-amylase immobilization on the surface of metal oxide nanoparticles ZnO-a-amylase and Fe 3 O 4 -a-amylase materials were obtained. In the processes of immobilization of the enzyme on the ZnO surface, the initial concentration of the enzyme, process time and its temperature showed to be important parameters. The most effective parameters of a-amylase immobilization on zinc oxide were determined, obtaining 96.20% efficiency of the enzyme sorption process and 100.8 mg/g carrier. The process of enzyme immobilization is complex-there is chemical adsorption and multilayer physical adsorption. Antimicrobial properties of prepared amylase from ZnO result from synergic action of zinc oxide nanoparticles with the enzyme, and a-amylase itself did not show antimicrobial properties.
In Fe 3 O 4 -a-amylase systems the obtained iron oxide particles had magnetic properties. By using iron oxide nanoparticles with magnetic properties, it is possible to separate the biocatalyst from the reaction system. The most favourable parameters for sorption of a-amylase to Fe 3 O 4 were: temperature 20°C, concentration of enzyme solution 1 g/dm 3 , sorption time 120 min. In the studies on the deposition of the enzyme on the surface of metal oxide nanoparticles, the equilibrium and kinetics parameters of the process were determined, based on which the chemical character of the enzyme immobilization was found. The model of pseudo-second order kinetics was characterized by the best fit (R 2 ) for Fe 3 O 4 -a-amylase. This indicated that chemisorption was taking place. The Langmuir isotherm showed the best fit for the study. The parameters of starch hydrolysis process indicated the catalytic activity of the enzyme immobilized on Fe 3 O 4 nanoparticles.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.
Compliance with ethical standards
Conflict of interest The authors report no declarations of interest. A B A B A B A B A B A B A B A B 24 2 2 2 1 0 0 0 1 0 1 0 0 3 3 3 3 3 3 48 2 2 2 1 0 0 2 2 2 3 2 2 3 3 4 4 4 4 72 2 2 2 1 0 0 3 3 2 3 3 2 4 4 2 3 3 3 3 3 3 3 3 3 3 3 3 48 2 2 2 2 2 2 3 3 4 3 4 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. | v3-fos-license |
2017-11-19T10:58:25.736Z | 2010-10-01T00:00:00.000 | 54447577 | {
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} | pes2o/s2orc | Synthesis and Reactivity of [1,2,4]Triazolo-annelated Quinazolines
This paper reports the synthesis of phenyl-substituted 2-alkoxy(methylsulfanyl)-1,2,4-triazolo[1,5-a]quinazolines starting from N-cyanoimidocarbonates and substituted hydrazinobenzoic acids as building blocks. Thionation or chlorination of the inherent lactam moiety in the target compounds followed by treatment with multifunctional nucleophiles provided access to a variety of derivatives.
Introduction
Triazolo-annelated quinazolines are known to constitute a pharmacologically interesting class of compounds. For instance, the novel compound Ia is effective adenosine antagonist whereas the related compound Ib was found to be benzodiazepine receptor antagonist [1][2][3]. The recently reported 1,2,4triazoloquinazolines of type II were also found to exhibit promising antihistaminic activity against histamine induced bronchospasms and showed negligible sedation, compared to chlorpheniramine maleate, and could therefore serve as lead molecules for further modification to obtain a clinically useful class of non-sedative antihistamines [4,5]. Furthermore, some triazoloquinazolines IIIa which originated from N-cyanoimidocarbonates as synthons, have been described as potent protein kinase inhibitors [6].
In our previous paper on the 1,2,4-triazolo [1,5-a]quinazolines series IIIb, the corresponding alkylated derivatives have been proven as excellent agents for controlling the plant growth diseases OPEN ACCESS caused by fungal pathogens, and some chlorinated compounds have shown an interesting affinity towards adenosine receptors [7].
Results and Discussion
The cornerstone of the strategy for the synthesis of our target products was the preparation of compounds 5a-h (Scheme 1, Table 1). The first step, the preparation of several dialkyl N-cyanoimidocarbonates 1 from equimolar amounts of cyanogen bromide and the corresponding alcohol was reported previously [8]. In addition, it has been found that, the reaction of cyanamide with carbon disulfide in the presence of KOH followed by the alkylation with methyl iodide gives dimethyl Ncyanoimidodithiocarbonate [9]. Scheme 1. Synthesis of [1,2,4]triazolo [1,5- Diazotization of the corresponding anthranilic acids [10] followed by the reduction with sulphur dioxide afforded the substituted 2-hydrazinobenzoic acids 2. Based on the high reactivity of Ncyanoimidocarbonates towards hydrazines to produce 1,2,4-triazole derivatives [11][12][13], reaction of 1 with 2 in ethanol in the presence of triethylamine under ice cooling analogously provided the intermediate 1,2,4-triazole derivatives 4, which upon treatment with hydrochoric acid produced the target [1,2,4]triazolo[1,5-a]quinazolin-5-ones 5a-h in 50-68% yield [14]. The structures of the novel compounds 5a-h have been established on the basis of their IR, 1 H-NMR and 13 C-NMR spectra and microanalysis.
The IR spectra of compounds 5a-h are characterized by a strong (C=O)-stretching band at 1,685-1,712 cm −1 .
Similarly, the corresponding thioxo derivatives 13a,b could be obtained in 56 and 61 % yield from the reaction of 10 with carbon disulfide in a molar ratio of 1:10 in refluxing pyridine for 2 h [26]. The IR spectra of 12 display strong (C=O) absorption bands at 1,702 and 1,711 cm −1 , and the 13 C-NMR spectra of 13 are characterized by a (C=S) resonance at 185.05 and 185.73 ppm. Replacement of the chlorine in compounds 9 by different hydrazides occurred smoothly in refluxing toluene to produce the [1,2,4]triazoloquinazolin-5-yl-carbohydrazides 14a,b in 65 and 76% yield [27]. The IR spectra of 14 are characterized by a strong (C=O) absorption band at 1,660, 1,673 and a weak (NH) absorption band at 3,184, 3,207 cm −1 , respectively. Like the reaction with hydrazides, the corresponding reaction of compounds 9 with carbazides according to literature [27] produced the respective [1,2,4]triazoloquinazolin-5-yl-hydrazine-carboxylic acid esters of type 15a,b in 75 and 80% yield as colorless solids. The IR spectra of 15 display a strong (C=O) absorption band at 1,708, 1,718 and a weak (NH) absorption band at 3,198, 3,261 cm −1 .
After having successfully elaborated the synthesis of the carbohydrazides 14, we became interested in seeing whether these compounds could be cyclo-condensed to the novel bis [1,2,4]triazoloquinazolines of type 16. In fact when amidrazones 14 were treated with phosphorus oxychloride at refluxing temperature for 2 h, followed by subsequent neutralization with saturated potassium carbonate solution or aqueous ammonia, the desired compounds 16a,b were obtained in 70 and 75% yield [28]. The completion of the internal cyclization was monitored by IR spectroscopy: disappearance of the (C=O) and (NH) absorption bands at 1,660, 1,673 and 3,184, 3,207 cm −1 signaled complete conversion of 14 to the tetracyclic compounds 16. When 5-chloro[1,2,4]triazoloquinazolines 9 were reacted with sodium azide in a molar ratio of 1:1. Table 2) [29].
The aforementioned facile nucleophilic displacement of the chlorine atom in 9 prompted us to investigate the reaction of 9 with methyl 3-amino-thiophene-2-carboxylate, which theoretically should provide access to the novel pentacyclic compounds of type 18. Thus, when compounds 9 were reacted with methyl 3-amino-thiophene-2-carboxylate in absolute dioxane in a molar ratio of 1:1.6, followed by addition of sodium hydride, the target compounds 18a,b could be isolated from the reaction mixture in 69 and 81% yield [27]. The IR spectra of compounds 18 are characterized by (C=O) stretching bands at 1,670 and 1,677 cm −1 . Table 2. Prepared compounds 10-18.
General
Melting points (ºC) were determined on open glass capillaries using a Mettler FP 62 apparatus and are uncorrected. Elemental analyses (C, H, N, S) were in full agreement with the proposed structures within ± 0.4% of the theoretical values, and were carried out with a Heraeus CHN-O-Rapid Instrument. The IR (KBr) spectra were recorded on a Shimadzu FT-IR 8300. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded on a Bruker AMX 400 spectrometer and chemical shifts are giving in a (ppm) downfield from tetramethylsilane (TMS) as an internal standard, DMSO is used as solvent. Mass spectra were recorded on a Finnigan MAT 311A and on a VG 70-250S (VG Analytical) instrument. Follow up of the reactions and checking the purity of compounds was made by TLC on DC-Mikrokarten polygram SIL G/UV 254, from the Macherey-Nagel Firm, Duren Thickness: 0.25 m. Column chromatography was conducted on silica gel (ICN Silica 100-200, active 60 Å)
Chemistry
3.2.1. Synthesis of compounds 5a-h 10 mmol of substituted hydrazinobenzoic acid 2 was added portionwise to a stirred solution of 1 (10 mmol) in EtOH (20 mL) at 0 o C. Afterwards triethylamine (30 mmol) was added dropwise over a period of 30 min. After the addition was complete, the reaction mixture was left to stir overnight at room temperature. Acidification of the mixture was performed by conc. HCl under ice cooling followed by refluxing for 1-3 h. After cooling, the mixture was poured into ice/water, the resulting solid was filtered, washed with water and dried. Recrystallization from THF gave analytically pure colored cpmpounds 5a-h. [1,2,4]
Synthesis of compounds 6a-d
To a solution of 5 (1 mmol) in DMF (5 mL) was added potassium carbonate (1.2 mmol) portion wise over a period of 10 min at room temperature. After stirring for 20 min, the appropriate alkyl halide (1.5 mmol) was added dropwise and the reaction mixture was stirred for 18 h at room temperature. The mixture was poured into ice/water, the precipitate was filtered off, washed with water and dried. Analytically pure products 6a-d were obtained after recrystallization from THF. [1,2,4]
Synthesis of compounds 7a-d
A solution of 5 (1 mmol) in dry THF (5 mL) was added dropwise to a stirred suspension of LiAlH 4 (3 mmol) in dry THF (10 mL). After stirring at room temperature for 3 h, water (0.4 mL) was added carefully and the mixture was stirred for an additional 30 min. The reaction mixture was filtered and the solvent removed under reduced pressure, the residue was dissolved in THF and passed through a short column chromatography, the solvent was removed under reduced pressure, and the obtained solid was recrystallized from EtOAc/n-hexane. [1,2,4]triazolo [1,5-a] [1,2,4]triazolo [1,5-a]
Synthesis of compounds 15a,b
A mixture of 9 (1 mmol) and benzyl carbazate or ethyl carbazate (2.2 mmol) was refluxed in benzene (10 mL) for 2.5 h. The solvent was removed under reduced pressure, the resulting solid was filtered off and recrystallized from MeOH.
Synthesis of compounds 16a,b
A mixture of 14 (0.5 mmol) and POCl 3 (5 mL) was refluxed at 100 o C for 2 h. After cooling, the excess of POCl 3 was removed under reduced pressure and the residue was treated with saturated aqueous solution of K 2 CO 3 under ice cooling. The resulting solids were collected by filtration and recrystallized from MeOH to afford 16a,b as colored pure products. [1,2,4]triazolo [1,5- | v3-fos-license |
2018-04-03T02:22:38.834Z | 2003-04-25T00:00:00.000 | 22145859 | {
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} | pes2o/s2orc | A single site-specific trans-opened 7,8,9,10-tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct induces mutations at multiple sites in DNA.
Site-specific mutagenicity of trans-opened adducts at the exocyclic N(2)-amino group of guanine by the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-enantiomers of a benzo[a]pyrene 7,8-diol 9,10-epoxide (7-hydroxyl and epoxide oxygen are trans, BPDE-2) has been determined in Chinese hamster V79 cells and their repair-deficient counterpart, V-H1 cells. Four vectors containing single 10S-BPDE-dG or 10R-BPDE-dG adducts positioned at G(0) or G(-1) in the analyzed 5'-ACTG(0)G(-1)GA sequence of the non-transcribed strand were separately transfected into the cells. Mutations at each of the seven nucleotides were analyzed by a novel primer extension assay using a mixture of one dNTP complementary to the mutated nucleotide and three other ddNTPs and were optimized to quantify levels of a mutation as low as 1%. Only G --> T mutations were detected at the adducted sites; the 10S adduct derived from the highly carcinogenic (+)-diol epoxide was 40-50 and 75-140% more mutagenic than the 10R adduct in V79 and V-H1 cells, respectively. Importantly, the 10S adducts, but not the 10R adducts, induced separate non-targeted mutations at sites 5' to the G(-1) and G(0) lesions (G(0) --> T and C --> T, respectively) in both cell lines. Neither the T 5' to G(0) nor sites 3' to the lesions showed mutations. Non-targeted mutations may enhance overall mutagenicity of the 10S-BPDE-dG lesion and contribute to the much higher carcinogenicity and mutagenicity of (+)-BPDE-2 compared with its (-)-enantiomer. Our study reports a definitive demonstration of mutations distal to a site-specific polycyclic aromatic hydrocarbon adduct.
Mutagenicity of BPDE adducts depends upon efficiency of cellular repair systems to remove the adducts from DNA (mainly nucleotide excision repair (17,18)) and fidelity of DNA polymerases replicating residual adducted sites. Although the normal replicative DNA polymerases pol and pol⑀ are blocked by bulky adducts, members of newly discovered Y superfamily of bypass DNA polymerases such as pol, pol, and pol (19) were found to replicate past sites of various DNA lesions, albeit with low fidelity and low processivity. The frequency of nucleotide misincorporation of pol (20), pol (21), and pol (22) replicating an undamaged template in vitro is in the range of 10 Ϫ3 -10 Ϫ2 . Assuming that the enzymes replicate not only the site of a lesion but possibly also a short stretch of DNA around the lesion with such a low fidelity suggests that besides incorporation of mismatched nucleotides opposite the adducted site, the bypass DNA polymerases could also introduce secondary mutations in the region flanking the adducted site, especially if its DNA structure is perturbed by the presence of the adduct. Multiple mutations in a shuttle vector treated with (Ϯ)-BPDE, believed to be generated by an error-prone polymerase, have been observed (23) in random mutagenesis experiments.
In the present study, we have examined the mutagenicity of 10S-BPDE-dG and 10R-BPDE-dG lesions both at adducted sites and at unmodified flanking sites. We constructed doublestranded plasmid vectors bearing cDNA of the HPRT gene of Chinese hamster V79 cells containing a single adduct positioned at each of the two adjacent sites in the non-transcribed strand of the gene and transfected them separately into V79 cells and also into their nucleotide excision repair-deficient derivative, V-H1 cells (24). These cells are defective in the xeroderma pigmentosum complementation group D/ERCC2 gene encoding for an ATP-dependent DNA helicase (25), an essential subunit of transcription and nucleotide excision repair complex TFIIH (26). They are 9-fold more sensitive to cytotoxic effects of (ϩ)-BPDE-2 than V79 cells and have ϳ50% lower capacity for removal of (ϩ)-BPDE-2-induced adducts from DNA compared with V79 cells (27). We qualitatively and quantitatively evaluated mutagenic effects of both 10R and 10S adducts in a sequence of seven nucleotides by a novel quantitative minisequencing method, and we compared their differences with respect to 10R/10S stereochemistry, position of the adducts at two adjacent sites, and also cellular DNA repair status. The most striking observation was that, in addition to mutations at the adduct sites, significant numbers of mutations were induced 1 or 2 bases 5Ј to these sites by 10S but not 10R adducts.
MATERIALS AND METHODS
Oligonucleotides-All non-adducted oligonucleotides were prepared at IDT (Coralville, IA). The 19-mer oligonucleotide and primers for quantitative minisequencing (Fig. 2) were gel-purified. Adducted oligonucleotide 18-mers (5Ј-AAACTG 0 G Ϫ1 GAAAGCCAAAT) containing a single trans-opened BPDE adduct at one or the other of the numbered dG residues were prepared by solid-phase synthesis using the mixed 10R/10S diastereomers of the appropriately protected adducted phosphoramidite as described (28,29), followed by high pressure liquid chromatography separation of the resultant pair of diastereomeric oligonucleotides. Synthesis and characterization of the diastereomeric pair of oligonucleotides bearing trans-opened BPDE adducts at G 0 with 10R and 10S configuration at the point of attachment of the base to the hydrocarbon have been reported (28). A second R/S pair of 18-mers with trans-opened BPDE adducts at G Ϫ1 was prepared (3-mol scale) by the same methodology and purified by high pressure liquid chromatography as described for the adduct at G 0 (28) (Hamilton PRP-1 column (7 ϫ 305 mm), eluted with a gradient from 0 to 35% solvent B in solvent A over 20 min, where solvent A is 0.1 M (NH 4 ) 2 CO 3 , pH 7.5, and solvent B is a 1:1 mixture of solvent A with CH 3 CN adjusted to the same pH). The late eluting oligonucleotide (t R 19.0 min) was assigned as contain-ing the trans-opened 10S-BPDE dG adduct and the early eluting oligonucleotide (t R 17.6 min) as containing the 10R adduct, on the basis of the CD spectrum (14,30) of the known nucleoside adducts obtained upon enzymatic hydrolysis (12) of the late eluting adduct. The CD spectra of the 18-mer oligonucleotides themselves exhibited bands at 320 -350 nm that were positive for the early eluting (10R) isomer and weakly negative for the late eluting (10S) isomer, consistent with previous observations (31) of other oligonucleotides containing transopened BPDE dG adducts of known absolute configuration as follows: 18-mer modified with the 10S or 10R adduct at the position G 0 , 5Ј-AAACTGGGAAAGCCAAAT; 18-mer modified with the 10S or 10R adduct at the position G Ϫ1 , 5Ј-AAACTGGGAAAGCCAAAT; 19-mer, 5Ј-CATATTTGTGTCATTAGTG; 59-mer scaffold, 5Ј-GAATTCTCATCTT-AGGCTTTGTATTTGGCTTTCCCAGTTTCAGTAATGACACAAATA-TG; primer A, 5Ј-TGCGGGATCCCTCCTCACACCGCT; primer B, 5Ј-C-TGCTTTCCCTGGTCAAGCGG; primer C, 5Ј-GAAATTAATACGACTC-ACTATAGGG; primer D, 5Ј-GCAGATTCAACTTGAATTCTCATC. Sequence of primers used for mutation analysis by quantitative minisequencing is shown in Fig. 2.
Scaffold-directed Extension of the Adducted 18-Mers to the Adducted 37-Mers-The adducted oligonucleotides and the non-adducted control were extended on their 5Ј termini with the 19-mer to the final 37-mers by scaffold-directed ligation. Adducted 18-mers or the corresponding non-adducted 18-mer (10 pmol), 5Ј-labeled with 32 P, were incubated with the 19-mer and 5Ј-32 P-labeled 59-mer scaffold in a molar ratio 1:2:1.5 at 65°C for 5 min and cooled to room temperature over 10 min. Ligase reactions (30 l) containing 66 mM Tris-HCl, 5 mM MgCl 2 , 1 mM dithioerythritol, 1 mM ATP, and 0.5 units of T4 DNA ligase were incubated at 25°C for 2 h at pH 7.5 and terminated by adding 15 l of stop solution containing 90% formamide, 10 mM NaOH, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF. Reaction products were separated on 10% PAGE and detected by autoradiography. The 37-mer bands were excised from the gel and briefly washed with water. The gel was repeatedly treated with water by three rounds of heating and cooling (65°C for 5 min and 0°C for 5 min) to elute the DNA, and the DNA was precipitated from the eluate with ethanol. The total yield of the ligation product was ϳ40% for the non-adducted oligonucleotide and ϳ25% for the adducted oligonucleotides.
Preparation of Plasmid Vectors Containing the Full-length Hamster HPRT cDNA-The HPRT cDNA was prepared by RT-PCR from total mRNA of V79 cells using primer A and primer D as described before (32). Primer A and primer D create BamHI and EcoRI sites, respectively. Double-stranded pCR3 vector (5.1 kb, Invitrogen) containing the HPRT cDNA was prepared by TA cloning (Invitrogen) of the PCR product. The orientation of the HPRT cDNA insert was screened by HindIII digestion of the plasmid DNA. The construct with the antisense orientation of the insert with respect to the cytomegalovirus promoter (pCR3/HPRT antisense ), used for preparation of adducted vectors, yields a 289-bp fragment, whereas the construct with the sense orientation (pCR3/HPRT sense ) yields a 596-bp fragment. Sequence of the insert in pCR3/HPRT antisense was further verified by dideoxy sequencing using ThermoSequenase Cycle Sequencing Kit (U. S. Biochemical Corp.).
Preparation of Plasmid Vectors Containing Site-specific 10S-or 10R-BPDE Adducts in the HPRT cDNA-Four plasmid vectors adducted with BPDE and a non-adducted control were prepared by the enzymatic extension of the BPDE-adducted and control 37-mer oligonucleotides (see above for preparation) and annealed to the single-stranded circular pCR3/HPRT antisense DNA as shown in Fig. 3. The single-stranded DNA was isolated from the supernatant of an Escherichia coli culture transformed with pCR3/HPRT antisense plasmid after co-infection with M13K07 helper phage (33). Double-stranded DNA was prepared as follows: a mixture (72 l) containing 0.5 pmol of single-stranded pCR3/ HPRT antisense , 0.5 pmol of 37-mer oligonucleotide, 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , and 50 mM NaCl was incubated at 95°C for 3 min and then at 65°C for 3 min and cooled slowly (15 min) to room temperature. After adding 1 mM DTT, 1 mM ATP, 500 M dNTPs, T4 gene 32 protein (5 g), T4 DNA polymerase (5 units), and T4 DNA ligase (2.5 units), the reaction (100 l) was incubated at 25°C for 2 h. Following extraction with phenol/chloroform/isoamyl alcohol (25:24:1), DNA was precipitated with 2 volumes of cold ethanol after adding 0.3 M sodium acetate, pH 5.2, and 2 g/ml tRNA. After centrifugation, the DNA pellet was dissolved in 50 mM Tris-HCl, pH 7.6, 5 mM MgCl 2 , 5 mM DTT, and 50 g/ml bovine serum albumin (100 l) and digested with 20 units of exonuclease III for 1 h at 37°C to remove partially extended reaction products. Extraction (phenol/chloroform/isoamyl alcohol) and ethanol precipitation was repeated. The pellet was dissolved in 10 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 100 g/ml bovine serum albumin, pH 7.9 (25 l). The DNA was cut with BamHI ϩ EcoRI (5 units each) for 1 h at 37°C. Reaction products, including two BamHI-EcoRI fragments (15-and 31-mer) and a 24-mer EcoRI-EcoRI fragment, were separated on a 0.9% preparative agarose gel. Bands corresponding to 713 bp (HPRT cDNA) and ϳ5 kb were eluted from the gel (QIAquick Gel Extraction kit, Qiagen, Valencia, CA) and were re-ligated at 16°C for 16 h in a 50-l mixture containing 66 mM Tris-HCl, 6.6 mM MgCl 2 , 10 mM DTT, 66 M ATP, and 1 unit of T4 DNA ligase. This step changes the orientation of the insert from the antisense to sense and also separates residual single-stranded DNA. The reaction was stopped by 500 mM EDTA (1 l), and products were applied on the Qiagen QIAquick column (Nucleotide Removal kit, Qiagen). DNA was eluted in a sterile 10 mM Tris-HCl buffer, pH 8.5. The final concentration of pCR3/ HPRT antisense -BPDE and the non-adducted control used for cell transfections was 20 ng/l. All four plasmid vectors were prepared at least three times.
Transfection of V79 and V-H1 Cells with Adducted Plasmid Vectors-The Chinese hamster V79 cells were obtained from the ATCC and propagated in minimum Eagle's medium containing 10% dialyzed and heat-inactivated fetal bovine serum in an incubator with controlled humidified atmosphere containing 5% CO 2 . Before transfection, the cells were seeded at a density 2 ϫ 10 5 per 60-mm dish. Following 24 h of incubation, cells were transfected with 0.3 g of one of the modified plasmids (10S or 10R adduct at the G 0 or G Ϫ1 position) or the control plasmid using Effectene (Qiagen). The cells were incubated for 24 h, washed with phosphate-buffered saline, and supplied with fresh medium. After 48 h following the transfection, the cells were harvested by trypsinization and seeded in the same medium supplemented with 500 g/ml G418 (Invitrogen) at a density 3 ϫ 10 4 per 60-mm dish to select permanently transfected cells. Ten dishes were prepared from each transfection. Cellular colonies developed after an 11-day incubation with G418 were harvested by trypsinization from each dish and collected by centrifugation. To ensure analysis of at least 100 independent cellular clones from each plasmid transfection, 10 plates containing at least 60 cellular colonies in a random mixture were harvested. Thus, each transfection experiment yielded 5 ϫ 10 pooled samples, which were separately processed and analyzed.
The repair-efficient Chinese hamster V-H1 cells (kindly provided by Dr. M. Zdzienicka, Leiden University Medical Center, Leiden, The Netherlands) were grown and transfected in the same way with several changes. The cells were seeded at a density 2.6 ϫ 10 5 per a 60-mm dish, transfected with 0.5 g of the plasmid, and seeded after the transfection at a density 1 ϫ 10 5 per 60-mm dish.
Preparation of DNA Samples for Mutation Analysis-Chromosomal DNA from harvested cells was isolated using High Pure PCR Template Preparation kit (Roche Molecular Biochemicals). DNA fragment (337 bp) encompassing the examined region was prepared by PCR amplification using primer B corresponding to region 437-457 of the HPRT gene (exon 6) (32) and primer C corresponding to the T7 promoter region of the plasmid construct; the endogenous cellular HPRT gene is not amplified using this set of primers. The reaction was carried out in a 50-l mixture containing 200 ng of DNA, 200 nM primers, 200 M dNTP, and Taq DNA polymerase (0.025 unit/l, Qiagen). Following 30 cycles (95°C for 30 s, 55°C for 10 s, and 72°C for 10 s) with the initial 3 min at 95°C and the final 7 min at 72°C, the residual dNTPs were removed by a 30-min alkaline phosphatase treatment (1 unit per each 20 l of the reaction). The product was purified using QIAquick PCR Purification kit (Qiagen). Concentration of the product was estimated based on absorbance at 260 nm, and the purity was checked on 1.5% agarose gel containing 0.5 g/ml ethidium bromide. Standard 337-bp fragments used as positive and negative controls for quantitative minisequencing were amplified from plasmids pCR3/HPRT antisense containing all four nucleotides at each of the seven examined positions (Fig. 2). These plasmids were prepared by site-directed mutagenesis using a QuikChange Kit (Stratagene, La Jolla, CA).
Mutation Detection Assay Using Quantitative Minisequencing-The method is based on a single nucleotide primer extension assay (34,35) modified to quantify mutations in a known sequence context at levels lower than 10%. A primer annealed to the DNA template (337-bp fragments), one nucleotide before the analyzed site (Fig. 2), is extended with a mixture of one dNTP and three other ddNTPs. When annealed to a fragment carrying a specific analyzed nucleotide (mutation), the primer can be extended with the complementary dNTP before being terminated by ddNTP incorporation at the subsequent nucleotide, whereas elongation of the primer annealed to fragments not containing the analyzed nucleotide is terminated immediately with ddNTP. The level of mutation is determined by calculating the ratio of differently extended primers following their separation on denaturing polyacrylamide gel and analysis with PhosphorImager (see for 30 s, 50°C for 10 s, and 72°C for 10 s) with the initial 1 min at 95°C were used. The reaction was stopped with 1 volume of 95% formamide, 20 mM EDTA, 10 mM NaOH, 0.05% bromphenol blue, and 0.05% xylene cyanol FF. Reaction products were separated on 15% PAGE in 1ϫ TBE buffer and visualized by autoradiography. To increase the throughput of the assay, up to four sets of samples were subsequently loaded into the same gel. For quantitative evaluation (Fig. 5), the signal from the gel was transferred onto a screen and scanned by Cyclone Phos-phorScreening System (Packard Instrument Co.), and the amount of radioactivity associated with each spot was quantified using software provided with the system.
Construction of Site-and Stereospecifically Adducted
Plasmid Vectors-BPDE-adducted plasmid vectors were constructed by the in vitro enzymatic extension of 37-mer oligonucleotides after their annealing to a single-stranded plasmid template as shown in Fig. 3 and described under "Materials and Methods." The plasmid contains the HPRT gene inserted in the antisense orientation toward the plasmid promoter. The 37-mers were prepared from 18-mer oligonucleotides modified with 10S or 10R adducts at the G 0 or G Ϫ1 sites (see "Materials and Methods" and Fig. 4A). The sequence of the 18-mers corresponds to region 629 -646 of the non-transcribed strand of the HPRT gene in Chinese hamster cells. The use of 37-mers instead of 18-mers in the reaction substantially increases the yield of the covalently closed circular reaction products (data not shown). After the reaction, covalently closed circular DNA was digested with restriction enzymes EcoRI and BamHI and re-ligated yielding a vector with the HPRT gene in the sense orientation and a single BPDE adduct in the non-transcribed strand of the gene (Fig. 3). Purity of the final products was checked by digestion with restriction enzymes EcoRI and MslI and separation of the 32 P-labeled restriction fragments on denaturing PAGE (Fig. 4B). The presence of BPDE adducts retards mobility of the adducted fragments through the gel, which translates into the shift of the corresponding bands (Fig. 4B). Scanning of the gel using the Cyclon system detected at least 99.3% of a band corresponding to the adducted fragment in adducted samples. This indicates that the adducts were very stable during the preparation of the plasmid vectors and that the adducted vectors contain less than 0.7% of contaminants.
Transfection of Cells with Adducted Plasmid Vectors-Four adducted plasmids and a non-adducted control were separately transfected into repair-proficient V79 cells and repair-deficient V-H1 cells. Each experiment was repeated at least twice with a different plasmid preparation. Doubling times of exponentially growing V79 and V-H1 cells were ϳ14 and ϳ18 h, respectively. After transfection, doubling times of V79 and V-H1 cells increased to ϳ19 and ϳ34 h, respectively. There was no difference in proliferation of the cells transfected with different plasmid constructs. For the selection with antibiotics, cells were seeded in a density ensuring approximately the same yield of cellular colonies per plate from both cell lines. Because the cells kept proliferating at a time between the transfection and seeding (48 h), and their proliferative rate was different, it is reasonable to expect that 5.8 of the V79 cellular colonies and 2.7 of the V-H1 cellular colonies on average originated from a single cell. Thus, samples harvested for mutation analysis contained a random mixture of cellular colonies (pools) that were not necessarily independent. A sufficient total number of colonies (ϳ600) in random pools was harvested from each plasmid transfection to ensure that at least 100 of these were independent clones. Assuming that permanent transfection decreases the proliferative rate of the affected cells more than the nonaffected cells, which are not selected, this number of analyzed independent colonies is the lowest possible estimate.
Quantitative Minisequencing and Calculation of the Mutation Level at a Specific Site-Chromosomal DNA from pooled cellular samples was used for amplification of the 337-bp DNA template encompassing a region originally containing the adducted nucleotide. The amplified template is a mixture of fragments with a DNA sequence reflecting the mutagenicity of the adduct at the region. Quantitative minisequencing was used to assess both the type and the level of mutations at the region as described under "Materials and Methods" and Fig. 5. To validate accuracy and sensitivity of the assay model, binary mixtures of 337-bp fragments differing in a single nucleotide at each site of the region (pseudo-pools) were analyzed. These fragments were prepared by PCR amplification of 337-bp region from the HPRT cDNA manipulated in plasmid vectors by site-directed mutagenesis. The relative content of one fragment in the other was 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19, and 0%. Composition of these binary mixtures represented all potential single nucleotide mutations in the analyzed region (5Ј-A ϩ3 C ϩ2 T ϩ1 G 0 G Ϫ1 G Ϫ2 A Ϫ3 ). The ratio between a level of the fragment detected by the assay (f measured ) and a predicted level of the fragment in the mixture (f predicted ) represents a correction factor (k). The means Ϯ S.D. of the correction factor were calculated at each analyzed point from at least three independent experiments and plotted. The relationship between k and the level of the analyzed fragment was found linear between 0.78 -50%. Fig. 8 is included as Supplemental Material and shows the results from analyses of model binary fragment mixtures differing by a nucleotide in positions found mutated in the study (C ϩ2 , G 0 , and G Ϫ1 ). The average k values FIG. 5. Quantitative minisequencing of binary mixtures of DNA fragments with a single nucleotide difference at the position G 0 (see Fig. 2). A DNA fragment containing A, C, or T instead of G at the G 0 position (A, C, or T fragment) was mixed with the fragment containing G (G fragment) at different ratios as indicated. A 32 P-labeled primer annealed one nucleotide before the analyzed site was extended by a cycling reaction with ThermoSequenase DNA polymerase in a mixture containing one dNTP complementary to the nucleotide at the analyzed site and the three other ddNTPs. Reaction products were separated on denaturing 15% PAGE, and the signal was transferred from the gel onto a screen and scanned by Cyclone PhosphorScreening System (Packard Instrument Co.). The primer extended more than once (with dNTP ϩ ddNTP) generates the upper spot, whereas the primer extended only once (with ddNTP) generates the lower spot. Radioactivity of the upper spot corresponds to the amount of the analyzed fragment, whereas radioactivity of the lower spot corresponds to the amount of the second fragment in the mixture. The amount of radioactivity associated with each spot was quantified using software provided with the system. Plotted numbers were calculated according to the formula: (S 2 Ϫ(C n2 S 1 )/(C n1 ))/((S 1 ϩ S 2 ) Ϫ(C n2 S 1 )/(C n1 )) ϫ 100 ϭ mutation (%). S 1 and S 2 indicate the amount of radioactivity associated with the lower and upper spots of the analyzed samples, respectively; C n1 and C n2 indicate the same for the negative control containing 0% of the analyzed fragment (only pure G fragment).
FIG. 6. Determination of G 3 T mutations at the G 0 position by quantitative minisequencing. PCR fragments (frgmt.) were amplified from DNA of V79 cells transfected with plasmid vectors adducted with 10S-or 10R-BPDE or nonadducted at G 0 position (A) or G Ϫ1 position (B). 32 P-Labeled primer annealed one nucleotide before the analyzed site (G 0 primer, see Fig. 2) was extended by a cycling reaction with ThermoSequenase DNA polymerase in a mixture containing dATP, ddGTP, ddTTP, and ddCTP. Reaction products were separated on denaturing 1.5% PAGE; the signal was transferred from the gel onto a screen and scanned by Cyclone PhosphorScreening System (Packard Instrument Co.).
(0.85-1.05) calculated for each mutation in the region from model binary mixtures (the rest of the results not shown) were used to re-calculate levels of mutations found in unknown samples. The results demonstrate accuracy and linearity of the assay in tested binary mixtures containing more than 1% of the analyzed fragment. Accuracy of the assay decreases sharply when levels of the analyzed fragment are lower than 1% and limits sensitivity of the assay to 0.3% (see Supplemental Material Fig. 8). Thus, mutagenic changes at levels higher than 0.3% can be qualitatively detected and at levels higher than 1% can be accurately quantified by the assay.
Mutagenicity of BPDE Adducts in Repair-proficient and Repair-deficient Cells-Mutagenicity of 10S-BPDE-dG and 10R-BPDE-dG lesions in the sequence 5Ј-A ϩ3 C ϩ2 T ϩ1 G 0 -G Ϫ1 G Ϫ2 A Ϫ3 was examined at the adducted sites (G 0 and G Ϫ1 ) and also at their 5Ј-and 3Ј-flanking sites (from ϩ3 to Ϫ3, Fig. 2 and also Fig. 6). G 0 corresponds to the hotspot (G-634) of the non-transcribed strand of the gene found in random mutagenesis studies of (ϩ)-BPDE-2 in V79 cells (10,32).
In repair-proficient V79 cells, the guanine adduct derived from (ϩ)-BPDE-2, 10S-BPDE-dG, induced 2.5 Ϯ 1.3 and 2.8 Ϯ 1.1% of G 3 T mutations of the adducted nucleotides at the G 0 and G Ϫ1 sites, respectively. The guanine adduct derived from (Ϫ)-BPDE-2, 10R-BPDE-dG, induced 1.8 Ϯ 1.0 and 1.9 Ϯ 0.7% of G 3 T mutations of the adducted nucleotides at the G 0 and G Ϫ1 sites, respectively (Fig. 7A). Analysis of secondary mutations of nucleotides at sites flanking the lesion on the 5Ј-end revealed that the 10S adduct, but not the 10R adduct, induced C 3 T mutations (0.7 Ϯ 0.4%) at the (ϩ2) position when the G 0 site was adducted, and G 3 T mutation (1.3 Ϯ 0.5%) at the G 0 position when the G Ϫ1 site was adducted. Interestingly, adducts at the G 0 or G Ϫ1 site did not induce mutations of the T nucleotide located at the (ϩ1) position. Also, no mutations were found in the 3Ј-vicinity of the adducted sites (up to 3 nucleotides) or further upstream in the 5Ј-direction (3 and 4 positions analyzed overall from the adducted G 0 and G Ϫ1 sites, respectively).
The effect of repair deficiency in the V-H1 cells on the observed frequency of mutations is relatively small (a factor of 2 or less) and is only significant for the constructs containing 10S adducts at G 0 (Fig. 7B). For example, in the VH-1 cells, the 10S adduct induced 4.6 Ϯ 1.5 and 2.1 Ϯ 0.6% of G 3 T mutations of the adducted nucleotides at G 0 and G Ϫ1 sites, as compared with 2.5 and 2.8%, respectively, in the V79 cells (see above). The 10R adduct induced 1.9 Ϯ 0.8 and 1.2 Ϯ 0.6% of G 3 T mutations at the adducted nucleotides when at the G 0 and G Ϫ1 sites, respectively. Analysis of secondary mutations of nucleotides at sites flanking the lesion on the 5Ј-end showed the same pattern of mutations found in V79 cells; the 10S adduct, but not the 10R adduct, induced C 3 T mutations (1.7 Ϯ 0.6%) at the (ϩ2) position when the G 0 site was adducted and G 3 T mutations (1.5 Ϯ 0.7%) at the G 0 position when G Ϫ1 was the adducted site. As with the V79 cells, no other mutation type or other mutated nucleotide was found in the analyzed region. The formation of secondary mutations depends on the presence of the 10S adduct but probably not on the formation of primary mutations. If so, tandem mutations (two mutations on the same analyzed DNA fragment) would be detected. In the primer extension assay tandem T mutations would give rise to multiple A incorporations in the primers used. No such multiple extensions of the primer were observed. Although we cannot exclude the possibility of formation of tandem mutations in levels below the detection limit of the assay (0.3%), random formation of secondary mutations independent of the primary mutations is a more probable scenario. Large standard deviations of the means of the presented data originate in large differences in the individual data from each of the 10 samples containing pools of cellular colonies, not from irreproducibility of the assay. Repeated analyses of the same samples showed remarkable reproducibility (maximum scatter did not exceed 10% of the calculated values, data not shown).
FIG. 7. Mutagenic effects of 10S-and 10R-BPDE adducts in the vicinity of the lesion in V79 (A) and V-H1 cells (B).
Note the difference in vertical axis scales for the two cell lines. Types of mutations and their levels were determined by quantitative minisequencing at each indicated site as described under "Materials and Methods" (detection limit 0.3%). Data shown are means Ϯ S.D. of values acquired from at least two experiments. In each experiment, 10 samples were analyzed from transfection of each plasmid construct. Adduct position is indicated by an asterisk.
DISCUSSION
At the target sites (G 0 and G Ϫ1 ), the 10S adduct and the 10R adduct induced only G 3 T mutations. This mutation was found to be dominant in previous studies examining mutagenicity of site-specific adducts both in prokaryotic (31,36,37) and eukaryotic cells (37), although G 3 C and G 3 A mutations have also been observed in some sequences. The sequence context of our adducted G 0 site is identical to the ϳTGGϳ sequence examined in simian kidney cells (37). There was no significant difference between mutagenicity at the adducted site for the 10S-BPDE-dG lesion relative to the 10R-BPDE-dG lesion, and a preponderance of G 3 T mutations was observed, as in the present study. However, the level of mutation induced by both adducts was substantially higher than in our study (13 versus 1.8 -2.5%), and low levels (Ͻ1%) of G 3 C and G 3 A mutations were also detected. These differences may stem mainly from differences in the experimental systems; the single-stranded vector system used by Page et al. (31) and Moriya et al. (37) eliminates the involvement of DNA repair, whereas the double-stranded vector system used in our study is sensitive to DNA repair (both in V79 and in V-H1 cells, which have ϳ50% remaining capacity to remove adducts derived from the (ϩ)-(7R,8S,9S,10R)-enantiomer of BPDE from DNA compared with V79 cells (27)). Consequently, the results of our study show substantially lower mutagenicity of the examined adducts and reflect both DNA repair of the adducts and fidelity of their bypass.
Results from DNA repair-proficient (V79) and DNA repairdeficient (V-H1) cells show only minor differences. It is possible that the defect of V-H1 cells in the xeroderma pigmentosum complementation group D/ERCC2 gene (25) influences mainly transcriptional coupled repair (18) and causes a decreased ability of the cells to preferentially repair BPDE adducts from the transcribed strand of active genes (27), whereas the efficiency of global genomic repair (18), which is responsible for the repair of the non-transcribed strand (location of the adducts in this study), may be affected only marginally. Both lesions in both adducted G sites generated the same kind of mutation (G 3 T), and the 10S adduct induced identical secondary mutations in these two cell lines. Quantitatively, no significant difference in mutagenicity of the 10R adduct was observed in corresponding sites in V79 and V-H1 cells. However, mutagenicity of the 10S adduct was significantly higher at the G 0 site in the V-H1 cells than in the V79 cells (p Ͻ 0.006), although similar at the G Ϫ1 site in both cell lines (p Ͼ 0.1). The data suggest that repair of the 10S adduct is more efficient than the 10R adduct in the non-transcribed strand but also that repair efficiency of the 10S adduct may differ from site to site. These results are in accordance with a study by Custer et al. (29) who demonstrated a remarkable resistance of the 10R adduct to DNA repair in vitro compared with 10S adduct using a whole cell extract. However, in a different sequence context, no difference in repair efficiency of these two adducts in vitro was observed (38).
Mutations remote from a specifically modified base in DNA were first described by Lambert et al. (39) for frameshifts induced by an acetylaminofluorene adduct. The present study reports a definitive demonstration of substitution mutations remote from the target site induced by a single, site-specific polycyclic aromatic hydrocarbon DE adduct. These non-targeted mutations were observed only when the adduct has the 10S configuration and were found within two nucleotides on the 5Ј-side of the adducted base, namely at C 2 when G 0 was modified and at G 0 when G Ϫ1 was modified (for sequence see Fig. 2). Notably, the absence of non-targeted mutations with 10R adducts at either position provides strong internal evidence that these mutations with the 10S adducts do not result from any artifact of the oligonucleotide synthesis, since for each sequence the 10R and 10S adducted oligonucleotides were prepared together from the mixed 10R/10S diastereomers of the phosphoramidite (see "Materials and Methods"), and thus the two diastereomeric oligonucleotides underwent identical treatment prior to final chromatographic purification. The nontargeted mutations at both sites represented mutagenic changes of the original nucleotides to T, suggesting their bypass with a mismatched A. Furthermore, the T that is immediately 5Ј to the G 0 lesion or separated by one base from the G Ϫ1 lesion was not found to be mutated in the presence of either lesion. An attractive explanation for these data is that translesion synthesis results in A being inserted (either correctly or incorrectly) as the preferential nucleotide at the adducted site and at the two additional 5Ј-flanking nucleotides. In vitro studies with isolated bypass DNA polymerases (pol , , , and ) on templates containing a single 10S-BPDE-dG or 10R-BPDE-dG lesion identified pol as involved in low fidelity insertions at these lesions in vitro (40,41). The enzyme incorporates mostly A and G opposite the lesion site (40,41) and thus is likely to be responsible at least in part for mutations induced by BPDE-dG lesions in mammalian cells (mostly G 3 T with minor G 3 C and G 3 A). Although the enzyme extends the primer beyond the lesion site rather inefficiently, its preference to extend mispaired primers containing a purine opposite the adduct and its high misincorporation frequency on non-damaged templates (10 Ϫ3 -10 Ϫ2 ) (41) are features that may contribute to the formation of non-targeted mutations and would be worthy of further exploration by studies in vitro and in cells lacking functional pol.
The 10S BPDE adducts at the G 0 and G Ϫ1 sites induced identical non-targeted mutations in both repair-proficient (V79) and repair-deficient (V-H1) cell lines, and the frequency of the non-targeted mutations correlated with the frequency of targeted mutations. The increased level of the targeted mutation at the G 0 site (G 3 T) in V-H1 cells was accompanied by a similar increase in the level of the non-targeted mutations (C 3 T) in these cells compared with V79 cells, whereas no difference in the frequency of non-targeted mutation (G 3 T at the G 0 site) between V-H1 and V79 cells was observed when the levels of the targeted mutation (G 3 T at the G Ϫ1 site) were similar in both cell lines. Assuming that the differences in the targeted mutation frequency between these two cell lines reflect only the efficiency of the 10S adduct removal from a particular site, it seems likely that the adduct level determines the frequency of not only targeted but also non-targeted mutations.
The present observation that mutations are induced 1 or more bases away from the target site by a polyaromatic hydrocarbon DE lesion is consistent with results of our recent study (42) of mutations in an E. coli-M13 system induced by several cis-opened adducts derived from both BPDE-2 (the diastereomer used in the present study, whose benzylic hydroxyl group and epoxide oxygen are trans) and BPDE-1 (benzylic hydroxyl group and epoxide oxygen cis). More limited data in E. coli by Jelinsky et al. (36) using a single trans-opened 10S adduct of BPDE-2 had also led to the tentative suggestion of non-targeted mutations at a base immediately adjacent to this adduct. In our previous study (42) using the sequence ϳG 6 C 5 G 4 G 3 G 2 G 1 G 0 ϳ with cis-opened adducts at G 0 , non-targeted substitution mutations 4 and 6 bases remote from the target site were described, but their significance was not fully recognized. No significant non-targeted substitutions had been detected in this DNA sequence containing trans-opened BPDE-1 or BPDE-2 dG adducts in the same experimental system (31). The most prevalent non-targeted mutations (fre-quency 2-4%) induced by the cis-opened BPDE-1 and BPDE-2 dG adducts were T substitutions at G 6 and G 4 , and they occurred both in the absence of mutations at the target site (G 0 ) and in combination with G 0 3 T mutations at this site. These non-targeted mutations were not observed with the control (non-adducted) sequence, and most significantly they were observed only when the adducts at G 0 had 10S but not when they had 10R configuration, in analogy to our present observations. Because no significant non-targeted mutations were observed in a different (ϳG 6 C 5 G 4 T 3 T 2 C 1 G 0 ϳ) sequence containing BPDE-1 or -2 adducts at G 0 (42), the above mutations were most likely related to the run of 5 guanines in the ϳG 6 C 5 G 4 G 3 G 2 G 1 G 0 ϳ sequence. Despite the specific sequence effect, as well as the differences in experimental systems, the similarity between these results in E. coli and the present observations in mammalian cells is intriguing and suggests that mutations remote from the lesion site induced by polyaromatic hydrocarbon DE adducts may constitute a not uncommon mechanism of polyaromatic hydrocarbon mutagenesis whose significance has not been appreciated previously.
A limited number of site-specific mutagenesis studies with other types of bulky DNA adducts has demonstrated induction of non-targeted substitution mutations at non-adducted sites in the vicinity of the lesion. They include studies of the N7-guanyl adduct of aflatoxin B 1 8,9-epoxide and the adduct's ring opened formamidopyrimidine form in SOS-induced E. coli (43,44) as well as N-deoxyguanosin-8-yl)-2-acetylaminofluorene and N-(deoxyguanosin-8-yl)-2-aminofluorene in COS-7 cells (45). Although it is difficult to make any generalization about the mechanism of these non-targeted mutations by comparing the data of these studies to those presented here (different experimental models, structure of the adducts, and sequence context), it is clear at this point that the formation of various non-targeted mutations is likely related to the bulky character of the adducts and that the orientation of the adducts may play a significant role. The hydrocarbon of the 10S BPDE-dG adduct orients toward the 5Ј-side of the modified base in the minor groove of duplex DNA in the sequence 5Ј-CGC (15) as well as 5Ј-TGC (46) and could thus cause structural perturbations 5Ј to the lesion in our study (5Ј-TGG and 5Ј-GGG), resulting in the observed non-targeted mutations 5Ј to the adduct. In contrast, the 10R BPDE-dG adduct, which orients in the opposite direction toward the 3Ј-end of the modified strand in duplex DNA (16), was not found to cause any mutations at non-target bases on either side of the adduct. Interestingly, about 13% of the total mutations in SOS-induced E. coli caused by a dG adduct of aflatoxin B 1 8,9-epoxide (43), which intercalates into the helix on the 5Ј-side of the modified G base (47,48), were primarily C 3 T mutations at the dC immediately 5Ј to the lesion (G) in a 5ЈϳCGAϳ sequence. In contrast, an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene lesion, whose hydrocarbon moiety displaces the modified G and intercalates in its place opposite the complementary C (49), gave rise to base misincorporation on the 3Ј-side of the lesion site (50).
The dependence of non-targeted mutations on adduct configuration is of particular interest in light of the marked differences in mutagenicity and carcinogenicity between (ϩ)-and (Ϫ)-BPDE-2 enantiomers. Higher tumorigenic activity of (ϩ)-BPDE-2 compared with (Ϫ)-BPDE-2 has been demonstrated. (ϩ)-BPDE-2 induced lung tumors and skin tumors when injected intraperitoneally into newborn mice and applied topically to the skin of adult mice, respectively; but (Ϫ)-BPDE had little or no carcinogenic activity (7,8). Although the present mutagenesis study showed little difference in the mutagenicity of (ϩ)-and (Ϫ)-BPDE-2 dG adducts at specific sites, (ϩ)-BPDE-2 was ϳ11 times more mutagenic than (Ϫ)-BPDE-2 in the HPRT gene of V79 cells on a per dose basis in a random mutagenesis study (11). Many factors can contribute to these different results, including more efficient adduct formation from (ϩ)-relative to (Ϫ)-BPDE-2 (12), differences in relative proportions of dG and dA adducts formed from the two enantiomers (12,13), as well as sequence effects on adduct formation, repair (51), and bypass (41). Furthermore, the higher mutagenicity of the (ϩ)-BPDE-2 adducts observed in random mutagenesis studies may result in part from their mutagenic effects on the whole region (i.e. non-targeted mutations in the vicinity of the adduct) rather than only at a particular adducted site. Because not all DNA mutations lead to amino acid substitutions, and some substitutions may be silent in terms of function, it is obvious that induction of a mutation at more than one site by a single adduct increases the probability of an amino acid change that could generate a protein with a compromised function. Consequently, the mutagenic and tumorigenic potential of a BPDE adduct leading to "multiposition" mutations would be significantly higher. In summary, results of this study contradict the intuitive notion that point mutations arise exclusively by erroneous replication of the modified base and suggest that the higher mutagenic and carcinogenic activity of (ϩ)-BPDE-2 compared with (Ϫ)-BPDE-2 may partly stem from the capability of its major dG adduct to induce DNA mutations at multiple sites. | v3-fos-license |
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} | pes2o/s2orc | Characteristics of PCDD/Fs in a Particles Filtration Device with Activated Carbon Injection
Although numerous investigations have monitored polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran (PCDD/F) emissions from large municipal solid waste incinerators (MSWIs) and undertaken laboratory experiments to identify the formation mechanisms of PCDD/F, the PCDD/F profiles inside an air-pollution control device have seldom been determined. This study presents the outcome of a dioxin abatement program that injected particulate activated carbon (PAC) into an MSWI. The fly ashes collected from different locations in a bag filter were examined and the mass distribution was determined. Emissions from the stack were sampled to analyze PCDD/F content after injections of 10, 13 and 17 kg/h PAC. The concentration of PCDD congeners decreased from 117.00 to 0.32 ng/Nm and that of PCDF decreased from 94.84 to 0.19 ng/Nm. The concentrations of filter cake ashes in different chambers and at different locations varied at 105.11-147.53 ng/g. Based on mass balance evaluation, the flow rate of PCDD/Fs in filter cake ash was 3.33 ± 0.50 ng/min; this value was roughly six times higher than that of fly ash in the disposal pit, indicating that filter cake ash treatment warrants considerable attention due to the policy for controlling PCDD/Fs.
INTRODUCTION
Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) were discovered in flue gases and fly ash from municipal solid waste incinerators (MSWIs) in 1977 (Olie et al., 1977).Investigations revealed that PCDD/F emission in flue gases and fly ash from MSWIs were discovered by the same token in Taiwan.These PCDD/Fs are hydrophobic and do not metabolize; thus, they persist in the environment and bioaccumulate in the fatty tissues of animals and humans (US EPA, 2000).
The dioxin formation mechanism is based on the condition of source, temperature, location, and reaction type.It is normally divided into two parts.This study focuses on the bag filter to identify the temperature, source and influence.Thus, this study divided the dioxin formation mechanism into three parts.The following three mechanisms account for PCDD/F emissions from incinerators: 1) High-temperature gas phase formation (300-600°C) (Everaert and Baeyens, 2002); 2) Formation from precursors, such as chlorophenols, polychlorinated diphenyl ethers, and chlorobenzenes (Lustenhouwer et al., 1980;Hutzinger et al., 1985).For instance, chlorophenols are good surrogates for the toxicity equivalence (TEQ) of PCDD/Fs during different incineration processes (Tuppurainen et al., 2000); 3) Formation by de novo synthesis in the low-temperature post-combustion zone (200-400°C) through residue carbon or metal catalysts in the fly ash reaction (Dickson et al., 1989;Everaert and Baeyens, 2002).
Air pollutants, such as particulate matter, heavy metals, polycyclic aromatic hydrocarbons, and dioxins generated from combustion processes adversely affect human health (Lin et al., 2008;Wang et al., 2008).Taiwan currently has 24 large MSWIs in operation.Due to concern regarding their adverse health effects, the government has established guidelines that regulate air pollutant emissions.For instance, Taiwan's Environmental Protection Administration (EPA) has set the dioxin emission limit in flue gas for MSWIs at 0.1 ng I-TEQ/Nm 3 ; thus, removing PCDD/Fs from flue gases is necessary.Various combinations of air-pollution control devices (APCDs) have been examined.A dry scrubber combined with a bag filter with powder activated carbon (PAC) injected is the most effective technique for controlling PCDD/F emissions (Blumbach and Nethe, 1994;Buekens and Huang, 1998;Lee et al., 2004;Wang et al., 2005).Notably, PAC injection is followed by various types of APCDs to enhance the removal of PCDD/Fs, which can approach 92-99% for MSWIs (Tejima et al., 1996;Dong et al., 2001a;Dong et al., 2001b;Abad et al., 2003).After dioxins have been adsorbed onto the PAC, the PAC with fly ash is then captured by the bag filter and removed as fly ash.
Taiwan's government has set regulations for ashes generated by MSWIs; roughly 2 × 10 6 tons of incinerator residue is produced annually.Fly ash, including filter cakes, fabric filters and fly ash pits, has the highest dioxin concentration produced by MSWIs (Huang and Buekens, 1995;Lin et al., 2008).Although PAC injection technology can effectively decrease the flue gas dioxin concentration, PAC injection transfers the dioxin in gas phase to a particle phase, which increases total dioxin emissions (including those in fly ash and flue gas) from MSWIs (Chang and Lin, 2001;Giugliano et al., 2002).The memory effect increases the dioxin concentration in flue gas after PAC injection, i.e., the dioxin or precursor desorbs slowly to flue gas and increases the dioxin concentration in the stack, and reduces removal efficiency for PAC injection to a level lower than that expected (Chang and Lin, 2001).In other studies, together with injecting PAC into the front of the bag filter, these two measures reduce PCDD/F concentrations in the stack flue gas from 145 ng I-TEQ/Nm 3 to 3.38-7.73ng I-TEQ/Nm 3 .Even with high PAC usage (40 kg/h), the PCDD/F concentration in flue gas still exceeds the regulatory limit of 1 ng I-TEQ/Nm 3 (factory process).This may be due to low PAC utilization efficiency (< 3%) in conventional activated carbon injection for a single bag filter system (Chi et al., 2007;Kim et al., 2007;Li et al., 2007).
Due to its simple engineering and high efficiency, PAC injection has become a popular retrofit technology for reducing dioxin emissions at most MSWIs.The PAC injection has been widely used with different APCDs for dioxin removal.However, few studies have focused on obtaining optimal dioxin control with PAC injection and a filter for MSWIs.In this study, different amounts of PAC were injected and the PCDD/F concentrations in stack flue gases were measured to investigate control of PCDD/F emissions.Additionally, the PCDD/F content in the bag filter ashes at different locations was determined.
EXPERIMENTAL APPROACH
The MSWI used in this study, which has been operating in southern Taiwan for 7 years, has two incineration units, with a total capacity of 900 tons of waste daily.The incinerator operating temperature is 850-1050°C.Each incinerator is equipped with a semidry scrubber (230-250°C) and bag filter (160-180°C) for controlling gaseous and particulate emissions.The PAC is injected between the semidry scrubber and bag filter.Mean flue gas generation rate was 95.11 KNm 3 /hr.The rate at which fly ash and cake ash were generated was estimated at 785.63 kg/h.Fig. 1 shows the operational parameters, a flow diagram, and sampling points in the MSWI.
Flue gas was sampled at the bag filter inlet and across every process unit of the flue gas cleaning system (PAC injection and bag filter).Sampling was performed during operation with PAC injection of 10, 13 and 17 kg/h and the performance of PAC in reducing PCDD/F emissions was evaluated.
Fly ashes were collected from the storage pit and bag filters.The bag filter cake ashes were sampled in chambers D, H and J.The bag filter, an APCD, has 10 chambers, each with 150 glass fiber filters (6 m × 12 cm i.d.) coated with Teflon.The samples from the D, H and J chambers were taken from random filter cakes in the bag filter.Every sample collect filter cakes over 10 filters.Fig. 1 presents a diagram of this process.
Analyses of stack flue gas and fly ash samples followed the US EPA Modified Method 23 and Modified Method 1613, respectively.All chemical analyses were conducted at the Super Micro Mass Research and Technology Center, Cheng-Shiu University, an accredited laboratory in Taiwan for analyzing PCDD/Fs.Prior to analysis, each sample was spiked with a known amount of the 13 C 12 -labeled internal standard to the extraction thimble.After extraction for 24 h in a Soxhlet extractor, the sample extract was concentrated and then treated with concentrated sulfuric acid.A series of sample cleanup and fractionation procedures followed.The eluate was concentrated to approximately 1mL and transferred into a vial.The concentrate was further concentrated to near dryness using a nitrogen stream.Prior to analysis, the standard solution for recovery checking was added to the sample.
A high-resolution gas chromatograph/high-resolution mass spectrometer (HRGC/HRMS) was used for PCDD/F analyses.The HRGC (Hewlett Packard 6970 Series Gas, Agilent, CA, USA) was equipped with a DB-5MS fused silica capillary column (L = 60m, ID = 0.25 mm, film thickness = 0.25 m) (J & W Scientific, CA, USA) with splitless injection.The HRMS (Micromass Autospec Ultima, Manchester, UK) was equipped with a positive electron impact (EI+) source.The analyzer mode of the selected ion monitoring (SIM) was used with a resolving power of 10,000.Details of analyses can be found in Wang et al. (2007).
To evaluate the partitioning of the PCDD/F concentration for each sample from the bag filter system, the results obtained for the PCDD/Fs formed were further evaluated using mass balance calculations.Mass fluxes of PCDD/Fs in the flue gas around the bag filter were obtained using concentrations and flow rate measurements, whereas the fluxes in the bag filter cake ash and fly ash were calculated based on the PCDD/F content in collected fly ash samples and their corresponding production rates.The mass balance was based on the concentration of PCDD/Fs, including flue gas before the bag filter (no PAC), stack flue gas, cake ash, and fly ash.At the time of sampling, flue gas production rate was 95.11 KNm 3 /hr, sampling volume was 2.19 Nm 3 , and fly ash and cake ash amounts were 10.0195 g and 3 g over 120 min.
PCDD/Fs Desorbed in Flue Gases with PAC Injection
Table 1 shows the concentrations of total PCDD and PCDF congeners when the PAC injection rate increased from 10 to 17 kg/h in flue gas, and the values for desorbed flue gas relative to PAC injection.The degree of chlorination of both PCDDs and PCDFs decreased markedly as the PAC injection amount increased, indicating that chlorination become increasingly important.Total PCDD congeners decreased from 117 to 0.32 ng/Nm 3 , and total PCDF congeners decreased from 94.84 to 0.19 ng/Nm 3 .Huang and Buekens (1995), who reviewed research regarding the mechanisms of PCDD/F formation, concluded that "de novo synthesis" can produce PCDD/Fs with a PCDF/PCDD ratio > 1, while precursor formation produces PCDD/Fs with a PCDF/PCDD ratio << 1.In this study, the PCDD/PCDF ratio was 1.25-13.93and the toxic equivalence was 0.32-0.65,demonstrating the significance of the PCDD/F concentrations as the precursor mechanism.The toxic equivalence is the de novo mechanism for PCDD/F formation.
Experimental results show that the maximum PCDD/F concentration and toxic equivalence were for a PAC injection of 10 kg/h, and the minimum PCDD/F concentration and toxic equivalence were for a PAC injection of 17 kg/h.However, the PCDD/F concentrations in flue gas were markedly decreased with PAC injection.The PAC injection was followed by various types of APCDs to enhance removal of PCDD/Fs, which approached 92-99% (Tejima et al., 1996;Dong et al., 2001a;Dong et al., 2001b;Abad et al., 2003).However, PAC injection only helps adsorb PCDD/Fs when it does not decompose PCDD/Fs.To meet the PCDD/F emissions standard of 0.1 ng I-TEQ/Nm 3 , the amount of PAC injected is typically excessive; thus, it is a costly treatment process.In addition to the quantity of PCDD/PCDFs desorbed, PAC injection also impacts the congener profile of desorbed PCDD/PCDF species found in adsorption traps.Fig. 2 shows data for desorbed PCDD and PCDF congener group profiles.The desorbed PCDD/PCDF species trapped in stack flue gas were mainly HpCDD/OCDD-HpCDF/OCDF (Lee et al., 2003;Kao et al., 2006), resulting from the dechlorination of PCDDs/PCDFs, and following similar trends relative to PAC injection as the total PCDD/F content.There are different influences in the HpCDD and OCDD.Experimental results indicate that I-TEQ decreased markedly when PAC was injected of 17 kg/h.Everaert et al. (2003) and Li et al (2008) indicate that increasing the quantity of activated carbon has a limited effect on the overall PCDD/F removal efficiency.
PCDD/F Content in the Bag Filter Ashes from Different Locations
Generally, PAC injected into the bag filter can adsorb PCDD/F pollutants in the flue gas.The PAC injection and removal efficiencies are significant.To quantify the residues on the bag filter, PCDD/F concentrations on filter ashes from different locations were measured.Table 2 shows the mean PCDD/F contents in bag filter ashes obtained from the fly ash storage pit and filter cake ashes from different chambers.The concentration of total PCDD/Fs in fly ash was 7.58 ng/g, and the PCDD/PCDF ratio was 1.39.The concentrations of filter cake ashes in different chambers and locations were 105.11-147.53ng/g, and the PCDD/PCDF ratio was 8.67-25.06.A number of different mechanisms influence the PCDD/PCDF content in ashes when flue gas passes across the bag filter.The amount of PACs injected may cause changes in the characteristics of the fly ash matrix (i.e., specific area and active sites).Comparing the filter ash and particulate phase of stack flue gas indicates that PAC injection technology can reduce flue gas PCDD/F concentrations; however, the technology increases the total amount of PCDD/Fs discharged (including those in fly ash and flue gas) from MSWIs.Activated carbon provides the basic organic material and catalytic surface for PCDD/F formation.
The possibility of a homogeneous gas phase reaction was also investigated.Fig. 3 shows the data for the PCDD and PCDF congener groups.The main congener profiles of the concentration are HpCDD and OCDD.The I-TEQ are PeCDF and HxCDF in fly ash, and HxCDD and PeCDF in filter cake ash.These analytical results show that samples from different chamber of bag filters have different concentrations and ratios.The congener profiles and the PCDD/PCDF ratio of flue gas and ashes also differed.
PCDD/Fs Mass Balance in the Bag Filter
To determine the content and distribution of PCDD/Fs in the bag filter system, flue gas and ashes were sampled from different locations, and the mass balance of PCDD/Fs in the bag filter was evaluated.
A detailed evaluation of experimental results is based on mass balance calculations (Fig. 4).Mass fluxes of PCDD/Fs in flue gas around the PAC injection and bag filter sites were obtained from actual concentrations and flow rate measurements, while the fluxes in solid and liquid residues were calculated using their PCDD/F content and corresponding mass production rates.In flue gas without PAC injected before the bag filter section, the PCDD/F flow rate was 3.87 0.25 ng/min.After the bag filter, the PCDD/F flow rate reduced to 0.01 0.004 ng/min and the fly ash was 0.63 0.24 ng/min.These findings indicate that stack gas levels constitute a minor contribution to total PCDD/Fs emitted by the MSWI; thus, complying with the regulatory limit of 0.1 ng I-TEQ/Nm 3 .The highest concentration of PCDD/Fs was found in fly ash responsible for PAC injection, and the PCDD/Fs shifted to solid phase.The PCDD/F levels of the filter cake ash flow rate were 3.33 0.50 ng/min, with a concentration roughly six-fold higher than that in fly ash.Surprisingly, the most significant contribution to the total PCDD/Fs released was filter cake ash (83.90%), followed by fly ash (15.87%); the contribution of stack flue gas was almost negligible.These analytical results have important implications for PCDD/Fs in filter cake ash.
Generally, an APCD combined with PAC injection can control PCDD/F emissions.Comparing the input (flue gas) and output (filter cake ash, fly ash, and stack flue gas), 2.78% of PCDD/Fs was produced.Many studies have indicated that fly ash (including filter cakes, fabric filter, and fly ash pit) contains the highest dioxin emissions from MSWIs (Huang and Buekens, 1995;Lin et al., 2008).The partitions were mainly on filter ash (about 83.9%).Relatively in fly ash, filter ash is comparatively steady (38.05% and 14.99%, respectively).
Although the operating temperature of the bag filter was only 160-180°C, which is below the favorable range of 250-400°C for PCDD/F formation, PCDD/Fs were captured by the active carbon in the bag filter.Furthermore, the duration fly ash remained in the bag filter was longer than that for other incinerator units, thereby increasing the amount of PCDD/Fs downstream.Similar trends were observed by Giugliano et al. (2002), Abad et al. (2003) and Wevers and De Fr´e (1998).The PAC injection system was located between the semidry scrubber and bag filter.Additionally, the PAC injection system is a batch system; the bag filter was purged for 30 sec every 30 minutes.This purging involved passing air through the bag filter to remove dust, and then coating it with activated cake adsorbents to remove dioxins and other noxious pollutants downstream.Most filter ashes dropped from the filter into the fly ash pit.Mistakes in the sampling techniques may have altered experimental results.Notably, obtaining a sample in the bunker in which 50% of incineration occurs is difficult.Since wastes cannot be mixed perfectly, samples may not be completely representative.The experimental results suggest that the treatment should be studied extensively to control PCDD/F formation in fly ash.
CONCLUSIONS
For PAC injection of 10, 13, and 17 kg/h, the total concentration of PCDD congeners decreased from 117 to 0.32 ng/Nm 3 ; the total concentration of PCDF congeners decreased from 94.84 to 0.19 ng/Nm 3 ; and the PCDD/PCDF ratio was 1.25-13.93.Experimental results indicate that the PCDD/F concentration and toxic equivalence were minimal at a PAC injection of 17 kg/h.Additionally, filter cake ash concentrations in samples obtained from different chambers and locations in the bag filter were 105.11-147.53ng/g, and the PCDD/PCDF ratio was 8.67-25.06.A number of different mechanisms influence the PCDD/PCDF content in ashes when flue gas passes across the bag filter.
The PCDD/F flow rate in the flue gas without PAC injection before the bag filter section was 3.87 0.25 ng/min via mass balance calculations.After the bag filter, the PCDD/F flow rates reduced to 0.01 0.004 ng/min and the fly ash was 0.63 0.24 ng/min.The highest concentration of PCDD/Fs was in the fly ash responsible for PAC injection, and the PCDD/Fs shifted to solid phase.The PCDD/F concentration of the filter cake ash flow rate was 3.33 0.50 ng/min, roughly 6-fold higher than that in fly ash.Compared with fly ash, filter ash was comparatively steady (38.05% and 14.99%, respectively).These experimental results suggest that the filter cake ash and filter treatment should be investigated extensively to control PCDD/F formation in fly ash.
Fig. 4 .
Fig. 4. PCDD/Fs mass balance across the PAC injection and bag filter.
Table 1 .
Mean PCDD/F content in flue gas by injection PAC.
Table 2 .
Mean PCDD/F content in ashes.Mix cake ash containing different chambers of D, J, and H. a | v3-fos-license |
2018-01-18T22:33:09.732Z | 2016-06-23T00:00:00.000 | 40032416 | {
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} | pes2o/s2orc | Removal of odorous sulphur compounds from industrial gases by biotrickling filters
A pilot plant for the treatment of Sulphur-based odorous gases was installed in a line of a phosphoric acid plant located in Skhira, Tunisia. The air pollution control system train consisted of a first stage, including a chemical scrubber operating with an alkaline solution containing caustic soda, followed by a two-stage biotrickling filter (BTF) filled with Mytilus edulis shells. This study evaluated the performance of the dual-stage BTF in removing hydrogen sulphide (H2S), sulphur dioxide (SO2) and dimethyl sulphide (DMS) from the phosphoric acid reactor’s exhaust air current. Concentrations of H2S, SO2 and DMS at the inlet of the two-stage BTF were 10-30 ppm, < 1-20 ppm and 16-30 ppm, respectively. All of the respective concentrations at the outlet of the biological step were < 1 ppm, except for the H2S in the outlet during the first day of operation (10 ppm). Removal efficiencies were generally higher than 95% for all compounds, and remained high even with an increase of the off-gas flow rate. Mass-removal capacity was at least 2.0 g m -3 h -1 , 0.5 g m -3 h -1 and 6.2 g m -3 h -1 , for H2S, SO2 and DMS, respectively. The removal efficiencies of the process were satisfactory, especially considering the already low inlet concentrations, due to the high quality of the raw phosphate used.
INTRODUCTION
Exposure to odorants is more a nuisance than an hazard to human health.However, prolonged exposure to odorants may cause adverse reactions ranging from emotional stress, such as anxiety, headache, or depression, to physical symptoms, such as eye irritation, respiratory problems, nausea or vomiting (Capodaglio et al., 2002;Shareefdeen and Singh, 2004).For these reasons, industrial activities that emit odorous compounds need appropriate removal systems in order to limit their presence in exhaust gases.
Control of pollution in large air flows with low concentrations of pollutants (generally < 100 ppm) is generally not economically viable when removal technologies require large amounts of fuel (electric energy), or when chemicals and adsorbents are adopted, due to the low achievable efficiencies (Hunter and Oyama, 2014).For this reason, the application of biologically-based technologies, which turn out to be more cost-effective than conventional ones (e.g., activated carbon adsorption, thermal or catalytic oxidation, absorption, selective catalytic or non-catalytic reduction), is becoming a popular alternative for industrial air pollution control.
Among biological technologies, biotrickling filters (BTFs) have recently gained increased attention due to their comparatively low cost and energy efficiency.In particular, BTFs have proven very effective in the treatment of industrial air streams (Rada et al., 2014), and have shown several advantages compared to the more common biofilters.These advantages are their limited size, longer life, lower pressure drop with subsequent lower power consumption, easy control of temperature, pH, salt concentration and metabolite accumulation, wide range of treatable pollutants, greater stability of operation and better atmospheric dilution of the plume (Xue et al., 2013).BTFs have been widely tested and applied in the treatment of exhaust air pollutants from pharmaceutical (Chen et al., 2006), paint (Mathur and Majumder, 2008), refinery (Viotti et al., 2015) and livestock industries (van Groenestijn and Kraakman, 2005), as well as wastewater (Lebrero et al., 2014) and solid waste treatment facilities (Gutierrez et al., 2014).BTFs are reactors partly filled with inert materials, such as lava rock, shells, and plastic spheres or rings, which serve as a physical support for the growth of biomass.Water is sprayed at the top of the reactor, creating a liquid biofilm on the filler for the absorption of pollutants, providing the microorganisms with the required nutrients and removing excess sludge or decomposition products from the reactor by scouring.The water is collected at the bottom of the reactor and recirculated back.Therefore, both pollutant absorption into the biofilm and biodegradation occur in BTFs.
A pilot treatment line for the removal of odorous sulphur gases, including hydrogen sulphide (H 2 S), sulphur dioxide (SO 2 ) and dimethyl sulphide (DMS), was established at a phosphoric acid production facility in Skhira, Tunisia.Phosphoric acid is primarily used in fertilizer production and, secondarily, in the production of synthetic detergents, animal feed and pesticides.In this application, phosphoric acid is obtained by a wet process, consisting of the attack of ground phosphate rocks with sulphuric acid, the filtration of phosphoric acid from calcium sulphate (a byproduct of the chemical reaction), and the concentration of the phosphoric acid to the desired level.The chemical reaction leads to the formation of carbon monoxide, nitrogen oxides, SO 2 , hydrogen fluoride, ammonia, H 2 S and DMS gases.
The adopted air pollution control system was designed to remove sulphur pollutants by means of a scrubber followed by a dual-stage BTF, and was operated for two months on site.This paper evaluates the performance of the dual-stage BTF in degrading the H 2 S, SO 2 and DMS contained in the phosphoric acid reactor's off-gas.The paper presents the biological processes used in the test, the operating and testing procedures, and the results achieved in the abatement of these compounds.
MATERIALS AND METHODS
The treatment system studied is shown in Figure 1 and consists of: The exhaust air under treatment is drawn by a centrifugal blower, located downstream of the entire system.The blower operates with a power of 3 kW, allowing a maximum flow rate of 1800 Nm 3 h -1 .Air passes first through the scrubber, and reaches the BTF unit at a temperature lower than 45°C, a relative humidity around 80-100% and solid particle content lower than 20 mg m -3 .Throughout the whole experimental period, both the scrubber and the dual-stage BTF were operated continuously (24/7).
Scrubber operation
The scrubber used in this system consists of a vertical polypropylene cylinder with internal diameter of 0.8 m and height of 6.3 m.The scrubber operates with alkaline washing of caustic soda (NaOH solution < 30% v/v)).
The main role of the chemical scrubber is to absorb the soluble pollutants in the exhaust gas stream into water, resulting in attenuation of the H 2 S and SO 2 peak concentrations.In addition, due to its buffering capacity, the alkaline NaOH washing solution allows for the control of the pH of the gas prior to the biological treatment.The scrubber also serves as a backup system to control gas temperature and to protect the biological stage.
The washing solution is introduced from the top of the scrubber through spray nozzles, flowing by gravity through the absorption column to the bottom, where it is collected in a reservoir (diameter of 1.55 m and height of 0.33 m).The reservoir is equipped with a tap for NaOH addition, and another for water replenishment, ensuring a constant level and concentration of the washing solution in the tank.The washing solution is recirculated to the top of the scrubber with a pump, while the exhaust gas flows counter-currently from the bottom to the top of the absorption column, which contains plastic fillers with large specific surface area to improve the contact between the gas and liquid phases, thus enhancing chemical exchange.This, combined with a high ratio between liquid and gas flows, results in a high elimination capacity of the incoming pollutants.Finally, a high-efficiency droplet separator ensures good aerosol retention before transition to the biological stage.
Dual-stage BTF operation
The unit consists of a dual-stage biological filter and could be considered a biological washing tower.The incoming air from the chemical scrubber is passed vertically through an area filled with calcareous sea shells, continuously humidified by the water recirculated within the system via a centrifugal pump.
The filling material is a bed of natural shells belonging to blue mussels of the Mytilus edulis species, pre-treated prior to installation by an exclusive process (Monashell ® , patented).The filter bed has different functions, such as supporting the growth of the microorganisms that biologically oxidize DMS, H 2 S and sulphuric acid; supplying additional nutrients to the biomass; humidity retention; forming a surface where pollutants can be absorbed; and stabilizing the pH.The latter is made possible through chemical reactions between the filter bed itself and the sulfates in the air stream.The Mytilus edulis shells are rich in calcium carbonate, present high affinity with weak acids, and provide a high buffering power to the system.The choice of this filling material offers several economical and process-related advantages: from an economic perspective, it is a recovered waste material and is thus available at low cost (compared to the costs required for its disposal); from the biological perspective, it facilitates absorption, since many odorous compounds are not easily soluble in the natural state.An additional advantage is low operating costs; for example, less water is necessary for pH restoration than in a bioscrubber, since pH is controlled by the filter bed itself.
The dual-stage unit consists of a modular bioreactor with a volume of 17 m 3 , where the filling material occupies a volume of 8 m 3 , placed within a container with a flat roof, all supported by a concrete platform.The recirculation of the washing solution is supported by two pumps, each with a power of 1.1 kW, and connected with two nozzles working at the pressure of 0.8 Bar, with total flowrate of about 20 L min -1 .The treated emissions are released to the atmosphere through a stack (diameter of 0.2 m), providing a maximum outlet velocity of 16 m s -1 .
In the first stage, the recirculated water sprayed on the top of the filter bed flows through the filling material and exits through a PVC pipe, collecting the water to repeat the cycle.The first-stage effluent air is sent to the second BTF stage, characterized by separate circulation water and spray systems, where the cycle is repeated.The water levels in the storage tanks of both stages are controlled with level sensors.
In order to keep the solution clean and avoid a pH decrease of the recirculation water in the second stage, part of the washing solution is periodically wasted through a time-controlled (4 s h -1 ) solenoid valve.An equal amount of water from the first stage is automatically transferred to the second stage, and fresh make-up water is drawn into the first stage tank from the supply network.
The BTF was initially seeded with a bacterial inoculum of Thiosphaera pantotropha, a selected bacterial culture capable of assimilating reduced-sulphur compounds.
Test Procedure
The pilot system was operated continuously for about 10 weeks.During this period, chemical, physical and microbiological analyses were carried out on the off-gas, in addition to microbiological analyses on the cell count of the mesophilic bacteria, both in the water solution and in the filtering material.
Off-gas analyses included temperature and H 2 S, DMS, SO 2 concentrations; the latter were measured at the sampling points located upstream of the dual-stage BTF (A) and at the outlet (C).These are compounds of interest due to their abundance in the exhausts and to the low odor threshold values, equal to 0.00041 ppm, 0.0030 ppm and 0.87 ppm, respectively (Nagata, 2003).Tests were performed by varying the gas flow rate between 1200 and 1500 Nm 3 h -1 , corresponding to an empty bed residence time (EBRT) between 16 and 24 s.In addition, the off-gas temperature was monitored at A, C and at a sampling point located upstream of the scrubber (B).The pressure drop between B and A and between A and C was measured also.Dräger tubes (Drägerwerk AG & Co. KGaA, Germany) were used for the analysis of H 2 S, DMS and SO 2 concentrations.Analyses of recirculation water of both stages concerned pH, sulphate (SO 4 2-) concentration, and temperature.SO 4 2-is an oxidation product of H 2 S, SO 2 and mercaptans in the circulation water (Zhang et al., 2015); its concentration was measured with colorimetric strips (Merck KGaA, Germany).The presence of SO 4 2-in the BTF recirculation water is an indicator of biodegradation effectiveness.
During the ordinary running period, air concentrations of H 2 S, SO 2 and DMS, and pH, SO 4 2-content and the temperature of the BTF recirculation water were sampled at least once per week.
RESULTS AND DISCUSSION
The combined scrubber-BTF system was initially tested at different airflow rates, with a maximum of 1800 Nm 3 h -1 , as reported in Table 1.The purpose of this initial test was the quantification of the pressure drop at increasing flow rates.The total pressure drop, measured between the sampling points B and C, ranged from 70 to 245 mm H 2 O when the air flow rate increased from 1200 to 1800 Nm 3 h -1 .This airflow increase induced an increase in the pressure drop over the BTF that was anyway limited to between 10 and 45 mmH 2 O. Vincenzo Torretta et al.
In addition to the removal of the initial pollutants, a positive effect deriving from the scrubber unit operation is the reduction of the off-gas temperature from 44.0-53.6 °C in B to 32.0-36.7 °C in A (Table 1), favoring the growth of the biofilm.
After inoculation, the dual-stage BTF was started up.During the tests, thanks to the replenishing scheme of the recirculation water, its pH never fell below 6.5, from a maximum of 7.5 (Table 2).Three days after the BTF start-up, the biological bed was fully active; however, an acclimation period of about four weeks was necessary to reach full efficiency (Table 3).The concentrations of H 2 S, DMS and SO 2 at the inlet of the dual-stage BTF (sampling point A) were in the ranges between 10 and 30 ppm, 16 and 30 ppm, < 1 and 20 ppm, respectively.The respective concentrations at the outlet of the biological step (sampling point C) were all < 1 ppm, except for the H 2 S concentration during the very first day of operation (10 ppm).Removal efficiencies were generally higher than 95% for all compounds.Elimination rates were, at minimum, 2.0 g m -3 h -1 , 0.5 g m -3 h -1 and 6.2 g m -3 h -1 , for H 2 S, SO 2 and DMS, respectively.The analytical methodology used did not allow for the determination of concentrations < 1 ppm; therefore, the effective elimination rates may be higher than those here reported.The removal efficiencies of the incoming pollutants were high even when higher off-gas flow rates were used.Concentration of SO 4 2-in the BTF recirculation water ranged between 20 and 90 ppm, indicating the presence of microbial activity degrading sulphur compounds (Table 2).
Other BTF pilot-and laboratory-scale applications for the removal of sulphur compounds reported in the literature generally referred to higher inlet concentrations: Chen et al. (2006) worked with H 2 S inlet concentrations about 10 times higher than those reported in this paper, obtaining removal efficiencies > 90% at an EBRT of 9 s.Removal efficiencies > 95%, and elimination rates of about 50 g m -3 h -1 were obtained by a BTF operating with an EBRT of 29 s, and inlet concentrations 10 times higher than those observed here (Montebello et al., 2013).Biodegradation of SO 2 was documented in a recent publication (Zhang et al., 2015) where a biofilter, operating with EBRT of 18 s degraded SO 2 in an air stream at 100-200 ppm, showing a removal efficiency > 93% and a maximum elimination rate of 50.67 g m -3 h -1 .DMS removal was the object of a study on a BTF application operating with EBRT of 120 s, which achieved a removal efficiency > 90% and a maximum elimination rate of 7.2 g m -3 h -1 , from an initial concentration of 117 ppm (Sercu et al., 2005).
a chemical scrubber, operating with an alkaline solution for SO 2 reduction and H 2 S partial abatement; a dual-stage BTF, downstream of the chemical scrubber, for the removal of the remaining sulphur odorous compounds; water recirculation pumps; an off-gas centrifugal pump; a control cabinet.
Figure 1 .
Figure 1.Exhaust air plant views: a) horizontal and b) vertical, with indication of sampling points.
Table 1 .
Velocity, temperature and pressure drop of the exhaust gas measured at sampling points A, B and C with different gas flow rates.
Table 3 .
Pollutant concentrations measured at sampling points A, B and C during the operational period of exhaust-treatment plant. | v3-fos-license |
2019-12-28T14:03:20.486Z | 2019-12-27T00:00:00.000 | 209488823 | {
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"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/jcmm.14903",
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} | pes2o/s2orc | Baicalein inhibits mitochondrial apoptosis induced by oxidative stress in cardiomyocytes by stabilizing MARCH5 expression
Abstract Abnormal mitochondrial fission and mitophagy participate in the pathogenesis of many cardiovascular diseases. Baicalein is a key active component in the roots of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. It has been reported that baicalein can resist cardiotoxicity induced by several stress, but the mechanisms of baicalein operate in the protection of cardiomyocytes need to be researched further. Here we report that baicalein can promote cell survival under oxidative stress by up‐regulating the expression level of MARCH5 in cardiomyocytes. Pre‐treatment cells or mice with baicalein can stabilize the expression of MARCH5, which plays a crucial role in the regulation of mitochondrial network and mitophagy. Overexpressed MARCH5 is able to against H2O2 and ischaemia/reperfusion (I/R) stress by suppressing mitochondrial fission and enhancing mitophagy, and then attenuate cells apoptosis. Altogether, our present study investigated that baicalein exerts a protective effect through regulating KLF4‐MARCH5‐Drp1 pathway, our research also provided a novel theoretical basis for the clinical application of baicalein.
transport by ubiquitinating different proteins. 8,9 Recent findings advocate that MARCH5 plays a critical role in regulating mitochondrial morphology and apoptosis. 10 Loss of MARCH5 enhances apoptosis and promotes mitochondrial fission in HEK293, HeLa cells 11 and HCT116 cells. 12 Abundantly expressed MARCH5 in COS7 cells promotes the formation of long tubular mitochondria 13 and BC cells growth. 14 Mitophagy is responsible for eliminating of damaged mitochondria and regulating apoptosis in HeLa cells, which is also regulated by the MARCH5 level. 15 However, it is not yet clear whether MARCH5 participates in the regulation of mitochondrial dynamics in cardiomyocytes and how mitophagy links with the mitochondrial fission.
Baicalein (5,6,7-trihydroxyflavone) is one of the major phenolic flavonoids extracted from the root of Scutellaria baicalensis Georgi, 16,17 which has been widely applied in traditional Chinese medicine. It has been reported that baicalein has the effects of anti-inflammatory, antioxidant and anti-cancer. [18][19][20] Recently, studies have shown that the antioxidant activities of baicalein can inhibit lung mitochondrial lipid peroxidation during ROS stress 21 and decrease myocardial tissue injury undergo I/R in rats. However, whether baicalein is involved in the regulation of mitochondrial dynamics and mitophagy need further in-depth studies.
Our present work reveals that MARCH5 plays a key role in regulating mitochondrial dynamics and mitophagy. Overexpression
| Cell cultures and treatment
H9C2 cells were cultured in DMEM (Gibco) with 10% foetal bovine serum (TransGen), and 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen) in a humidified 5% CO 2 incubator at 37°C. When the cultured cells reached approximately 70% confluently, they were treated with H 2 O 2 (100 μM) incubated at 37°C for 3-24 hours in complete culture medium. For tested the protective effect of baicalein (Invitrogen), we pre-treated cells with baicalein (50 μM) for 4 hours and then incubated with H 2 O 2 as above described.
| The MARCH5 plasmid construction
The expression plasmids for MARCH5 was generated by amplifying the corresponding cDNA by PCR with Phanta Max Super-Fidelity DNA Polymerase (Vazyme), and then cloning it into pcDNA3.1 expression vector by using ClonExpress Ultra One Step Cloning Kit (Vazyme). We used Lipofectamine 3000 (Thermo Fisher) for transfection vector. The procedures were in accordance with the kit instructions.
We used Lipofectamine 3000 (Thermo Fisher) for transfection siRNA. The procedures were in accordance with the kit instructions.
| Mitochondrial staining and analysis of mitochondrial fission
Mitochondrial staining was performed as others described with modifications. 1, 22 Briefly, cells were plated onto the poly-L-lysine coated coverslips. After treatment, they were stained for 30 minutes with 0.02 µM MitoTracker Red at 37°C. Mitochondria were imaged using a laser-scanning confocal microscope (Zeiss LSM510 META).
| Apoptosis assays
Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) using a kit from TransGen. The detection procedures were in accordance with the kit instructions.
| Immunoblotting
Immunoblot was carried out as others described. 23 Briefly, the cells were lysed for 20 minutes on ice in RIPA lysis buffer containing a F I G U R E 1 H 2 O 2 exposure induces cardiotoxicity. H9C2 cells were exposed to 100 μm H 2 O 2 for the indicated time. (A) Mitochondrial morphology was stained with MitoTracker Red and observed using a laser-scanning confocal microscope. (B) shown the percentage of cells undergoing mitochondrial fission. (C) Apoptotic cells were detected by TUNEL assay and the percentage of apoptotic cells shown in (D). (E) Autophagy flux was assessed with transduced Ad-RFP-GFP tandem-tagged LC3. Autophagy flux was observed using a laser-scanning confocal microscope the numbers of autolysosomes and autophagosomes in H9C2 cells (F). mRFP dots (red) indicated autolysosomes, and the merged (yellow) dots indicated autophagosomes. The expression level of MARCH5 was detected by Western blotting (G) and RT-qPCR (I). LC3 protein expression level was detected by Western blotting (G), densitometry (H). All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 The samples were rotated at 4°C overnight. The beads were washed three times with 1 mL of low-salt NP40 lysis buffer (300 mM NaCl) and twice with 1 mL of high-salt lysis buffer (500 mM NaCl). The beads were then boiled for 10 minutes in the presence of 25 μL 2× sample buffer, and the released proteins were fractionated in 12% SDS-PAGE gels. Proteins were detected by immunoblotting as described above.
| Real-time quantitative PCR
RT-qPCR for MARCH5 was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad). Total RNA was extracted using Trizol reagent. Reverse transcription reactions were carried out using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen) to make cDNA according to the manufacturer's guide. TransStart Green qPCR SuperMix (TransGen) was used for quantitative PCR (qPCR) analysis, and the procedures were in accordance with the kit instructions. The levels of MARCH5 analysed by RT-qPCR were normalized to that of GAPDH. MARCH5 primers were forward: 5′-ATGCCGGACCAAGCCCTT-3′ and reverse: 5′-TTATGCTTCTTCTTGCTCTGGATAATTTAGGAT-3′. GAPDH forward primer: 5′-GTCGTGGAGTCTACTGGCGTCTTCA-3′ and reverse: 5′-TCGTGGTTCACACCCATCACAAACA-3′.
| Autophagic flux
Autophagic flux experiment was performed as others described with some modifications. 24 Autophagy flux was assessed with transduced Ad-RFP-GFP tandem-tagged LC3. RFP retains its fluorescence even in the acidic environment of lysosomes where GFP loses its fluorescence. Thus, green LC3 puncta primarily indicate autophagosomes, while red LC3 puncta indicate both autophagosomes and autolysosomes. The red puncta that overlay with the green ones and appear yellow in merged images are indicators of autophagosomes, while the free red puncta that do not overlay with the green ones and appear red in merged images are indicative of autolysosomes.
The puncta were imaged using a laser-scanning confocal microscope (Zeiss LSM510 META). And mice were subjected to 30 minutes of left anterior descending coronary artery (LAD) ligation followed by 3 hours of reperfusion.
| Animal experiments
The heart was rapidly excised. The heart slices were used for cardiomyocyte apoptosis analysed.
| Statistical analysis
The results are expressed as mean ± SEM of at least three independent experiments. The statistical comparison among different groups was performed by one-way analysis of variance (ANOVA) for multiple comparisons. Statistical analyses were performed with GraphPad F I G U R E 2 Overexpression of MARCH5 reduces cardiotoxicity induced H 2 O 2 . (A) H9C2 cells were transfected with MARCH5-cDNA for 24 h, and the expression level of MARCH5 was detected by Western blotting. (B) H9C2 cells were exposed to 100 μM H 2 O 2 for another 24 h, and mitochondrial morphology was stained with MitoTracker Red and observed using a laser-scanning confocal microscope, (C) shown the percentage of cells undergoing mitochondrial fission. Apoptotic cells were detected by TUNEL assay (D) and the percentage of apoptotic cells shown in (E). Autophagy flux was assessed with transduced Ad-RFP-GFP tandem-tagged LC3. Autophagy flux was observed using a laser-scanning confocal microscope (F) and (G) shown the numbers of autolysosomes and autophagosomes. LC3 protein was detected by Western blotting (H) and densitometry (I). All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 Prism 5.0 (GraphPad Software, Inc, San Diego, CA). P < .05 was considered statistically significant.
| H 2 O 2 is able to induce mitochondrial fission, apoptosis and change mitophagy flux
To explore whether MARCH5 is involved in the regulation of oxidative stress-induced mitochondrial fission, mitophagy and apoptosis in cardiomyocyte, we treated the H9C2 cells with H 2 O 2 and determined the change of cell dynamics at different time points.
We observed a time-dependent increase in the mitochondrial fission ( Figure 1A LC3 is widely used as autophagy marker and up-regulated with autophagy occurrence. 25,26 We detected the expression level of LC3 in H9C2 cells treatment with H 2 O 2 at different time points and found that the tendency of LC3 is similarly to mitophagy flux. The expression level of LC3-II is little higher in 3 hours, and then lower upon H 2 O 2 exposure time ( Figure 1G,H). Since H 2 O 2 can induce mitochondrial fission, suppress mitophagy and then enhance cell apoptosis, we further want to understand whether MARCH5 links with these events. We noted that the expression level of MARCH5 markedly decreases with the growth over time ( Figure 1G,I). These data suggested that hydrogen peroxide could induce mitochondrial fission, inhibit mitophagy and a concomitant decrease in cell viability.
Time-dependent decreased of MARCH5 level revealed that it might tightly link with these cellular events.
| Overexpression of MARCH5 reduces H 2 O 2 induced cardiotoxicity
To systematically understanding the critical role of MARCH5 in H 2 O 2 induced cardiotoxicity, we transfected the cells with the plasmid construct of MARCH5-cDNA to induce MARCH5 overexpression. The expression level of MARCH5 was significantly increased by its plasmid but not by its control (Figure 2A). As observed in Figure 2B These results discovered that overexpressed MARCH5 in cardiomyocytes can protect cells from cardiotoxicity induced by H 2 O 2 .
| Knockdown of MARCH5 sensitizes H9C2 cells to H 2 O 2 caused cardiotoxicity
To examine wether MARCH5 is the key factor of attenuating H 2 O 2 caused cardiotoxicity, we knocked down the endogenous MARCH5 expression using MARCH5-siRNA. The expression level of MARCH5 was significantly decreased by MARCH5-siRNA but not by its control ( Figure 3A). Lower dose of H 2 O 2 (50 μM) was used for treatment cardiomyocytes, and we explored whether MARCH5 knockdown is able to enhance the sensitivity of cardiomyocytes to H 2 O 2 induced cardiotoxicity. As shown in Figure 3B
| Protective effect of baicalein on H 2 O 2 induced cardiotoxicity
Baicalein is a key active phenolic flavonoids in the roots of Scutellaria baicalensis Georgi, 16 which has been reported to have multi-functions.
F I G U R E 4
Protective effect of baicalein on H 2 O 2 induced cardiotoxicity. H9C2 cells were pre-treated with 50 μM baicalein for 4 h, then H9C2 cells were exposed to 100 μM H 2 O 2 for 24 h, and the expression level of MARCH5 was detected by Western blotting (A). The percentage of cells undergoing mitochondrial fission is shown as (B). The percentage of apoptotic cells is shown as (C) detected by TUNEL assay. The numbers of autolysosomes and autophagosomes were observed as (D). LC3 protein was detected by Western blotting (E) and densitometry (F). All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 To determine whether baicalein protects the cardiomyocytes against oxidative stress injury, we pre-treated H9C2 cells with baicalin for
| The protective mechanism of baicalein on H 2 O 2 induced cardiotoxicity
To understand the underlying protective mechanism of baicalein, based on the results shown in Figure 4, we first treated the H9C2 cells with H 2 O 2 after pre-treatment with baicalein and transfection with MARCH5-cDNA. We found that in baicalein pre-treatment group and MARCH5 overexpression group, the number of mitochondrial fission cells was remarkably reduced ( Figure 5C), mitophagy was enhanced ( Figure 5A,E), and the number of apoptotic cells was significantly decreased ( Figure 5D). These data revealed that baicalein had the similar protective function with overexpression MARCH5 via increasing the MARCH5 expression ( Figure 5A).
We also observed the synergetic protective effect of baicalein and MARCH5 on H 2 O 2 induced cardiotoxicity ( Figure 5C-E). Most recent studies have revealed that ubiquitination can promote apoptosis, 27 we then tested the modification of MARCH5 by ubiquitin.
As shown in Figure 5B, the mount of ubiquitin-MARCH5 noticeable increased compared with control under oxidative stress. In baicalein groups, the ubiquitination level striking decreased as compared with its related control ( Figure 5B). All the data illuminated that baicalein can attenuate the ubiquitination level of MARCH5 and consequently stabilize intracellular MARCH5 level. Stably expression of MARCH5 is essential for maintaining the homoeostasis of mitochondrial dynamics and mitophagy.
F I G U R E 5
The protective mechanism of baicalein on H 2 O 2 induced cardiotoxicity. H9C2 cells were exposed to 100 μM H 2 O 2 for 24 h after pre-treated with 50 μM baicalein for 4 h, transfected with MARCH5-cDNA for 24 h, pretreated with baicalein and transfected with MARCH5-cDNA, respectively. MARCH5 and LC3 proteins were detected by Western blotting. (A) Ubiquitylation assays were performed as described in methods. The ubiquitylation level of MARCH5 was detected using an Ub antibody (B). (C) shown the percentage of cells undergoing mitochondrial fission. (D) shown the percentage of apoptotic cells. (E) shown the autolysosomes and autophagosomes in H9C2 cells. All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 To further investigate the protective effect of baicalein via MARCH4 pathway, we detected the expression level of KLF4, which is a transcriptional regulatory factor of MARCH5. 28 As showed in Figure 6A The ubiquitylation level of Drp1 in normal and pre-treated with baicalein after treatment with H2O2. All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 apoptosis. 13,29 The expression level of Drp1 was markedly increased after exposure to H 2 O 2 ( Figure 6A,C) , and treatment with baicalein can signally reduce the expression level of Drp1 ( Figure 6D,F). There is remarkable negative correlation between Drp1 and MARCH5 ( Figures 1A and 5A). Drp1 can be ubiquitylated by ubiquitin ligases and then degraded. 9 In order to probe wether MARCH5 can F I G U R E 7 Baicalein attenuate myocardial I/R injury. Mice were undergoing I/R, and the myocardial tissue sections were used for evaluation cells apoptosis (A) by TUNEL assay. (B) shown the percentage of apoptotic cells. Mice were undergoing I/R, and the myocardial tissue was used for assessment proteins expression level by Western blotting (C) and densitometry (D-G). All of the data were expressed as the mean ± SEM of three independent experiments. *P < .05, **P < .01, ***P < .001 ubiquitylate Drp1, we performed the immunoprecipitation assay and the result showed in Figure 6G. In the MARCH5 knockout group, the expression level of Drp1 was significantly higher, but the ubiquitination level was the lowest in the H 2 O 2 treated three groups. In the MARCH5 overexpression group, the expression level of Drp1 was remarkably reduced, but the ubiquitination level was the highest in the H 2 O 2 treated three groups. In baicalein treatment group, the expression level of Drp1 was significantly decreased and the ubiquitination level was higher than other groups ( Figure 6H). Altogether, we investigated that MARCH5 played crucial role in the ubiquitination of Drp1, MARCH5 could decrease the expression level of Drp1 via promoting it ubiquitination.
| Baicalein attenuates myocardial ischaemia reperfusion injury
The oxidative stress induced by superoxide production during I/R is one of the main causes of cardiomyocytes death. 30 Damaged mitochondria removed by mitophagy is an essential process in mitochondrial quality control. 15 In this study, we found that oxidative stress can inhibit mitophagy, which led to the accumulation the accumulation of damaged mitochondria, and then induce cells apoptosis.
| D ISCUSS I ON
Recent research reported that MARCH5 plays an anti-apoptotic role against ER stress. 11 In this study, we identify MARCH5 as a key factor to maintain mitochondrial homoeostasis and mitophagy. Loss intracellular MARCH5 enhanced mitochondrial fission, inhibited mitophagy and then induced cells apoptosis (Figure 3).
Baicalein as a bioactive component present in Chinese herbal medicine, possesses a wide range of pharmacological activities with excellent oxidant scavenging capability. It has been reported to exert a cardioprotective effect by effective oxidant scavenging. 32 Baicalein can protect cardiomyocytes by reduction oxidative stress, myocardial inflammatory responses and apoptosis in LPS-induced sepsis. 33 It also can attenuate LPS-induced TNF-a, IL-6, NO and iNOS expression in neonatal rat cardiomyocytes. 34 Here we suggested a novel point, ba-
ACK N OWLED G EM ENTS
This work was supported by the National Science Foundation of China (grant numbers 81071246).
CO N FLI C T O F I NTE R E S T
No conflicts of interest exist.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. | v3-fos-license |
2018-04-03T01:27:24.727Z | 2017-03-23T00:00:00.000 | 16524590 | {
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} | pes2o/s2orc | Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro
While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid–gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.
Scientific RepoRts | 7:44077 | DOI: 10.1038/srep44077 and ultrasound-triggered particles. Ultrasound-triggering provides the benefits of non-invasiveness, deep penetration (more than cm-order) and sub-millimetre to millimetre-order spatial controlling capability of ultrasound-beam-focusing that enables high spatial-temporal control of therapeutic activation. Active targeting is a potential approach to achieve intracellular delivery of the nanomedicine. An antibody possessing strong and specific antigen recognition ability often induces endocytosis upon binding to the antigen expressed on the surface of cancer cells 22,23 . Epiregulin (EREG), the cell-membrane-expressed ligand of epidermal growth factor receptor, is expressed and integrated into the plasma membrane at relatively high levels in a variety of human cancers, including colorectal and breast cancer 26 . This ligand has been intensively investigated as a therapeutic target 26 . The anti-EREG antibody 9E5 was conjugated as the active targeting moiety to submicron particles called phase-change nano-droplets (PCNDs), acoustic droplets composed of a phospholipid shells and liquid perfluorocarbon (PFC) core (Fig. 1a). These nano-sized PFC droplets have attracted attention as multi-modal imaging contrast agents and drug carriers [27][28][29][30] because they vaporise into microbubbles upon exposure to ultrasound 31 . We attempted to utilise this feature to physically kill cancer cells by intracellular vaporisation. Once 9E5-conjugated PCNDs were internalised to cells, ultrasound exposure vaporises PCNDs and those liquid-to-gas transition phenomena is considered to induce significant damage to cells (Fig. 1b). Here, we succeeded in demonstrating the selective targeting and cytotoxic effects in vitro with direct observation of intracellular vaporisation by high-speed imaging.
Results
Synthesis of 9E5-conjugated PCND. 9E5-conjugated PCNDs consists of a PFC liquid core (a mixture of perfluoropentane and perfluorohexane), a phospholipid shell and antibody 9E5. The 9E5 human anti-EREG antibody was selected for active targeting of PCNDs. In a preliminary experiment, fluorescent-labelled 9E5 antibody clearly bound to high-EREG-expressing cells, followed by rapid internalisation into intracellular compartments within a few hours ( Supplementary Fig. 1). 9E5 was conjugated to PCNDs using the biotin-streptavidin-biotin binding technique (see Methods). The 9E5 was purified from the peritoneal fluid of mice transplanted with hybridoma cells secreting 9E5. Biotinylated-9E5 and Alexa Fluor 647-conjugated streptavidin (SA-AF647) were bound to biotinylated PCND. The mean diameter of 9E5-conjugated PCNDs was 140 ± 120 nm (Fig. 1c).
Targeting capability and 9E5-conjugated PCND internalisation. Conjugation of 9E5 to PCNDs permits the selective targeting of PCNDs to high-EREG-expressing cells and induces internalisation by endocytosis. The human colonic adenocarcinoma cell line DLD1 and the human gastric cancer cell line AGS were selected as high and low EREG-expressing cancer cell lines, respectively. To demonstrate the selective targeting capability of 9E5-conjugated PCNDs to DLD1 cells, we used SA-AF647 as a probe. The targeted cells were observed by confocal laser scanning microscopy (CLSM), and the number of PCNDs attached and the fraction of bound DLD1 cells were quantitatively measured by flow cytometer. Figure 2a,b shows CLSM observation of antibody-mediated accumulation and internalisation of AF647-labelled 9E5-conjugated PCNDs to DLD1 and AGS cells. The pink fluorescent signals from AF647 were only observed from DLD1 cells ( Supplementary Fig. 2, Iijima et al. in preparation). To further investigate the location of 9E5-conjugated PCNDs, cell membranes were stained with DiO dye, which exhibits green fluorescence. As shown in Supplementary Fig. 3, cell membranes labelled with green and pink fluorescent signals from AF647 were observed inside DLD1 cells (Supplementary Movies 1 and 2). These CLSM images clearly show the internalisation of 9E5-conjugated PCNDs into DLD1 cells, whereas no clear fluorescence signals were observed from the other types of cells. Figure 2c-e shows the results of flow cytometry analysis to evaluate the targeting capabilities of 9E5-conjugated PCNDs toward DLD1 cells (N = 5). Results indicate that the 97.8 ± 0.5% of DLD1 cells were targeted by 9E5-conjugated PCNDs, whereas 1.4 ± 0.3% of DLD1 cells were targeted by non-9E5-conjugated PCNDs, similar to that of the control (4.4 ± 1.6%). Furthermore, the ratio of AGS cells targeted by 9E5-conjugated (8.7 ± 1.0%) and non-9E5-conjugated PCNDs (6.5 ± 1.0%) were close to the levels of the control. Pre-treatment of DLD1 cells with free 9E5 and co-treatment with 9E5-conjugated PCNDs significantly decreased the targeting efficiency of 9E5-conjugated PCNDs (5.4 ± 0.9%), indicating that recognition of EREG by 9E5 antibody plays an important role in the targeting of PCNDs to DLD1 cells (Fig. 2f).
These results show that 9E5-conjugated PCNDs selectively targeted and were internalised by DLD1 cells (high EREG expression) but not AGS cells (low EREG expression). Moreover, PCNDs non-conjugated to 9E5 antibody displayed no targeting capability toward DLD1 cells.
Intracellular vaporisation of 9E5-conjugated PCND.
We showed that the 9E5-conjugated PCNDs accumulate selectively inside DLD1 cells. Next, PCND-accumulated cells were exposed to ultrasound and its cytotoxic effects were visualised. The intracellular vaporisation of 9E5-conjugated PCNDs in DLD1 cells was observed by a high-speed imaging system recording 101 subsequent frames at 1000000 frames per second (fps). Figure 3 shows a typical example of intracellular vaporisation processes. Upon exposure to 100 cycles at 4 MHz of ultrasound at a peak negative pressure of 1.5 MPa, the droplets vaporised and cell membranes were ruptured or broken into several parts during this initial stage of vaporisation ( Supplementary Movies 3 and 4). Finally, the vaporised PCNDs gushed out of the cells, rupturing the cell membranes. High-speed images clearly show that intracellular vaporisation caused a significant disturbance in cell morphology and destroyed the cells. These results are the first direct evidence that ultrasound exposure of cells after PCND uptake dynamically destroys cells by intracellular vaporisation.
Cytotoxic efficacy of vaporised 9E5-conjugated PCND. We showed that ultrasound treatment destroys cancer cells that have accumulated 9E5-conjugated PCNDs. Next, we quantitatively evaluated the cytotoxic capabilities of vaporised PCNDs using the ultrasound exposure system shown in Fig. 4a. Cell viability was measured by flow cytometry. Five cycles at 5 MHz of pulsed ultrasound with a peak negative pressure of 4.6 MPa was applied using an ultrasound imaging probe. Figure 4b-d shows the fraction of viable cells after ultrasound exposure (N = 5). Cell viability was significantly reduced to 43.0 ± 5.6% for 9E5-conjugated PCND-treated DLD1 cells, while there was no significant cell viability decrease for PCNDs without 9E5 conjugation and without ultrasound exposure ( Fig. 4b-g). Furthermore, the viability of AGS cells did not decrease. These data indicate that PCND conjugated with 9E5 can sufficiently kill DLD1 cells with high selectivity. The addition of free 9E5 to DLD1 cells before treating/co-treating with 9E5-conjugated PCNDs significantly increased the number of PI− unstained cells (89.5 ± 10.2%). This result is consistent with the decrease in the number of 9E5-conjugated PCNDs taken up by DLD1 cells (Fig. 2e). It is apparent that overexpression of EREG on the target cell surface and 9E5 are essential for the PCND targeting therapy. It is noteworthy that a conventional low-energy ultrasound imaging probe could be used for such treatment.
The 9E5-conjugated PCNDs attached and internalised 97.8 ± 0.5% of DLD1 cells. However, it should be noted that 43.0 ± 5.6% of the ultrasound-exposed cells were viable. In the aforementioned experiments that evaluate the cytotoxic efficacy of intracellular vaporisation, PI was added immediately after ultrasound sonication because we considered that intracellular vaporisation would cause significant cellular morphological changes and immediate cellular death. However, it is possible that some cells endure intracellular vaporisation and then undergo apoptosis. Thus, the proportion of apoptotic cells was measured by Annexin V and PI dual-staining. Figure 4h,i shows the fraction of apoptotic (PI− ; Annexin V+ ) and necrotic cells (PI+ ) after ultrasound exposure to DLD1 cells treated with or without 9E5-conjugated PCND (N = 3). It is apparent that intracellular vaporisation causes **Indicates p < 0.01 by one-way ANOVA, followed by the Tukey method. (h-j) Flow cytometry analysis of apoptotic and necrotic cells by multi-staining with PI and Annexin V. DLD1 cells treated with (h) and without 9E5-conjugated PCNDs (i) were exposed with ultrasound and then enzymatically harvested from the dishes. (j) After ultrasound exposure, cells detached during exposure were collected from the medium before enzymatic harvesting. The PI-negative and Annexin V-positive cells and double-positive cells were identified as apoptotic and necrotic cells, respectively. necrosis (66.7 ± 3.8%) and not apoptosis (1.4 ± 3.8%). Furthermore, as shown in Fig. 4j, flow cytometry analysis of the supernatant medium showed that most ruptured cells were dead (viability = 0.7 ± 0.1%; N = 3).
Intracellular content release by intracellular vaporisation.
We showed the selective cytotoxic capabilities of vaporised PCNDs. Furthermore, our high-speed images ascertained that the treated cells release their contents into the extracellular space by intracellular vaporisation, which could potentially provoke an inflammatory response. Intracellular content release was determined using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, JP) that produces an orange formazan dye from extracellular lactate dehydrogenase (LDH), a cytoplasmic enzyme present inside the cell. The presence of formazan dye was confirmed by measuring the absorbance at 490 nm using a spectrophotometer (NanoDrop 1000 Spectrophotometer, Thermo Fisher, DE, USA). Figure 5 shows the normalised absorbance at 490 nm after exposing PCND-treated cells to ultrasound (black) and in cells without ultrasound (white). Results indicated that the absorbance at 490 nm, which is proportional to the extracellular LDH activity, of 9E5-conjugated PCND-treated DLD1 cells after ultrasound exposure (0.62 ± 0.19) is at the same level as that of cells treated with lysis buffer (0.61 ± 0.02) and 20-fold higher than that of cells without any treatment (0.03 ± 0.02). Thus, intracellular vaporisation can increase their internal contents into the extracellular space.
Discussion
Antibody-conjugated PCND is a new type of nanomedicine for ultrasound cancer therapy that could be used to treat cancer mechanically by ultrasound with high selectivity. In the present study, 9E5 was conjugated to PCNDs to allow for active targeting and internalisation into DLD1 cells. Both CLSM observations and flow cytometry analysis (Fig. 2) showed the selective targeting capability of 9E5-conjugated PCNDs. Without 9E5 conjugation to PCNDs, neither cell surface attachment nor endocytosis of PCNDs were observed. We achieved excellent targeting capability by conjugation of 9E5 alone, without any external force assistance.
Few studies have reported the conjugation of active targeting molecules to PFC droplets such as aptamer 32 , folate 33,34 and anti-vascular endothelial growth factor receptor 2 antibody with magnetism-assisted targeting 35 in order to direct them to cancer cells. To obtain cytotoxicity with droplet vaporisation, previous studies have combined anti-cancer drugs such as doxorubicin with droplets 32,34 . Marshalek et al. 36 recently demonstrated intracellular delivery and ultrasound activation of intracellular located droplets by decorating folate to the anti-cancer drug-free droplets. However, a cytotoxic effect could not be observed in their study. Ninomiya et al. 37 investigated the targeting ability and cytotoxic effect of anti-cancer drug-free liposomes containing nano-emulsions of perfluoropentane. They modified liposomes with avidin as a targeting ligand for cancer cells and the envelope of hemagglutinating virus of Japan to promote the fusion of liposomes to cells. However, they did not confirm the virus-mediated internalisation of liposomes containing perfluoropentane nano-emulsions toward cancer cells. Moreover, cytotoxic mechanisms were not studied.
Our high-speed imaging shows direct evidence that ultrasound treatment of cells with PCND uptake ruptures the cell membranes by intracellular vaporisation (Fig. 3). Although the location of PCNDs cannot be determined by high-speed imaging, we have confirmed the intracellular location of 9E5-conjugated PCNDs by CLSM observations. Thus, we confirmed that the recorded images are of intracellular vaporisation. This drastic cellular morphological alternation occurred during the initial stages of vaporisation. Therefore, length of the ultrasound duration (number of cycles) would have a less effect on the high-speed imaging results. However, the effect of intracellular vaporisation on surrounding cells would be influenced by the ultrasound parameters because of the alternation in the maximum size of the generated bubbles and oscillation and movement of the bubbles.
Investigations of the selectivity of the cytotoxic effects (Fig. 4) indicate that the decrease in cell viability generated by 9E5-conjugated PCNDs is selective. Ultrasound exposure to the targeted 9E5-conjugated PCNDs significantly decreased the viability of DLD1 cells to 43.0 ± 5.6%. Conversely, PCNDs without 9E5 did not decrease cell viability. Additionally, the cytotoxic effects of 9E5-conjugated PCNDs were not observed in AGS cells, in which EREG expression is relatively low compared to DLD1 cells. To the best of our knowledge, this is the first method reported to mechanically increase cytotoxicity against cancer cells in a highly controllable manner.
One issue that still remains is that the fraction of DLD1 cells targeted by 9E5-conjugated PCND (Fig. 2e) and cell viability after ultrasound exposure (Fig. 4d) were inconsistent. A possible reason is that some portion of intracellular PCNDs remained inert and did not cause a biological effect under the ultrasound exposure conditions. Most cells, in which the vaporisation of PCND occurred, were detached from the dish surface after ultrasound exposure to 9E5-conjugated PCND. Cells that would be included in the supernatant were mostly the necrotic cells, as shown in Fig. 4j (viability = 0.7 ± 0.1%; N = 3). Therefore, we assumed that once vaporisation was achieved, cells could undergo necrosis. Hence, further improvement or optimisation of exposure conditions is required to kill all PCND-incorporated cells, and this will be done in our future research.
We proposed the use of an antibody as a cancer cell-targeting tool in combination with PCND-vaporisation as a nanomedicine for ultrasound cancer therapy. 9E5-conjugated PCNDs have several benefits for cancer treatment owing to their active targeting functionality. Treatment strategies for the proposed ultrasound cancer therapy will not require precise identification of the treatment area owing to the selective adsorption and accumulation of 9E5-conjugated PCNDs inside the target cells. Furthermore, the therapeutic effects are exerted by physical actions, thus avoiding concerns about drug resistance and biological variability between cancer types. This approach can be potentially used to damage any type of cancer cell by selecting the appropriate antibody.
Beyond its use as a dynamic therapeutic agent, it could potentially activate the human-inherent immune system to further aid in the death of cancer cells 38,39 . Our high-speed images and extracellular LDH activity analysis ascertain that the treated cells release their contents into the extracellular space by intracellular vaporisation, which could potentially provoke an inflammatory response 40 . This acute inflammation caused by vaporisation-induced necrosis might improve cancer immunity through antigen presentation 41 . Therefore, 9E5-conjugated PCNDs could be the next generation of both ultrasound-activated dynamic nanomedicine and cancer immunotherapy. The effects of intracellular vaporisation on the immune system must be considered in future studies. It is noteworthy that the proposed ultrasound cancer therapy can be conducted using a conventional ultrasound imaging probe with low energy, as this factor will promote easy clinical translation. Furthermore, it has the potential to treat cancer cells without affecting adjacent normal cells. As shown in Fig. 2a and Supplementary Fig. 3, PCNDs are transported inside cells in a short period of time. The ultrasound-induced vaporisation of intracellular PCNDs allows for high selectivity in cell killing. Hence, it would beneficial for treating cancers such as glioblastoma, in which important tissues are involved. It also might be effective for treating cancers such as peritoneal metastasis and hepatoma, where scattered micro-masses make the conservation of vital organs difficult.
Methods
Preparation of Biotinylated 9E5 antibody. We chose anti-epiregulin antibody (9E5) as the active targeting agent, with the potential to induce internalisation of PCND. 9E5 hybridoma cells were intraperitoneally implanted in BALB/c nude mice, and ascites were obtained and purified on a Protein G column. The 9E5 monoclonal antibody was generated as previously described 26,42 . All animals were maintained in accordance with the regulations set by The University of Tokyo, and all animal experiments were conducted following the institutional guidelines. Animal study protocol was approved by The University of Tokyo (#RAC120101). A solution containing 9E5 (250 μ L) was dialysed against 1 L of 100 mM boric buffer (pH 8.3) at 4 °C overnight, and the concentration of 9E5 in the dialysed solution was estimated by measuring absorbance at 280 nm. A molar extinction coefficient of 220000 M −1 cm −1 was used to calculate the concentration of 9E5. One mM of Sulfo-NHS-activated biotin derivatives (EZ-Link Sulfo-NHS-LC-Biotin, Life Technologies, Carlsbad, CA, USA) in dry DMSO, freshly prepared right before the experiment, was then added to the antibody solution at a final concentration of 5 eq. of 9E5 to conduct biotinylation of the antibody. The reaction took place overnight at 4 °C. To quench the remaining activated biotinylated compound, 100 μ L of 100 mM Tris-acetate (pH 7.0) was added and allowed to react for 1 hour at 4 °C. Finally, biotinylated 9E5 was concentrated by centrifugation using an ultra-membrane filter of 100 kDa MWCO (Amicon Ultra 0.5 mL, Millipore, US); the buffer was exchanged to PBS. The concentration of biotinylated 9E5 was measured as described above.
Preparation of AF647-labelled 9E5-conjugated PCND. The biotinylated-PCNDs (internal composition, (1:1) mixture of perfluoropentane and perfluorohexane) were prepared as described elsewhere 43,44 and provided by Central Research Laboratory Hitachi (Tokyo, JAPAN). The mixture composition ratio of 1:1 was chosen to ensure thermal stability (boiling point calculated to be 40 °C) and sensitivity to activation by ultrasound 44 . Biotinylated-9E5 antibody and Alexa Fluor 647 conjugated streptavidin (SA-AF647) (Life Technologies, US) were mixed in PBS at concentrations of 0.4 μ M each in a total volume of 50 μ L and incubated for 15 min at room temperature to form AF647-labelled 9E5-SA conjugates. The biotinylated PCND dispersion was diluted 20-fold with cold PBS and mixed with the solution containing AF647-labelled 9E5-SA conjugate at a volume ratio of 1:1. The mixture was then incubated for 30 min on ice. The unconjugated 9E5 and SA-AF647 were removed by centrifugation at 3000 × g for 5 min and the supernatant was discarded. The precipitated AF647-labelled 9E5-conjugated PCNDs were dispersed in 100 μ L of cold PBS containing 20% glycerol and centrifuged again under the same conditions as described above to wash PCNDs. This washing step was repeated twice. Finally, the AF647-labelled 9E5-conjugated PCNDs were dispersed in 1 mL of cold RPMI and kept on ice until use. The particle-size distribution of the AF647-labelled 9E5-conjugated PCND suspensions was measured using a laser diffraction particle analyser (LS13320, Beckman Coulter, US). For negative control experiments, non-9E5-conjugated (SA-biotin conjugated) AF647-labelled PCNDs were prepared using biotin instead of biotinylated-9E5 as described above.
Scientific RepoRts | 7:44077 | DOI: 10.1038/srep44077 Cell culture. DLD1 and AGS cells were cultured in 35-mm cell culture dishes at an initial density of 2 × 10 5 cells/dish with RPMI medium supplemented with 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere with 5% CO 2 . All the experiments were conducted one day after seeding on 35-mm culture dishes. The same procedure was used when cells were cultured in the 35-mm glass-bottomed dishes.
Targeting ability: CLSM and flow cytometry analysis. DLD1 and AGS cells were cultured in 35-mm glass-bottomed dishes as described above. Intracellular delivery of 9E5-conjugated PCNDs into DLD1 cells was verified using CLSM (LSM510 META-ConfoCor 3, Carl Zeiss, DE). The use of AF647 probes permits the detection of internalised 9E5-conjugated PCNDs. After a 24-hour incubation, all the culture medium in the dish was aspirated, the cells were washed with PBS and 1 mL of RPMI-diluted 9E5-conjugated PCNDs solution was added to the cells. After a 3-hour incubation with/without 9E5-conjugated PCNDs, all the solutions were removed. Approximately 10 mL of RPMI was added to the dish to fill it with the media. A dish cap was placed on top, and the dish was inverted so that non-attached PCNDs would settle out, since the density of PCND (1.6 g/mL) is higher than that of the media. This washing procedure was repeated twice; lastly, 2 mL of fresh RPMI was added, and cells were observed by CLSM. For cellular membrane labelling, Vybrant Dio (Invitrogen Corporation, Carlsbad, CA) was used (see Supplementary Section 'Cell membrane labelling').
Additionally, the targeting capability of AF647-labelled 9E5-conjugated PCNDs to DLD1 cells was quantitatively measured by flow cytometry (BD FACSCalibur, BD Biosciences, SanJose, CA, USA). DLD1 and AGS cells were cultured in 35-mm cell culture dishes (2 × 10 5 cells/dish). The AF647-labelled 9E5-conjugated PCNDs were introduced to the DLD1 cells in the same manner as for the CLSM observation and analysed by flow cytometer (see Supplementary Section 'Flow cytometry').
As additional negative controls for the experiments, the cells with non-9E5-conjugated AF647-labelled PCNDs and those without PCNDs (cells alone) were incubated in the same manner for both CLSM observation and flow-cytometry analysis. Furthermore, we considered that AF647-labelled 9E5-conjugated PCND uptake might be inhibited by the addition of free 9E5 to DLD1 cells, free 9E5 should bind to EREG and block the binding of AF647-labelled 9E5-conjugated PCNDs. Hence, 1 mL of 0.5 μ M 9E5 antibody solution was added to the DLD1 cells and incubated for 30 min before adding AF647-labelled 9E5-conjugated PCNDs.
High-speed imaging of intracellular vaporisation. Intracellular vaporisation of 9E5-conjugated
PCNDs was observed using the high-speed imaging system shown in Fig. 3. A custom-made, nose-cone-shaped wave guide 45 mounted 4-MHz single element piezoelectric transducer with a focal length of 25 mm (17.5 × 7.9 mm, Type C213, Fuji Ceramics, JP) was developed to enable sonication to DLD1 cells during high-speed imaging. The transducer was connected to a multi-function generator (WF1974, NF Corporation, JP) and a 50-dB broadband amplifier (2100 L, Electronics & Innovation, US). The wave number was set to 100 cycles at 4 MHz (pulse duration, 25 μ s). The waveguide was filled with ultrasonic gel, and the nose was fully covered with a paraffin film to ensure ultrasound propagation through the waveguide. DLD1 cells were exposed to a peak negative pressure of 1.5 MPa. The multi-function generator was also connected to a high-speed imaging camera (Hyper Vision HPV-1, Shimadzu, JP) coupled with an inverted microscope (Eclipse Ti-U, Nikon, JP) equipped with an oil immersion 60× objective lens to synchronise the sonication and the recording of images by high-speed camera. A super high-pressure mercury lamp (C-LHG1, Nikon, JP) connected to a power supplier (C-SHG1, Nikon, JP) was used for the illumination light source. The high-speed camera was set to a frame rate of 0.25, 1Mfps and an exposure time of 0.5 μ s. A total of 101 subsequent frames was recorded, and the image resolution was 312 × 260 pixels. The 9E5-conjugated PCND-targeted DLD1 cells were prepared in glass-bottom dishes and placed on top of the objective lens. The transducer was positioned by a three-axis motorised stage, and the angle was set to 45° with a custom-made holder. PBS was added inside the dish until the tip of the waveguide was fully covered.
Flow cytometry analysis of cytotoxic efficacy. The cytotoxic efficacy of vaporisation of 9E5-conjugated PCNDs was investigated in cultured DLD1 and AGS cells using the experimental system shown in Fig. 4a. An ultrasound imaging probe (EUP-L73S, Hitachi Aloka Medical, JP) connected to a programmable ultrasound imaging system (V1, Verasonics, Kirkland, WA, USA) was used for delivering vaporisation pulses to the PCNDs. The cell culture dishes were positioned 36 mm from the transmission surface of the ultrasound imaging probe with a custom-made dish holder connected to a single axis motorised stage (ALZ-115-E1P, Chuo Precision Industrial, JP). The pulse length was set to 5 cycles at 5 MHz with a peak negative pressure of 4.6 MPa (measured at the bottom of the dish), and the focus of the transmitted ultrasound was set at 38 mm from the transmission surface of the ultrasound imaging probe. Such short pulses were used to avoid unintended cavitation that damages cells non-specifically. The acoustic profile measured at the bottom of the dish is shown in Supplementary Fig. 4. The focus was electronically scanned from − 1.20 mm to 1.20 mm (centre of the probe was set to 0 mm) with a 0.20-mm pitch in the lateral direction of the probe; the pulse repetition frequency was set to 0.5 kHz. These sonication conditions are known to achieve successful vaporisation of PCNDs 31 . The probe was positioned by a two-axis motorised stage (ALD-604-E1P, Chuo Precision Industrial, JP) with a 0.30-mm pitch in the elevational direction and 1.20-mm pitch in the lateral direction, both at an interval of 100 ms. Trigger signals were transmitted to the programmable ultrasound imaging system at each position to expose 6 sets of the above-mentioned vaporisation pulses. A water tank was filled with degassed water, and the temperature was maintained at 37 °C. DLD1 and AGS cells were cultured in 35-mm cell culture dishes at a density of 2 × 10 5 cells/dish. The 9E5-conjugated PCNDs were introduced to the cells in the same manner as described for the targeting capability measurements. As a negative control for the experiments, cells were incubated with non− 9E5-conjugated AF647-labelled PCNDs and without PCNDs. All the samples were tested with and without ultrasound sonication. Furthermore, for DLD1 cells treated with AF647-labelled 9E5-conjugated PCNDs, EREG blocking was Scientific RepoRts | 7:44077 | DOI: 10.1038/srep44077 conducted in the same manner as described above. PI (25535-16-4, Dojindo Laboratories, JP) was added to the dishes just after the sonication, resulting in a final concentration of 1 μ L/mL. Cell viabilities were quantitatively measured by flow cytometry (see Supplementary Section 'Flow cytometry'). PI is used to distinguish between viable and non-viable cells.
The proportion of apoptotic cells was measured by Annexin V and PI dual-staining using the Annexin V-FITC Apoptosis Detection Kit (BioVision Incorporated, CA, USA). DLD1 cells treated with or without 9E5-conjugated PCNDs were exposed to ultrasound. The supernatant was collected in a tube after ultrasound exposure. To remove adhered cells, 1 mL of PBS was added to the dish and added to the same tube containing the supernatant and 100 μ L 0.25% EDTA-trypsin solution was added to the dishes. After 3 min of incubation, 2 mL of PBS was added, and all the medium was collected in the same tube. The tube was centrifuged at 1400 rpm for 3 min, and the supernatant was aspirated. The pellet was resuspended in 500 μ L of binding buffer (BioVision Incorporated, CA, USA), and 5 μ L of Annexin V-FITC and PI was added. After 5 min of incubation at room temperature, the samples were kept on ice until just before the measurement. For supernatant viability measurement, trypsinisations were excluded. Intracellular content release: LDH activity analysis. Intracellular content release was determined using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, JP). Extracellular LDH activity was quantified using a spectrophotometer (NanoDrop 1000 Spectrophotometer, Thermo Fisher, DE, USA) by measuring the absorbance of formazan dye at 490 nm. DLD1 cells were cultured in 35-mm cell culture dishes at a density of 2 × 10 5 cells/dish. The 9E5-conjugated PCNDs were introduced to the cells in the same manner as that described for the targeting capability measurements, and the cells were exposed to ultrasound in the same manner as that described for the cytotoxicity measurements. The supernatant was collected in a tube after ultrasound exposure. The tube was centrifuged at 1400 rpm for 3 min, and 100 μ L of supernatant was mixed with 100 μ L of the assay medium. After 30 min of incubation at room temperature without light exposure, 50 μ L of stop solution was added and the absorbance at 490 nm was measured using the spectrophotometer. As additional negative controls for the experiments, the cells without PCNDs (cells alone) were incubated in the same manner for both CLSM observation and flow cytometry analysis. All the samples were tested with or without ultrasound sonication. As additional positive controls for the experiments, the cells were treated with lysis buffer (Dojindo Laboratories, JP). All the culture medium in the dish was aspirated, the cells were washed with RPMI, and 2 mL of RPMI-diluted lysis buffer (RPMI = 1.8 mL; lysis buffer = 0.2 mL) was added to the cells. After 30 min of incubation at 37 °C in a humidified atmosphere with 5% CO 2 , an assay medium was added in the same manner as that described above.
Statistical analysis. Analysis of variance (ANOVA) was used to establish the significance between the different experimental groups. The Tukey method was applied to evaluate the significance of differences between the groups. | v3-fos-license |
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} | pes2o/s2orc | Removal of Organic Micropollutants from a Municipal Wastewater Secondary Effluent by UVA-LED Photocatalytic Ozonation
Numerous contaminants of emerging concern (CECs) have been found in different water bodies. Directive 2013/39/EU and Decision 2018/840/EU are consequently being implemented in the field of water policies. Twelve CECs (e.g., isoproturon, ciprofloxacin, and clarithromycin are among those listed) were detected in a municipal wastewater secondary effluent by means of solid phase extraction and ultra-high-performance liquid chromatography with tandem mass spectrometry (SPE-UHPLC-MS/MS). Different advanced oxidation processes (AOPs), based on the combination of ozone, UVA-LED and powdered TiO2, were investigated for their removal in a semi-batch operation. In addition, TiO2-coated glass rings (P25R) were characterized with different techniques (SEM, WDXRF) and used for continuous mode operation in a packed bed reactor (PBR). Among the AOPs studied, ozone-based processes were found to be more efficient than heterogeneous photocatalysis. A kinetic study was performed showing that direct ozonation is the main oxidation pathway for CEC removal. Ozone was successfully decomposed in combination with UVA-LED and P25R, resulting in an apparent rate constant of 3.2 × 10−2 s−1 higher than in the O3/LED system (1.0 × 10−3 s−1) or with ozone alone (8.6 × 10−5 s−1). Hydroxyl radical reaction could prevail over direct ozone reaction for the most refractory compounds (e.g., isoproturon).
Introduction
Contaminants of emerging concern (CECs) are continuously discharged into aquatic systems with little or no awareness of their consequences. Pharmaceuticals, pesticides, industrial compounds, natural hormones, and personal care products belong to this group of substances. Although their concentration levels are very low, usually in the ng·L −1 or µg·L −1 range [1][2][3], biological acute and chronic toxicity in aquatic organisms has been observed [4,5].
To date, the European Union (EU) has established a list of priority substances (Directive 2013/39/EU) and a watch list of CECs that have to be monitored to guarantee water quality (Decision 2015/495/EU, Decision 2018/840/EU) [6][7][8]. Other CECs are most likely to be included in the watch list in the near future.
Since CECs are difficult to remove by conventional treatments of municipal wastewater treatment plants (MWWTPs), alternative treatment methods such as adsorption, membrane separation, photocatalysis, or ozonation are being investigated [9,10]. Among the oxidation processes applied in this work, ozonation has been shown to be a very effective treatment due to ozone direct reactions with some organics and ozone decomposition (mainly at basic pH) into hydroxyl radical (HO • ), a strong non-selective oxidant radical oxygen species (ROS) [11]. However, decomposition of ozone in water also involves a series of reactions which prevent other less reactive ROS from being able to degrade most of the CECs. To enhance the efficiency of ozonation processes for the degradation of CECs, ozone may be used in combination with certain catalysts and/or radiation sources to promote ozone decomposition into HO • [12,13]. Reaction mechanisms of these oxidation processes are well known in literature [13] (see main reaction steps of these mechanisms in section S1 of the supplementary information).
In this sense, light emitting diodes (LEDs) represent an attractive and cost-effective alternative because of their long lifetime and high-energy efficiency. Regarding photocatalysts, TiO 2 in powder form has so far been the most commonly used material in photocatalytic oxidation of CECs. However, given the difficult separation of nanosized powered TiO 2 , a great deal of research is recently devoted to the use of immobilized TiO 2 [14].
This work focuses on the removal of some CECs detected in municipal secondary wastewater collected in an MWWTP in Northern Portugal. Combinations of ozone, UVA-LEDs, and P25 TiO 2 (Evonik P25), both in powdered form (P25) and immobilized on glass rings (P25R), were tested as treatment approaches. Two modes of ozonation operation were also investigated: semi-batch (P25 as catalyst) and continuous flow (P25R as catalyst). Some CECs examined in this study (e.g., isoproturon, ciprofloxacin, and clarithromycin) are among those included in Directive 2013/39/EU and Decision 840/2018/EU. The main objectives of this work were: (i) to assess efficiencies of semi-batch and continuous processes and (ii) to determine the importance of direct ozonation and HO • mediated-reactions in the degradation of the CECs.
CECs Detected in Secondary Effluent Samples
Two wastewater (WW) samples (namely WW1 and WW2) were collected at the outlet of the secondary settling tank of an MWWTP in Northern Portugal (see Table S1 for main WW characteristics). WW1 was used in semi-batch experiments and WW2 in continuous flow runs. Forty-five CEC candidates were analyzed by the solid phase extraction and ultra-high-performance liquid chromatography with tandem mass spectrometry (SPE-UHPLC-MS/MS) validated method (Table S2) and twelve compounds were found in both WW1 and WW2 samples (Table 1): the herbicide isoproturon, and eleven pharmaceutical compounds (bezafibrate, carbamazepine, ciprofloxacin, clarithromycin, clopidogrel, diclofenac, fluoxetine, metoprolol, propranolol, tramadol, and venlafaxine).
While clopidogrel was detected at concentrations below the quantification limit, carbamazepine, ciprofloxacin, diclofenac, isoproturon, tramadol, and venlaxafine were found at µg·L −1 concentration level, thus confirming that the conventional treatment applied at the MWWTP is not efficient enough to eliminate these micropollutants. Of special concern are isoproturon (included in Directive 2013/39/EU), ciprofloxacin (recently added to the latest 2018/840/EU Decision), clarithromycin (included in the first Decision 2015/495/EU and maintained in Decision 2018/840/EU) (Table S2). <MQL.: below method quantification limit. As can be observed, O 2 /LED/P25 (i.e., heterogeneous photocatalysis) barely reached 15% removal of any CEC. In contrast, elimination percentages achieved by ozonation processes were above 90% in all cases except for ciprofloxacin, metoprolol, and propranolol. For ciprofloxacin, percentage removals were 56%, 62%, and 100% by single ozonation, photolytic ozonation, and photocatalytic ozonation, respectively. For the beta-blockers propranolol and metoprolol, which were detected at much lower concentrations, random trends were observed for the different processes and removal efficiencies were much lower (for instance, 43% and 16% were respectively achieved with photocatalytic ozonation).
Continuous Flow Experiments
A preliminary residence time distribution (RTD) analysis was carried out to model flow pattern in the packed photo-reactor used for continuous flow experiments. Two possible scenarios were hypothesized regarding the flow model to be used ( Figure S3): a) a perfectly mixed reactor and b) two perfectly mixed reactors in series corresponding to the column reactor and the recirculation With regard to the substances on the priority and watch lists, isoproturon and clarithromycin were completely removed by the three ozone-based processes, whereas ciprofloxacin was totally removed by photocatalytic ozonation. The effect is strongly marked due to its higher concentration in WW1. Similar trends are seen in particular for those CECs whose concentrations were above 1000 ng·L −1 (Table 1).
To ascertain the effect of suspended matter on removal efficiency of CECs, a series of runs with filtered WW1 samples were carried out. As has been reported [15], filtration did not enhance the performance of these processes in terms of removal of CECs (see Figure S1).
Continuous Flow Experiments
A preliminary residence time distribution (RTD) analysis was carried out to model flow pattern in the packed photo-reactor used for continuous flow experiments. Two possible scenarios were hypothesized regarding the flow model to be used ( Figure S3): a) a perfectly mixed reactor and b) two perfectly mixed reactors in series corresponding to the column reactor and the recirculation column. Figure S4 shows the experimental F curve obtained together with the calculated F curves inferred from the two assumed models. The experimental F curve perfectly agrees with the calculated one corresponding to a perfectly mixed reactor. Taking this information into account, the mean residence time was expected to be 39 min (see Section S2 of Supporting Materials for details). (Table S3) show that ca. 0.16 wt. % of TiO 2 was immobilized on the glass rings. This composition was similar in fresh P25R, and after the photocatalytic oxidation and photocatalytic ozonation processes. Figure 2 presents the results obtained in terms of removal of CECs (%), with the exception of bezafibrate, which was not considered in this part of the study due to its very low concentration in this sample (WW2), which was close to the method quantification limit. Photolysis led to different degrees of removal depending on the specific CEC: from no removal at all (in the case of clarithromycin and ciprofloxacin) to a maximum removal of 37% for fluoxetine. Photocatalytic oxidation improved these figures to some extent. With this system, ciprofloxacin reached nearly 100% elimination, though removals of the other CECs were moderate, between 58% for carbamazepine, and approximately 28% for propranolol and clarithromycin. In agreement with the results obtained in semi-batch experiments, the highest removals were obtained with the ozone-based processes (O 3 , O 3 /LED, and O 3 /LED/P25R), for which removal percentages of more than 90% were observed in all the cases. Photocatalytic ozonation was particularly effective with removal of any CEC with percentages higher than 99.9%, except for carbamazepine with 99.2% elimination.
SUVA 254nm (specific ultraviolet absorbance at 254 nm) is an important variable to be measured in wastewater since it can be considered a surrogate parameter of the presence of aromatic and unsaturated compounds [16]. Figure 3 shows the values of SUVA 254nm measured at steady state of continuous runs. A decrease in SUVA 254nm was found as a result of the application of ozone-based processes, especially those involving light, whereas photocatalytic oxidation had no effect on this parameter. Conversely, WW treated with UVA (no catalyst or ozone added) led to an increase in SUVA 254nm . In this case, some intermediates can be formed, which would absorb more radiation as a consequence of their direct photolysis. When both light and catalyst are simultaneously applied, this effect disappears probably due to the formation of HO • . In ozone-based processes, the fast reaction of this sample (WW2), which was close to the method quantification limit. Photolysis led to different degrees of removal depending on the specific CEC: from no removal at all (in the case of clarithromycin and ciprofloxacin) to a maximum removal of 37% for fluoxetine. Photocatalytic oxidation improved these figures to some extent. With this system, ciprofloxacin reached nearly 100% elimination, though removals of the other CECs were moderate, between 58% for carbamazepine, and approximately 28% for propranolol and clarithromycin. In agreement with the results obtained in semi-batch experiments, the highest removals were obtained with the ozone-based processes (O3, O3/LED, and O3/LED/P25R), for which removal percentages of more than 90% were observed in all the cases. Photocatalytic ozonation was particularly effective with removal of any CEC with percentages higher than 99.9%, except for carbamazepine with 99.2% elimination. Results previously reported of CECs removal in secondary urban wastewater doped at some mgL −1 , [17][18][19][20] also show similar CECs elimination rates when different ozone processes are applied. This confirms that, as far as CECs removal is concerned, direct ozone reactions are likely the main responsible mechanism of oxidation (see later Section 2.4). For instance, Encinas et al. [17] studied a mixture of nine CECs (metoprolol and diclofenac among them) in a secondary urban wastewater with O 2 /UVA/TiO 2 , O 3 , and O 3 /UVA/TiO 2 systems. CECs, doped at concentrations of 10 mgL −1 were removed with similar rates in less than 30 min in ozone processes while photocatalytic oxidation took 120 min to reduce CECs concentration in 20-30%. In another work, this time with solar radiation, Marquez et al. [18] studied the removal of four CECs (atenolol, hydrochlorotiazide, ofloxacine, and trimethoprim) in a secondary urban wastewater. The advanced oxidation process (AOP) applied were O 2 /Sun/TiO 2 , O 3 , O 3 /TiO 2 , O 3 /Sun, and O 3 /Sun/TiO 2 . With CEC concentrations of 10 mgL −1 and ozone dose of 20 mgL −1 ozone processes needed less than 25 min for total removal of CECs while O 2 /Sun/TiO 2 required more than 3 h with the exception of ofloxacin that needed 2 h. It should be noted that in these works [17][18][19][20], due to analytical equipment limitations, total removal of CECs was reducing their concentration below the quantification limit that was some µgL −1 .
processes, especially those involving light, whereas photocatalytic oxidation had no effect on this parameter. Conversely, WW treated with UVA (no catalyst or ozone added) led to an increase in SUVA254nm. In this case, some intermediates can be formed, which would absorb more radiation as a consequence of their direct photolysis. When both light and catalyst are simultaneously applied, this effect disappears probably due to the formation of HO • . In ozone-based processes, the fast reaction of ozone and HO • with unsaturated moieties present in the wastewater could explain the SUVA254nm decrease. Decrease of SUVA254nm with time clearly shows the decrease of intermediate concentrations and the beneficial effects of ozone processes application compared to photocatalytic oxidation. On the other hand, both pH and dissolved organic carbon (DOC) remain practically unaltered (not shown). Results previously reported of CECs removal in secondary urban wastewater doped at some mgL −1 , [17][18][19][20] also show similar CECs elimination rates when different ozone processes are applied. This confirms that, as far as CECs removal is concerned, direct ozone reactions are likely the main responsible mechanism of oxidation (see later section 2.4). For instance, Encinas et al. [17] studied a mixture of nine CECs (metoprolol and diclofenac among them) in a secondary urban wastewater with O2/UVA/TiO2, O3, and O3/UVA/TiO2 systems. CECs, doped at concentrations of 10 mgL −1 were removed with similar rates in less than 30 min in ozone processes while photocatalytic oxidation took 120 min to reduce CECs concentration in 20%-30%. In another work, this time with solar radiation, Marquez et al. [18] studied the removal of four CECs (atenolol, hydrochlorotiazide, ofloxacine, and trimethoprim) in a secondary urban wastewater. The advanced oxidation process (AOP) applied were O2/Sun/TiO2, O3, O3/TiO2, O3/Sun, and O3/Sun/TiO2. With CEC concentrations of 10 mgL −1 and ozone dose of 20 mgL −1 ozone processes needed less than 25 min for total removal of CECs while O2/Sun/TiO2 required more than 3 h with the exception of ofloxacin that needed 2 h. It should be According to results of Figures 1 and 2 the presence of TiO 2 and LED in an ozone process only adds a small advantage to remove some CECs. However, contributions of simultaneous ozone, radiation, and TiO 2 application is deduced from the higher elimination of total organic carbon (TOC) as reported in previous works [17][18][19][20]. In these works, TOC could be followed because of the higher concentrations of initial CECs (doped at mgL −1 ). To give an example, in the work of Marquez et al. [18], above mentioned, application of O 3 and O 3 /Sun/TiO 2 systems lead to 38% and 80% TOC removed, respectively, after 3 h treatment in ultrapure water. In our work, TOC measurements were mainly due the water matrix components because of TOC contribution of CECs present was negligible. Then, no conclusion can be made about TiO 2 and LED addition to improve TOC removal of CECs. Another advantage of photocatalytic ozonation, related to the economy of the process, is the lower consumption of ozone to remove a given amount of organic carbon compared to ozonation. For instance, Espejo et al. [19] reported a consumption of 36 and 17 mg O 3 /mg TOC with O 3 and O 3 /UVA/Fe 2 O 3 systems, respectively, after 30 min reaction, corresponding to the treatment of nine CECs in a secondary urban wastewater.
Kinetic Modelling Aspects
This section focuses only on ozone processes since these were the most efficient in removing the CECs. According to the literature, contaminants are mainly removed on reacting with ozone and HO • [11]. Given that the reacting system behaves as a continuous stirred tank reactor (CSTR), the mass balance of each CEC is given by Equation (1): where t m is hydraulic residence time (HRT), k D and k HO are the rate constants of the reactions of ozone and HO • with each CEC (see Table 2), C CECo is the inlet CEC concentration and C CEC , C O3 , and C HO are the outlet concentrations of CEC, ozone, and HO • , respectively.
Application of Equation (3) to the experimental results should permit determination of k T for each contaminant. However, this was only possible for some cases of single ozonation or photolytic ozonation, where full conversion of some CECs was not achieved (Figure 2), namely propranolol (ozonation, 90%, or photolytic ozonation, 93% conversions) and isoproturon (photolytic ozonation, 94% conversion). For these CECs, values of k T were 486, 1653, and 1736 M −1 ·s −1 , respectively. These values are lower than those reported in the literature for the rate constants of the ozone direct reactions (k D ) ( Table 2). These apparently contradictory results suggest that direct ozonation is the main mechanism for the removal of these contaminants. This agrees with the fact that ozone-based processes lead to similar conversions, regardless of the use of radiation and/or catalyst (see Figure 2). In addition, this conclusion was confirmed, as shown later, through determination of the ozone kinetic regimes and comparison of the oxidation rates of a given CEC with ozone and HO • .
Because of the high conversions of CECs reached in the continuous ozone-based processes, the mass balance of CEC was also applied to semi-batch experiments. The CEC mass balance in this case leads to Equation (4): Integration of Equation (4) after variable separation gives: According to Equation (5), k T can be obtained from the ratio between the left-hand side of Equation (5) and the ozone exposure ( C O3 dt). Nevertheless, Equation (5) could only be applied to ciprofloxacin, metoprolol, propranolol, and venlafaxine, owing to the fact that conversions lower than 95% were Catalysts 2019, 9,472 8 of 16 achieved ( Figure 1). As observed, values of k T obtained (Table 3) are also lower than those of k D reported for the different ozone-contaminant reactions, which leads us to the conclusion that direct reactions are mainly responsible for contaminant removals in the WW samples studied. Table 3. Apparent rate constant values of the ozonation process in semi-continuous operation. Following recently reported reasoning [33], kinetic regimes of ozone reactions with the contaminants studied and ozone initiation reaction for the formation of HO • were evaluated. This required the determination of the Hatta number (Ha) of these reactions ( Table 2). For second order irreversible reactions, such as those between ozone and the CECs, Ha D is defined as: where D O3 is ozone diffusivity in the liquid phase and k L is the individual liquid phase mass-transfer coefficient at the reacting conditions. According to Johnson and Davis (1996), ozone diffusivity in water is 1.5 × 10 −9 m 2 ·s −1 while a value of 10 −3 m·s −1 was given for k L [31,32].
On the other hand, Ha for the initial ozone decomposition reaction into HO • , a pseudo first order irreversible reaction, is calculated as follows: In this case, k d is the corresponding rate constant which varies according to the nature of the ozone process. Thus, for single ozonation, k d = 70 × 10 pH−14 s −1 , while for photolytic and photocatalytic ozonation apparent k d values were experimentally determined in this work. For that purpose, a series of continuous flow runs were performed in the absence of CECs. The ozone mass balance at steady-state is given by Equation (8): β being the liquid hold-up (0.98), V the total reaction volume, k L a the volumetric mass-transfer coefficient, C O3 * and C O3 the ozone concentrations in the interphase and in the liquid bulk, respectively, and ν o the liquid flow rate. Figure 4 shows the aqueous ozone concentration measured in some ozone absorption experiments in continuous mode. The dissolved ozone concentration reached at the steady-state of the single ozone absorption run (8.3 mg·L −1 ) was taken as the solubility (C O3 *) to be used in Equation (8). From Figure 4 it is apparent that radiation (LED) and, especially, the combination of LED and P25R, enhanced decomposition of the ozone.
According to Charpentier (1981) [31], values of k L a in packed bubble columns are between 0.005 and 0.12 s −1 , giving as a result different k d values for both photolytic and photocatalytic ozonation as shown in Table 4. coefficient, CO3* and CO3 the ozone concentrations in the interphase and in the liquid bulk, respectively, and νo the liquid flow rate. Figure 4 shows the aqueous ozone concentration measured in some ozone absorption experiments in continuous mode.
The dissolved ozone concentration reached at the steady-state of the single ozone absorption run (8.3 mg·L −1 ) was taken as the solubility (CO3*) to be used in Equation (8). From Figure 4 it is apparent that radiation (LED) and, especially, the combination of LED and P25R, enhanced decomposition of the ozone. According to Charpentier (1981) [31], values of kLa in packed bubble columns are between 0.005 and 0.12 s −1 , giving as a result different kd values for both photolytic and photocatalytic ozonation as shown in Table 4. Taking into account the maximum values of k d constants, the values of Ha I for the initial ozone decomposition into free radicals at the pH of WW from Equation (7) were all found to be lower than 7.0 × 10 −3 (taking the minimum k L a) or 3.4 × 10 −2 (maximum k L a) which, in any case, indicates slow kinetic regimes.
Values of Ha D for the ozone direct reactions with the contaminants studied here were calculated from Equation (6), and are also presented in Table 2. As can be observed, Ha D is always lower than 0.1, which indicates a slow kinetic regime for these reactions. Since both ozone reactions (direct and decomposition) develop in the same kinetic regime, there could be potential competition between them for consuming ozone. In order to clarify this point, and following the procedure described in a previous work [33], the ratio between reaction rates of contaminant removal with HO • and ozone was determined with Equation (9): C s and k HO being the concentration of any other substances present in water that scavenge HO • and the rate constant of this reaction, respectively. Equation (9) expressed in its logarithmic form [29] is: Reaction rate ratio (r HO /r O3 ) strongly depends on the scavenging factor ( C s k HO ). This factor was calculated from DOC and inorganic carbon (IC) values in wastewater as reported by Nöthe et al. (2009) [34]. Thus, for WW1 and WW2 values of C s k HO were found to be 6.7 × 10 5 s −1 and 8.2 × 10 5 s −1 , respectively Figure 5 shows the plot of the reaction rate ratio versus the rate constant ratio of contaminant-hydroxyl radical and ozone reactions in the ozonation processes. It can be seen from Figure 5 that in all cases the ozone direct reaction rate is much higher than the HO • reaction rate with the contaminant, even for compounds 1,8,9,11, and 12 (bezafibrate, isoproturon, metoprolol, tramadol, and venlafaxine), when treated with O 3 /LED/P25R. This confirms that the direct reaction prevails over the hydroxyl radical reaction. However, these compounds (1,8,9,11, and 12) treated with the O 3 /LED/P25R system with good mass-transfer coefficient (k L a = 0.12 s −1 ) would be removed mainly by hydroxyl radicals. In summary, the importance of free radical reactions depends on the presence of scavengers in the wastewater to treat. The increase of concentrations of these substances will make the free radical reactions less important compared to the direct ozone reaction and this will depend on the type of AOP. It can be said that the higher the number of hydroxyl radical formation ways, the higher the importance of free radical reactions compared to direct ozone reactions (see Section S1 for initiation reactions of hydroxyl radical formation in AOPs studied). Table 1 for meaning of numbers (Plots based on reference [29]).
Materials and Methods
Chemicals and solvents (>95% purity) were purchased from different companies and ultrapure water was supplied by a Milli-Q water system. TiO2 (P25, 80% anatase and 20% rutile crystalline phases) was obtained from Evonik Degussa GmbH. All reference standards (>98% purity) and the isotopically labeled compounds used as internal standards were purchased from Sigma-Aldrich (Steinheim, Germany).
Wastewater Effluents
Secondary wastewater (WW) samples were collected from a MWWTP located in Northern Portugal and frozen until further use. The main physicochemical parameters are summarized in Table S1, namely pH, dissolved organic carbon (DOC) and inorganic carbon (IC).
Photocatalysts
Titanium dioxide powder (P25) was used as photocatalyst in semi-batch experiments, and TiO2coated glass rings (P25R) were used in continuous flow runs. P25R was prepared as described elsewhere [35]. Briefly, glass rings were immersed in a 5% w/v P25-ethanolic solution at a constant rate of 30 mm/min, in order to create a homogeneous TiO2 layer. The coated rings were then dried overnight. Since three layers of P25 have been shown to be the optimal number for photocatalytic treatment of water pollutants, the process was repeated two more times and finally P25R were calcined in air at 450 °C for 2 h [35]. Table 1 for meaning of numbers (Plots based on reference [29]).
Materials and Methods
Chemicals and solvents (>95% purity) were purchased from different companies and ultrapure water was supplied by a Milli-Q water system. TiO2 (P25, 80% anatase and 20% rutile crystalline phases) was obtained from Evonik Degussa GmbH. All reference standards (>98% purity) and the isotopically labeled compounds used as internal standards were purchased from Sigma-Aldrich (Steinheim, Germany).
Wastewater Effluents
Secondary wastewater (WW) samples were collected from a MWWTP located in Northern Portugal and frozen until further use. The main physicochemical parameters are summarized in Table S1, namely pH, dissolved organic carbon (DOC) and inorganic carbon (IC).
Photocatalysts
Titanium dioxide powder (P25) was used as photocatalyst in semi-batch experiments, and TiO2coated glass rings (P25R) were used in continuous flow runs. P25R was prepared as described elsewhere [35]. Briefly, glass rings were immersed in a 5% w/v P25-ethanolic solution at a constant rate of 30 mm/min, in order to create a homogeneous TiO2 layer. The coated rings were then dried overnight. Since three layers of P25 have been shown to be the optimal number for photocatalytic treatment of water pollutants, the process was repeated two more times and finally P25R were calcined in air at 450 °C for 2 h [35]. Table 1 for meaning of numbers (Plots based on reference [29]).
Materials and Methods
Chemicals and solvents (>95% purity) were purchased from different companies and ultrapure water was supplied by a Milli-Q water system. TiO2 (P25, 80% anatase and 20% rutile crystalline phases) was obtained from Evonik Degussa GmbH. All reference standards (>98% purity) and the isotopically labeled compounds used as internal standards were purchased from Sigma-Aldrich (Steinheim, Germany).
Wastewater Effluents
Secondary wastewater (WW) samples were collected from a MWWTP located in Northern Portugal and frozen until further use. The main physicochemical parameters are summarized in Table S1, namely pH, dissolved organic carbon (DOC) and inorganic carbon (IC).
Photocatalysts
Titanium dioxide powder (P25) was used as photocatalyst in semi-batch experiments, and TiO2coated glass rings (P25R) were used in continuous flow runs. P25R was prepared as described elsewhere [35]. Briefly, glass rings were immersed in a 5% w/v P25-ethanolic solution at a constant rate of 30 mm/min, in order to create a homogeneous TiO2 layer. The coated rings were then dried overnight. Since three layers of P25 have been shown to be the optimal number for photocatalytic treatment of water pollutants, the process was repeated two more times and finally P25R were calcined in air at 450 °C for 2 h [35].
) in comparison with the maximum k L a ( and ) and semi-batch mode (O 3 ). See Table 1 for meaning of numbers (Plots based on reference [29]).
Materials and Methods
Chemicals and solvents (>95% purity) were purchased from different companies and ultrapure water was supplied by a Milli-Q water system. TiO 2 (P25, 80% anatase and 20% rutile crystalline phases) was obtained from Evonik Degussa GmbH. All reference standards (>98% purity) and the isotopically labeled compounds used as internal standards were purchased from Sigma-Aldrich (Steinheim, Germany).
Wastewater Effluents
Secondary wastewater (WW) samples were collected from a MWWTP located in Northern Portugal and frozen until further use. The main physicochemical parameters are summarized in Table S1, namely pH, dissolved organic carbon (DOC) and inorganic carbon (IC).
Photocatalysts
Titanium dioxide powder (P25) was used as photocatalyst in semi-batch experiments, and TiO 2 -coated glass rings (P25R) were used in continuous flow runs. P25R was prepared as described elsewhere [35]. Briefly, glass rings were immersed in a 5% w/v P25-ethanolic solution at a constant rate of 30 mm/min, in order to create a homogeneous TiO 2 layer. The coated rings were then dried overnight. Since three layers of P25 have been shown to be the optimal number for photocatalytic treatment of water pollutants, the process was repeated two more times and finally P25R were calcined in air at 450 • C for 2 h [35].
Experimental Set-Up and Procedures
Semi-batch and continuous flow experiments were carried out in this study. Semi-batch mode runs were performed using the experimental set-up schematically shown in Figure 6. As can be seen, the reactor was enclosed in a square box with four 10 W UVA LEDs. Each LED (with maximum irradiance at 390 nm, Figure S2) was provided with a fan for refrigeration purposes. For ozonation runs, ozone was produced from pure oxygen in a BMT 802X ozone generator, and its concentration was monitored with a BMT 964 analyzer. First, the reactor was charged with 750 mL of WW and 0.375 g of P25 (i.e., catalyst concentration of 0.5 g·L −1 ). The mixture was kept under magnetic agitation in the dark for 10 min to achieve adsorption equilibrium of CECs. Then the LEDs were switched on and a gas stream (150 mL·min −1 ) of either pure oxygen or an ozone-oxygen mixture (50 mg·L −1 ozone) was bubbled through a ceramic diffuser. After 10 min of reaction, the residual ozone (if ozone was applied) was removed by using an air pump and samples were withdrawn from the reactor to analyze the concentrations of CECs and DOC. In addition to photocatalytic oxidation runs, single ozonation (in the absence of P25 and with LEDs turned off) and photolytic ozonation (in the absence of P25) experiments were carried out for comparative purposes.
Catalysts 2019, 9, x FOR PEER REVIEW 12 of 17 a gas stream (150 mL·min −1 ) of either pure oxygen or an ozone-oxygen mixture (50 mg·L −1 ozone) was bubbled through a ceramic diffuser. After 10 min of reaction, the residual ozone (if ozone was applied) was removed by using an air pump and samples were withdrawn from the reactor to analyze the concentrations of CECs and DOC. In addition to photocatalytic oxidation runs, single ozonation (in the absence of P25 and with LEDs turned off) and photolytic ozonation (in the absence of P25) experiments were carried out for comparative purposes. For continuous operation runs the experimental set-up schematically shown in Figure 7 was used. This system consists of a WW reservoir, a glass packed bubble column filled with glass rings (coated with TiO2 for photocatalytic experiments and uncoated for single and photolytic ozonation runs), a recirculating loop and eight 10 W UVA LEDs positioned along the column (maximum wavelength of emission at 381 nm, Figure S2). Noted that a high concentration of ozone was applied because of the presence in wastewater of substances different to CECs that are at high concentration (see TOC and IC values in Table S1). These substances also consume ozone and/or hydroxyl radicals. In any case, in a practical situation concentration of ozone could also be in the order of tens of mg L −1 [36].
Positive step input tracer experiments were carried out for characterizing the reactor flow For continuous operation runs the experimental set-up schematically shown in Figure 7 was used. This system consists of a WW reservoir, a glass packed bubble column filled with glass rings (coated with TiO 2 for photocatalytic experiments and uncoated for single and photolytic ozonation runs), a recirculating loop and eight 10 W UVA LEDs positioned along the column (maximum wavelength of emission at 381 nm, Figure S2). Noted that a high concentration of ozone was applied because of the presence in wastewater of substances different to CECs that are at high concentration (see TOC and IC values in Table S1). These substances also consume ozone and/or hydroxyl radicals. In any case, in a practical situation concentration of ozone could also be in the order of tens of mg L −1 [36].
Catalysts 2019, 9, x FOR PEER REVIEW 13 of 17 samples were centrifuged at 4000 rpm for 5 min prior to the analyses. The removal efficiencies of CECs were evaluated at steady-state.
Analytical Methods
Concentrations of CECs were measured using an eco-friendly validated method of solid phase extraction followed by ultra-high performance liquid chromatography with tandem mass spectrometry (SPE-UHPLC-MS/MS) described in a previous work [37] where detailed information about recovery percentage of CECs by SPE and precision and accuracy of concentration measurements are given. Typically, before SPE, 100 mL of WW samples were filtered through 1.2 μm glass microfiber filters GF/C, 47 mm (Whatman TM ), acidified to pH 2 with sulfuric acid and 100 μL of a solution containing internal standards was added. Afterwards, samples were loaded through the conditioned Oasis® HLB cartridges (Hydrophilic-Lipophilic-Balanced sorbent, 150 mg, 6 mL) at a constant flow rate of 10 mL·min −1 , under vacuum. The sample cartridges were then washed with ultrapure water, dried under vacuum and eluted with ethanol. The extracts were evaporated to dryness in a vacuum concentrator and the residues were reconstituted in 400 μL of ethanol and filtered through 0.22 μm polytetrafluoroethylene (PTFE) syringe filters.
Chromatographic analysis was performed using a Shimadzu Corporation UHPLC (Tokio, Japan) equipment provided with two pumps ( Positive step input tracer experiments were carried out for characterizing the reactor flow pattern. A NaCl aqueous solution (2 g·L −1 ) was used as the tracer. The conductivity of the liquid stream leaving the column was continuously monitored using a Crison GLP 31 conductivity meter to equalize the conductivity value of the inlet solution.
In a typical degradation run, the system was filled with distilled water and then 15 mL·min −1 of WW were continuously pumped from the reservoir to the column, the recirculation flow rate being set at 166 mL·min −1 . At the same time, a gas flow rate of either oxygen or a mixture of ozone-oxygen (15 mL·min −1 and 20 mg·L −1 ozone) was continuously supplied to the column through a porous diffuser placed at its bottom. LEDs were also turned on (if required). The gas and liquid outlet streams left the system continuously through valves at the top of the reactor. The ozone concentration in the gas stream was continuously monitored while liquid samples were regularly taken from the outlet liquid and immediately bubbled with air to remove residual ozone before analysis. Then the collected samples were centrifuged at 4000 rpm for 5 min prior to the analyses. The removal efficiencies of CECs were evaluated at steady-state.
Analytical Methods
Concentrations of CECs were measured using an eco-friendly validated method of solid phase extraction followed by ultra-high performance liquid chromatography with tandem mass spectrometry (SPE-UHPLC-MS/MS) described in a previous work [37] where detailed information about recovery percentage of CECs by SPE and precision and accuracy of concentration measurements are given.
Typically, before SPE, 100 mL of WW samples were filtered through 1.2 µm glass microfiber filters GF/C, 47 mm (Whatman TM ), acidified to pH 2 with sulfuric acid and 100 µL of a solution containing internal standards was added. Afterwards, samples were loaded through the conditioned Oasis®HLB cartridges (Hydrophilic-Lipophilic-Balanced sorbent, 150 mg, 6 mL) at a constant flow rate of 10 mL·min −1 , under vacuum. The sample cartridges were then washed with ultrapure water, dried under vacuum and eluted with ethanol. The extracts were evaporated to dryness in a vacuum concentrator and the residues were reconstituted in 400 µL of ethanol and filtered through 0.22 µm polytetrafluoroethylene (PTFE) syringe filters.
Thickness of P25 layers on P25R samples was determined by scanning electron microscopy (SEM, Quanta 3D FEG (FEI), Billerica, MA, USA) with 20 kV accelerating voltage and a back-scatter electron detector (BSED). Wavelength dispersive X-ray fluorescence (WDXRF, Brucker SB Tiger 4K, Billerica, MA, USA) measurements were carried out in He atmosphere, using XS-5S, PET and LiF(200) crystals and a mask size of 28 mm.
Conclusions
CECs found in WW at concentration levels around ng·L −1 , and in some cases µg·L −1 , were efficiently removed from municipal secondary wastewater samples by the proposed ozonation-based processes (>90%) except ciprofloxacin, metoprolol, and propranolol in semi-batch experiments. Although few differences were observed between these processes, continuous experiments in deionized water showed that a synergic effect takes place for ozone decomposition with UVA-LED radiation and TiO 2 -coated glass rings, giving rise to a slight enhancement of contaminant removal in the treatment of this municipal wastewater.
In addition, the kinetic study clarified that direct ozone reaction is the main pathway responsible for the removal of CECs. Nevertheless, indirect and direct ozone reaction rates ratios indicate that the HO • reaction pathway could prevail over direct reaction in the case of the most refractory compounds for the O 3 /LED/P25 system (i.e., isoproturon).
Consequently, the use of P25 TiO 2 -coated rings and energy-efficient LEDs for photocatalytic ozonation could be an attractive way for wastewater treatment in continuous operation.
Additional recommended studies on the AOP treatment of CECs in wastewater are, among others, confirmation of intermediate removals and ozone consumption per TOC removed.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/5/472/s1, Table S1: Main characteristics of wastewater samples (WW), Table S2: List of compounds: (i) that can be identified by the SPE-UHPLC-MS/MS method; (ii) listed in 2013/39/EU Directive and/or (iii) 2018/840/EU Decision; and (iv) detected in WW samples, Figure S1: Comparison of removal percentage between WW1 filtered and unfiltered in different semi-batch experiments. Conditions: reaction time = 10 min; gas flow rate = 150 mL·min −1 ; P25 loading (when applied) = 0.5 g·L −1 ; ozone concentration = 50 mg·L −1 , Figure S2: Irradiance spectra of UVA LEDs used for semi-batch (solid line) and continuous operation (dotted line), Figure S3: Case A: The system behaves as one perfectly mixed reactor. Case B: The system behaves as two perfectly mixed reactors in series due to the reaction column and the recirculation column, Figure S4: F curve versus time from tracer experiment (solid line) and simulated F curves for the reactor set-up in case A (o) and B (∆), Figure S5: SEM images of fresh coated glass rings surface (A and B). Layer thickness measurements in a polished coated glass ring © and in a scratched coated glass ring surface (D), Table S3: WDXRF results of uncoated glass rings ®, coated glass rings before treatment (P25R) and after photocatalytic oxidation (O 2 /LED/P25R) and photocatalytic ozonation (O 3 /LED/P25R). Funding: This research was funded by ERDF-European Funds for Regional Development through "MINECO -MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD OF SPAIN, Project CTQ2015-64944-R", "NORTE 2020 (Programa Operacional Regional do Norte), NORTE-01-0145-FEDER-000006", "COMPETE2020 -Programa Operacional Competitividade e Internacionalização (POCI) and FCT -Fundação para a Ciência e a Tecnologia, POCI-01-0145-FEDER-006984". | v3-fos-license |
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