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} | pes2o/s2orc | Influence of Nonpolar Substances on the Extraction Efficiency of Six Alkaloids in Zoagumhwan Investigated by Ultra Performance Liquid Chromatography and Photodiode Array Detection
A reverse phase ultra performance liquid chromatography and photodiode array (UPLC-PDA) detection method was established for the determination of six alkaloids in Zoagumhwan (ZGW), and further for investigating the influence of nonpolar substances on the extraction efficiency of these alkaloids. The method was based on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) column and mobile phase of aqueous phosphoric acid and acetonitrile including 0.05% buffer solution under gradient elution. ZGW samples of ZGW I, II, III and IV were obtained and prepared by pre-processing the crude materials of Coptidis rhizoma and Evodiae fructus using four technologies, namely direct water decoction, removal of nonpolar substances in Evodiae fructus by supercritical fluid extraction (SFE), removal of nonpolar substances in ZGW by SFE and removal of nonpolar substances in ZGW by steam distillation. The developed and validated UPLC-PDA method was precise, accurate and sensitive enough based on the facts that the six alkaloids showed good regression (r > 0.9998), the limit of detections and quantifications for six alkaloids were less than 28.8 and 94.5 ng/mL, respectively, and the recovery was in the range of 98.56%–103.24%. The sequence of the total contents of six alkaloids in these samples was ZGW II > ZGW IV > ZGW III > ZGW I. ZGW II, in which nonpolar substances, including essential oils, were firstly removed from Evodiae fructus by SFE, had the highest content of the total alkaloids, indicating that extraction efficiency of the total alkaloids could be remarkably increased after Evodiae fructus being extracted by SFE.
Introduction
Traditional Chinese medicines (TCMs) and their compound preparations have become more and more popular around the World during the last decade for their therapeutic effects which are complementary to Western medicines, and their capability to deal with many essential problems that have not yet been solved by current medicinal practices [1]. Zoagumhwan (ZGW) is a well-known compound preparation composed of Coptidis rhizoma and Evodiae fructus at the ratio of 6:1 (w/w), and has been used for more than 1,000 years. It is one of the simplest compound preparations but retains the basic therapeutic features [2].
Today, ZGW has been officially listed in the Chinese Pharmacopoeia [3] for its satisfactory effects of healing hypochondric and costal pain, stomach ache, acid reflux and nausea. It is reported [4][5][6][7] that the major active components of Zoagumhwan are alkaloids, namely columbamine, epiberberine, jatrorrhizine, coptisine, palmatine and berberine, as shown in Figure 1. To date, many factors shown to affect the extraction efficiency of these alkaloids from Zoagumhwan, such as altering extraction solution [8], decoction conditions, sequence of decoction [9], etc., have been investigated. However, it is well-known that Evodiae fructus, one of the components in Zoagumhwan, contains a volatile fraction, and monoterpenes and sesquiterpenes are the main volatile components in the volatile sample of Evodia according to gas chromatography-mass spectrometry (GC-MS) analysis [10][11][12]. Nevertheless, the influence of the nonpolar substances, including essential oils and other substances, on the extraction efficiency of the alkaloids was neglected in previous studies. In decoction, it is very easy that volatile components are freed from the herb. A small amount of them can be dissolved in the extraction solution, and this might affect the extraction of alkaloids from Zoagumhwan. Therefore, it is significant to investigate the influence of nonpolar substances on extraction efficiency of the main alkaloids.
Supercritical fluid extraction (SFE) and steam distillation are the most commonly utilized methods to extract nonpolar substances from medicinal and edible plant foods. In this study, the two methods were employed to remove the nonpolar fraction. Four different protocols, including direct water decoction, removal of nonpolar substances in Evodiae fructus by SFE, removal of nonpolar substances in Zoagumhwan by SFE and removal of nonpolar substances in Zoagumhwan by steam distillation were used to pre-process crude materials of Coptidis rhizoma and Evodiae fructus for obtaining different Zoagumhwan samples. Then, a rapid ultra performance liquid chromatography and photodiode array (UPLC-PDA) detection method was developed for the simultaneous determination of the six alkaloids in these extracts.
Development of the UPLC Method
In the course of developing the rapid UPLC method, the basic chromatographic conditions such as mobile phase, flow rate, elution conditions, column temperature and detection wavelength were taken into consideration.
Mobile phase was a major factor for the good separation of the six tested alkaloids. Firstly, methanol, which was found to be less selective in the separation of the six alkaloids than acetonitrile, was discarded. According to the reports [13,14], the composition of acetonitrile and 20 mM ammonium acetate, which was adjusted to pH 10 by ammonia water, was investigated first. The results showed that the alkaloids could be separated well, but serious tailing was observed. Therefore, in order to improve the tailing, an acid solvent was considered according to the acid-base equilibrium theory. Choosing acetonitrile-0.5% (v/v) acetic acid could give an acceptable performance, but the separation between columbamine, jatrorrhizine, epiberberine and coptisine was not satisfactory. Finally, a more satisfactory chromatographic separation was acquired through the combination of acetonitrile and 0.05% (v/v) phosphoric acid water solution. A gradient elution procedure as shown in Table 1 was performed in order to decrease the total run time. Attempts to further improve the chromatographic performance, the flow rate and the column temperature were adequately investigated. The experiments showed that the influence of column temperature was very remarkable on the separation of COL, JAT, EPI and COP. When the column temperature was raised from 15 °C to 40 °C, resolution was notablely degraded and the number of theoretical plates was raised notably. Finally, it was preferred to the column temperature of 22 °C. Meanwhile, with a reduced flow rate, the tailing factor of the six alkaloids was increased.
Three different UPLC-PDA detection wavelengths of 220, 270 and 340 nm were monitored and compared. 270 nm was finally selected for the lowest baseline noise for berberine, which was observed to be the best detection wavelength for all the subsequent experiments. Under the optimized UPLC conditions, the satisfactory and rapid separation of the six tested alkaloids shown in Figure 2 was achieved in 6 min with a back pressure of 7,800 psi. Peaks: 1. columbamine; 2. epiberberine; 3. jatrorrhizine; 4. coptisine; 5. palmatine; 6. berberine.
Validation of the UPLC Method
The UPLC method was validated in terms of linearity, precision and accuracy according to ICH guidelines [15].
Calibration Curves
Methanol stock solutions containing six reference compounds were prepared and diluted to appropriate concentrations for the construction of calibration curves. At least six concentrations of the solution were analyzed in triplicate, and then the calibration curves were constructed by plotting the peak areas (y) versus the concentration (x) of each tested alkaloid. The results are shown in Table 2. The linearity of the calibration curves was verified by a correlation study and the correlation coefficients (r) were all better than 0.9998 within the test ranges.
Sensitivity
The methanol stock solution containing the six reference compounds was diluted to a series of appropriate concentrations with the same solvent, and an aliquot of the diluted solutions was injected into the UPLC system for PDA analysis. The limits of detection (LODs) and quantification (LOQs) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. Table 2 showed the data of LOD and LOQ for each investigated compound. The confidence LODs and LOQs for the six alkaloids were 25.8-31.8 and 85.0-104 ng/mL with 95% confidence level, respectively.
Precision
Intra-and inter-day variations were chosen to determine the precision of the developed method. For the intra-day variability test, six replicates of the mixed standards solution were analyzed within 1 day, while for the inter-day variability test, the solution was examined in duplicate for three consecutive days. For every calibration curve, the calibration concentrations were back-calculated from the peak area of the standard substances. The deviation from the nominal concentration was defined as accuracy. As shown in Table 3, variations, which were expressed by the intra-and inter-day relative standard deviations (RSD), were less than 0.9 and 1.2%, respectively.
Stability
With the temperature of both the laboratory and the control of the column and the auto-sample cell, being 22 °C, samples usually have to be stay at 22 °C for a long time, which should be considered carefully for unstable standard substances. In this study, the stability of the six alkaloids was tested by injecting standard solution at 0, 1, 2, 4, 8, 12 and 18 h at 22 °C. The results showed that all samples were stable under these conditions during the test time period (Table 3). Thus, the quantification of investigated compounds in all samples was preformed within 18 h.
Recovery
The recovery of the method was determined by spiking known amount of mixed standards in a certain amount (75 mg) of extract abtianed by direct water decoction. Three replicates were performed for the test. The mixture was extracted and analyzed using the method mentioned above. The results were calculated and are given in Table 4. The average recoveries of the five alkaloids were 98.56%-103.24%, with RSD values of less than 0.9%. All the above results illustrate that the developed UPLC-PDA method was precise, accurate and sensitive enough for simultaneous quantitative determination of the six alkaloids in Zoagumhwan samples.
Analysis of Real Samples
In this study, the contents of six alkaloids in four kinds of Zoagumhwan samples identified as ZGW I, II, III and IV from four different pre-processing methods were determined rapidly and simultaneously with the developed UPLC-PDA method. To reduce experimental error, each sample was extracted and analyzed in triplicate. The typical UPLC-PDA chromatograms of Zoagumhwan samples (ZGW I, II, III and IV) are shown in Figure 3. The target components were identified by comparing their retention times and UV spectra with those presented in the chromatogram of the mixture standard solution (Figure 2). The peak purity of target components from the four samples was verified using a photodiode array detector and found to be satisfactory. It could be seen from Figure 3 that the peak height and peak area of the same component had slight differences, though the number of predominant peaks from the four samples didn't change. Comparing the six characteristic peaks in the four chromatograms, one could find easily that peak 6 (berberine) is the most important active constituent in Zoagumhwan with the highest peak height. In order to further find the differences between the same components in the four Zoagumhwan samples, the contents (mg/g) of the six investigated alkaloids in the four kinds of Zoagumhwan samples (ZGW I, II, III and IV) were determined in triplicate and listed in Table 5. From the quantitative results, one could find that the contents of total alkaloids in the four samples showed significant differences. The sequence of the contents of total alkaloids in these samples was ZGW II > ZGW IV > ZGW III > ZGW I. ZGW II, in which nonpolar substances were firstly removed from Evodiae fructus by SFE-CO 2 , had the highest content of total alkaloids. The results indicated that removal of nonpolar substances from Evodiae fructus could remarkably increase the extraction efficiency of the total alkaloids. In addition, the contents of total alkaloids in ZGW III and ZGW IV were lower than that in ZGW II. The reasons might be that parts of alkaloids in ZGW III were extracted by SFE-CO 2 , and the extraction efficiency of alkaloids in ZGW IV was decreased by the remaining nonpolar substances. The increment, which was calculated by the formula: increment (%) = 100 × (the contents of total alkaloids in ZGW II, III or IV-ZGW I)/ZGW I, of ZGW II, III and IV were 10.9%, 0.8% and 4.3%, respectively.
On the other hand, the content of berberine was still the highest among the six major constituents in the samples, and the content of each alkaloid was influenced by nonpolar substances, but from Figure 4, it could be found that compared with the ZGW I sample, the content of columbamine and berberine was increased, while that of epiberberine and coptisine was decreased. The results indicated that the nonpolar substances existing in Zoagumhwan did not decrease the extraction yield of all alkaloids. The developed pre-processing method through removal of nonpolar substances in Evodiae fructus by SFE provided significant benefits in terms of extraction efficiency of the total alkaloids. Epiberberine, coptisine, palmatine and berberine all contain the -OCH 2 -group, which increases the dissolution rate under normal circumstance due to its polarity. However, with the absence of the role of the volatile oil when boiling, these four alkaloids in the solution vould reach new dissolution equilibrium. Epiberberin is the isomer of berberine, and would transfer into berberine for stability reasons. In the same way, the instability of -OCH 2 O-group would result in the decrease of content of coptisine. Thus, the above results were produced through a complicated interaction among chemicals, radicals, or ions in the extraction process. To confirm this deduction, further experiments should be conducted in the future.
Materials and Reagents
Coptidis rhizoma and Evodiae fruvtus were purchased from Lv-Ye Pharm. Co. Ltd China). The purity of all reference compounds was more than 98.0%. Phosphoric acid was analytical purity from Beijing Chemical Works (Beijing, China). Methanol and acetonitrile were all of chromatographic purity from Fisher Chemicals (Pittsburgh, PA, USA). The water was double distilled water.
Instruments
A HA221-50-06 supercritical fluid extraction apparatus was purchased from Nantong Hua-An Supercritical Fluid Extraction Co. Ltd. (Nantong, Jiangsu, China). UPLC analysis was performed on a Waters Acquity Ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA), equipped with a binary solvent delivery pump, an auto sampler and a photodiode array detector.
Chromatographic Conditions
The chromatographic separation of six alkaloids was performed on a Waters Acquity BEH C 18 (50 mm × 2.1 mm, 1.7 μm) column. The mobile phase consisted of phosphoric acid aqueous solution and acetonitrile, both including 0.05% (v/v) buffer solution. The gradient elution procedure has been listed in Table 1. The detection wavelength was set at 270 nm. The injection volume was 1 μL while the column was maintained at 22.0 ± 0.5 °C.
SFE Conditions
The SFE conditions of Zhang et al. [10] were used with slight modifications. The flow rate (CO 2 ) was kept at 4.7-15.7 L/h, extraction pressure was retained at 20-25 MPa, extraction time was 2 h, extraction temperature was maintained at 40.0 ± 2.0 °C and separation vessel II and vessel II were set 8 MPa and 6 MPa, respectively.
Pre-Processing by Direct Water Decoction
Coptidis rhizoma and Evodiae fructus were crushed to pieces and mixed together thoroughly at a ratio of 6:1 (w/w). Then, the mixture was macerated in warm water (40 °C) for 30 min and decocted with water three times, 2 h for the first time, 1 h for the second time and 0.5 h for the third time, respectively. The filtrates from each decoction were blended and concentrated to a thick solution using a rotary evaporator (BÜCHI, Flawil, Switzerland) at 75 °C. The concentrated sample was dried in a vacuum oven at 50 °C, and the percentage yield was 26.25%.
Pre-Processing through Removal of Nonpolar Substances in Evodiae Fructus by SFE
Essential oil was removed firstly from Evodiae fructus by SFE under the above-described conditions. Coptidis rhizoma and Evodiae fructus without nonpolar substances at ratio of 6:1 (w/w) was mixed thoroughly and decocted with the same procedure as that of direct water decoction. The concentrated sample was dried in a vacuum oven at 50 °C, and the percentage yield was 25.72%.
Pre-Processing through Removal of Nonpolar Substances in Zoagumhwan by SFE
Coptidis rhizoma and Evodiae fructus were mixed thoroughly at a ratio of 6:1 (w/w). Then, essential oil in the mixture was removed by SFE under the above-described conditions. Zoagumhwan without nonpolar substances was decocted with the same procedure as that of direct water decoction. The concentrated sample was dried in a vacuum oven at 50 °C, and the percentage yield was 27.54%.
Pre-Processing through Removal of Nonpolar Substances in Zoagumhwan by Steam Distillation
Coptidis rhizoma and Evodiae fructus were thoroughly mixed first at a ratio of 6:1 (w/w). Then, the extraction method of steam distillation described in the Chinese Pharmacopoeia [16] was applied to remove nonpolar substances from the mixture. Zoagumhwan without essential oil was decocted with the same procedure as that of direct water decoction. The concentrated sample was dried in a vacuum oven at 50 °C, and the percentage yield was 24.58%.
Preparation of Zoagumhwan Sample Solutions
The concentrated and dried powders, namely ZGW I (78.75 mg), ZGW II (77.16 mg), ZGW III (82.62 mg) and ZGW IV (73.74 mg) from the above-described pre-processing methods of crude materials were dissolved in 10 mL methanol, shaken thoroughly and transferred into a flask. After being making up to 25 mL with methanol, the solutions were filtered through a 0.22 μm filter prior to UPLC analysis. For convenience, the sample solutions from the above-described four different pre-processing methods were labeled as ZGW I, II, III and IV, respectively.
Data Analysis
The Waters Empower software was used to collect and process the chromatograms data. Through importing the peak area and retention time in mean chromatograms, all parameters, including linear equation, correlations, and RSD, were calculated and analyzed using SPSS 13.0.
Conclusions
An easy and economical UPLC-PDA chromatographic procedure has been developed and successfully applied to determine the contents of six major alkaloids in Zoagumhwan extracts after different pre-processing technologies were used on the crude materials. The UPLC-PDA method was selective, sensitive and reproducible for the simultaneous determination of six alkaloids with low LOD and LOQ. The results have shown that the pro-processing technology through removal of nonpolar substances in Evodiae fructus by SFE could greatly improve the extraction efficiency of the main active components in Zoagumhwan. Thus, in the future, this described pro-processing method combined with UPLC analysis should be preferred and applied to pharmaceutical products of Zoagumhwan in order to improve the production technology, and to evaluate the productivity. | v3-fos-license |
2018-04-03T06:11:11.012Z | 2018-01-31T00:00:00.000 | 23186355 | {
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} | pes2o/s2orc | Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits
Abstract Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem–spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICAN in vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.
Introduction
Members of the transient receptor potential (TRP) channel superfamily, which mediate cationic current fluxes and control cell excitability and intracellular signaling, are involved in diverse aspects of brain function. Here we investigated roles of two subtypes of TRP channels-TRPM4 of the melastatin TRPM channel family and TRPC3 of the canonical TRPC channel family-in generating respiratory motor activity in the rodent brainstem-spinal cord. We established that these channels are expressed at the molecular level in populations of respiratory interneurons and motoneurons, they are endogenously activated during circuit activity, and they have a fundamental role in respiratory motor pattern generation.
Prominent, but unproven, Ca 2ϩ -based theories involving TRPM4 have been proposed for respiratory rhythm generation (Mironov, 2009;Del Negro et al., 2010) by excitatory neurons in the mammalian brainstem pre-Bötzinger complex (pre-BötC), the established locus of interneurons in the medulla critical for inspiratory rhythm generation (Smith et al., 1991;Feldman and Del Negro, 2006). TRPM4, known to be a Ca 2ϩ -activated TRP channel (Guinamard et al., 2014), is postulated to be the molecular substrate of Ca 2ϩ -activated nonselective cation current (I CAN ) in respiratory neurons (Crowder et al., 2007;Del Negro et al., 2010). TRPM4-mediated I CAN is proposed to be importantly involved in rhythm generation by functionally coupling activity-dependent intracellular Ca 2ϩ signaling to neuronal depolarization and rhythmic neuronal activity generation (Del Negro et al., 2010;Guinamard et al., 2010). Previous studies (e.g., Koizumi and Smith, 2008) have shown that the rhythmically active neurons in pre-BötC circuits as well as neurons in downstream rhythmic drive transmission circuits exhibit large transient increases of intracellular Ca 2ϩ , potentially mediating TRPM4/ I CAN channel activity, during each respiratory cycle. TRPC3, on the other hand, is not directly activated by Ca 2ϩ , but mediates Na ϩ /Ca 2ϩ fluxes, and as a proposed Ca 2ϩ store-operated channel, may be involved in regulating neuronal Ca 2ϩ -related signaling (Talavera et al., 2008;Birnbaumer, 2009;Guinamard et al., 2013), potentially affecting TRPM4/I CAN in respiratory neurons. The role of TRPC3-mediated cationic currents/Ca 2ϩ -related signaling in generating respiratory neuron activity has not been investigated.
TRPM4 and TRPC3 channels have been identified by mRNA/protein expression in tissue harvested from the mouse pre-BötC region (Crowder et al., 2007;Ben-Mabrouk and Tryba, 2010), although they have not been demonstrated to be expressed in functionally identified respiratory neurons. Here we examined, by single-cell RT-PCR, expression of TRPM4 and TRPC3 mRNA in functionally identified glutamatergic, glycinergic, and GABAergic inspiratory pre-BötC neurons as well as inspiratory motoneurons. We also examined neuronal channel expression by antibody labeling in the pre-BötC region and motor nuclei. We then investigated whether these channels contribute to respiratory circuit activity by pharmacological experiments with the selective channel inhibitors 9-phenanthrol for TRPM4 (Guinamard et al., 2014) and 3-pyrazole for TRPC3 (Kiyonaka et al., 2009), in both neonatal rat and mouse slice preparations with rhythmically active pre-BötC and respiratory motor circuits in vitro. Comparative analyses for these two species was important because there are numerous studies on respiratory rhythm and pattern generation using rats (e.g., Gray et al., 2001;Onimaru and Homma, 2003;Koizumi and Smith, 2008) or mice (e.g., Thoby-Brisson and Ramirez, 2001;Peña et al., 2004;Del Negro et al., 2005), so it is necessary to establish commonality of Ca 2ϩ -based mechanisms for respiratory rhythm and pattern generation. We also analyzed perturbations of pre-BötC excitatory circuit activity by dynamic Ca 2ϩ imaging of inspiratory glutamatergic pre-BötC neurons with a genetically encoded Ca 2ϩ sensor (Chen et al., 2013) in transgenic mice. We show that amplitudes of inspiratory pre-BötC neuronal activity, and the correlated amplitudes of motoneuronal output in vitro, are significantly reduced by TRPM4 and TRPC3 channel inhibitors. The pharmacological profile of inspiratory activity attenuation by inhibiting TRPM4 activation matched that with another proposed blocker of I CAN , flufenamic acid (FFA), consistent with the concept that TRPM4 mediates I CAN (Launay et al., 2002). In all cases, the attenuation of inspiratory circuit activity occurred without significant perturbations of the frequency of the inspiratory rhythm.
We also demonstrate critical involvement of TRPM4 and TRPC3 channels in regulating the amplitude of pre-BötC population activity and motor output patterns in more intact respiratory circuits active in arterially perfused brainstem-spinal cord in situ preparations from mature rats and mice. The reduction, by the channel inhibitors, of pre-BötC and motoneuronal inspiratory activity amplitude recorded electrophysiologically was accompanied by reductions of post-inspiratory motoneuronal activity. These amplitude perturbations also occurred without disrupting rhythm generation. In general, our results indicate that endogenous activation of these two types of TRP channels are involved in generating respiratory motor patterns, but critically not rhythm generation, in both neonatal and mature rodents.
Animal procedures
All animal procedures were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke.
The medulla oblongata from neonatal and mature rats or mice was fixed in 4% paraformaldehyde (wt/vol) in PBS, cryoprotected overnight at 4°C in 30% sucrose and 0.1 M PBS solution, and sectioned coronally (25 or 50 m) with a freezing microtome. For fluorescent immunohistochemistry, floating sections were incubated with 10% donkey serum in PBS with Triton X-100 (0.3%) and incubated for 48 -72 h at room temperature with the following primary antibodies: polyclonal rabbit anti-TRPM4 (ab63080, Abcam ab63080, RRID: AB_956418, 1:1000) and polyclonal rabbit anti-TRPC3 (ACC-016, Alomone Labs, ACC-016, RRID: AB_2040236, 1:200). We verified the specificity of these TRPM4 and TRPC3 antibodies by confirming the absence of immunoreactivity in the mouse medullary tissue sections with the primary antibody that was preincubated for 1 h at room temperature with saturating concentrations (10:1) of the antigenic blocking peptide (TRPM4: ab65597, Abcam, TRPC3: ACC-016, Alomone Labs). We also note that the specificity of the same TRPM4 and TRPC3 antibodies as those we used has been confirmed in a TRPM4 knockout mouse (Schattling et al., 2012) and a TRPC3 knockout mouse (Feng et al., 2013), respectively. Individual sections were then rinsed with PBS and incubated for 2 h with the secondary antibody (donkey anti-rabbit, Dylight 647). Individual sections were mounted on slides and covered with an anti-fading medium (Fluoro-Gel; Electron Microscopy Sciences). Fluorescent labeling of neurons was visualized with a laserscanning confocal imaging system (Zeiss LSM 510). Motoneurons were identified by antibody labeling for choline acetyltransferase (ChAT; goat anti-ChAT, Millipore AB144, RRID: AB_90650, 1:200; donkey anti-goat-Dylight 488, 1:500). TRP channel expression in cell bodies of interneurons was identified by the presence of channel immunoreactivity without ChAT antibody labeling. All images were color/contrast enhanced and adjusted with a thresholding filter in Adobe Photoshop.
For tallying the numbers of TRPM4 or TRPC3 channel antibody-labeled neurons in adult (3-5-mo-old) transgenic mice with glutamatergic or glycinergic neurons labeled with tdTomato fluorescent protein as presented in Results, we counted labeled neurons within a region (300 -400 m diameter depending on animal size, ventral to the nucleus ambiguus) in the coronal plane of 25-mthick tissue sections obtained from the pre-BötC, BötC, or rVRG regions on both sides of the medulla. The locations and rostro-caudal extent of each region were defined from our established anatomic criteria based on previous electrophysiological recording/cell activity mapping studies (e.g., as well as the present neuronal population activity recordings in the adult transgenic mouse in situ brainstem-spinal cord preparations. The anatomic criteria included the location, in the ventrolateral medullary reticular formation, of the BötC region at the levels of the compact division of nucleus ambiguus (NAc), the pre-BötC region at the levels of the semicompact division of NA (NAsc), and the rVRG region extending from near the level of obex to the caudal end of the pre-BötC. The rostro-caudal dimension of the pre-BötC region was ϳ350 m; the BötC region, ϳ550 m; and the rVRG region, ϳ500 m in the 3-5-mo-old adult mice used for the analysis. We selected 4 -6 of the coronal sections from each region so that sections at different levels clearly within the region were included for the bilateral cell counting, and our sample included sections from both the caudal and rostral half of each region to produce the regional tally of labeled neurons presented.
Calcium imaging and identification of pre-BötC respiratory neurons in vitro
We employed Ca 2ϩ -sensors, either Ca 2ϩ -sensitive synthetic dye in slices from wild-type (WT) neonatal rats and mice or genetically encoded protein Ca 2ϩ sensor with fast kinetics (GCaMP6f; Chen et al., 2013) in transgenic mice, to dynamically image Ca 2ϩ activity of pre-BötC neurons for functional identification of respiratory neurons in vitro. In experiments with WT neonatal rats or mice, the Ca 2ϩ imaging was combined with whole-cell patch-clamp recording from the identified rhythmically active inspiratory neurons. In some of these experiments, the neurons were retrogradely labeled through their contralaterally projecting axons using membrane semipermeable acetoxymethyl (AM) dye (Oregon Green BAPTA-1 AM: OGB, Invitrogen) microinjected in the midline region of the slice (Koshiya and Smith, 1999;Koizumi et al., 2013). The slice was incubated overnight (12 h) in ACSF containing antibiotics (500 units/l penicillin, 0.5 mg/l streptomycin, and 1 mg/l neomycin) to allow labeling of pre-BötC neurons and used for optical and electrophysiological recordings throughout the next day. In other experiments, we microinjected the dyes directly into the pre-BötC to label cells nonselectively regardless of their axonal projections (Koizumi et al., 2013). The microinjection pipette (tip size: 2-3 m) was placed at a depth of 150 m in the slice and ϳ100 m away from the center of the pre-BötC to avoid excessive dye deposits and high background fluorescence in the imaging area of interest, and OGB was pressure injected (ϳ20 psi, 2 min). The slice was incubated (Ͼ1 h) for sufficient dye loading before the recording experiments.
In the set of imaging experiments with GCaMP6f in transgenic mice, we selectively expressed this Ca 2ϩ sensor in glutamatergic neurons using Cre-driven expression controlled by the VgluT2 promoter (VgluT2-GCaMP6f mice). These mice were produced by crossing the VgluT2iris-Cre strain and a Cre-dependent GCaMP6f expressing strain [B6;129S-Gt(ROSA)26Sor tm95.1(CAG-GCaMP6f)Hze /J, IMSR JAX: 024105, RRID: IMSR_JAX: 024105, Jackson Laboratory]. We imaged and quantified inspiratory-related GCaMP6f fluorescence transients in the pre-BötC glutamatergic neuronal population, which provides voltagedependent control of inspiratory frequency and functions as the critical rhythmogenic population .
In all experiments, optical imaging was performed with a Leica multiphoton laser scanning upright microscope (TCS SP5 II MP with DM6000 CFS system, LAS AF software), 20ϫ water-immersion objective (N.A. 1.0), beamsplitter (560 nm), and emission filter (525/50, Semrock). A two-photon Ti:sapphire pulsed laser (MaiTai, Spectra Physics) was used at 800 -880 nm for Ca 2ϩ -sensitive dye or 910 -920 nm for GCaMP6f, with DeepSee predispersion compensation. Dynamic Ca 2ϩ fluorescence images (16 kHz bidirectional, ϳ28 frames/s for 512 ϫ 512-pixel scan) were acquired in real time along with electrophysiological signals of inspiratory XII nerve activities (LAS AF acquisition hardware and software electrophysiology module v.2.60). Simultaneous recording of these signals allowed us to functionally identify pre-BötC inspiratory neurons that were rhythmically active in phase with inspiratory network activity monitored by XII discharge. The infrared excitation laser for two-photon fluorescence was simultaneously used for transmission bright-field illumination to obtain a Dodt gradient contrast structural image to provide fluorescence and structural images matched to pixels. This structural imaging also allowed us to accurately place a patch pipette on functionally identified neurons in experiments using whole-cell recording. For experiments performed with GCaMP6f analyzing changes in dynamic fluorescence signals during application of pharmacological channel inhibitors (below), average fluorescence intensities (F) of regions of interest were quantified for each frame, and dynamic fluorescence signals (⌬F) were represented as running baseline (F 0 )-subtracted values (F-F 0 ). We used ⌬F not ⌬F/F 0 for the activity quantification because ⌬F can be an order of magnitude brighter than F 0 at the laser wavelength optimal for GCaMP6f (910 -920 nm), and ⌬F was not proportional to F 0 .
Arterially perfused in situ brainstem-spinal cord preparations
To investigate contributions of TRPM4/TRPC3 channels in generation of respiratory activity in more intact systems, we also performed experiments with in situ arterially perfused brainstem-spinal cord preparations from mature (3-4-wk-old) rats of either sex (Sprague-Dawley, 45-100 g) or adult (3-5-mo-old) mice of either sex (C57BL/6, 25-35 g;Paton, 1996;. Preheparinized (1000 units, given intraperitoneally) rats/ mice were anaesthetized deeply with 5% isoflurane until loss of the paw withdrawal reflex, and the portion of the body caudal to the diaphragm was removed. The head and thorax were immersed in ice-chilled carbogenated ACSF solution containing the following (in mM): 1.25 MgSO 4, 1.25 KH 2 PO 4 , 5.0 KCl, 25 NaHCO 3 , 125 NaCl, 2.5 CaCl 2 , 10 dextrose, and 0.1785 polyethylene glycol. The brain was decerebrated at a precollicular level, and the descending aorta, thoracic phrenic nerve (PN), and cervical vagus nerves (VN) were surgically isolated. The dorsal brainstem was exposed by craniotomy and cerebellectomy. The preparation was transferred to a recording chamber and secured in a stereotaxic head frame with the dorsal side up. The descending aorta was cannulated with a double lumen catheter (DLR-4, Braintree Scientific) for ACSF perfusion with a peristaltic roller pump (505D, Watson-Marlow) and for recording of perfusion pressure with a pressure transducer. The ACSF perfusate was gassed with 95% O 2 -5% CO 2 and maintained at 31°C. Vecuronium bromide or rocuronium bromide (2-4 g/ml; SUN Pharmaceutical Industries) was added to the perfusate to block neuromuscular transmission. Vasopressin (200 -400 pM as required; APP Pharmaceuticals) was added to the perfusate to raise and maintain perfusion pressure between 70 and 80 mmHg (Pickering and Paton, 2006).
Electrophysiological recording in vitro and in situ
To monitor inspiratory network activity and motor output in the rhythmically active neonatal rat and mouse medullary slice preparations in vitro, XII motoneuron population activity was recorded from XII nerve rootlets with fire-polished glass suction electrodes (50 -100-m inner diameter). Extracellular recordings of inspiratory and post-inspiratory motoneuronal activity from VN, and inspiratory activity from PN, in the in situ perfused mature rat and mouse preparations were also obtained with suction electrodes (150 -200-m inner diameter). Signals in all cases were amplified (50,000 -100,000ϫ, CyberAmp 380, Molecular Devices), bandpass filtered (0.3-2 kHz), digitized (10 kHz) with an AD converter [Power Lab, AD Instruments or Cambridge Electronics Design] and then rectified and integrated digitally with Chart software (AD Instruments) for the in vitro slice preparations or Spike 2 software (Cambridge Electronics Design) for the perfused in situ preparations. Extracellular population activity from the pre-BötC in the perfused in situ preparations was also recorded by a dorsal approach with a fine-tipped glass pipette (3-5-M⍀ resistance) filled with 0.5 M sodium acetate (Sigma-Aldrich), or in some cases with a tungsten microelectrode (3-4 M⍀), which was positioned by a computer-controlled 3D micromanipulator (MC2000, Märzhäuser). The precise location of the pre-BötC was determined by the anatomic coordinates that we initially defined by mapping neuronal activity of medullary respiratory neurons within the ventral respiratory column. The pre-BötC is readily identified by a characteristic pattern of pre-inspiratory/inspiratory population activity and is distinguishable from the more rostral BötC region, which has a characteristic profile of post-inspiratory and augmenting expiratory activities, and from the more caudal rVRG region, which has an established profile of augmenting inspiratory population activity.
Analyses of TRP channel contributions to respiratory rhythm and motor pattern generation in vitro and in situ
We performed combined electrophysiological and pharmacological experiments to probe for the contributions of endogenously active TRPM4 and TRPC3 channels to respiratory rhythm and motor pattern generation in the in vitro slice preparations. We analyzed the time course of perturbations of the inspiratory burst frequency, amplitude, and duration of integrated XII inspiratory motor output after application to the slice bathing solution of the putative selective TRPM4 channel inhibitor (9-phenanthrol, Millipore, 10 -50 M; Guinamard et al., 2014), the selective TRPC3 channel inhibitor (Pyrazole compound-3: Pyr3, Millipore, 10 -50 M; Kiyonaka et al., 2009), and for comparison, the putative I CAN blocker flufenamic acid (FFA, Sigma-Aldrich, 20 -75 M;Teulon, 2000;Guinamard et al., 2013). In experiments with mouse slices expressing GCaMP6f in glutamatergic neurons, the dynamic Ca 2ϩ activity within the pre-BötC (regional and single-neuron ⌬F) was measured for 2.5-min intervals before and starting at 5, 10, 20, and 30 min after drug application to analyze local perturbations of neuronal activity within the pre-BötC rhythm-generating circuit accompanying perturbations of XII activity, which was continuously recorded throughout the experiments. Mean peak ⌬F values computed for the 2.5-min periods were normalized to the mean peak ⌬F values during control (before drug application) 2.5-min periods.
Contributions of TRPM4 and TRPC3 channels to respiratory rhythm and motor pattern generation in the mature rat and mouse arterially perfused brainstem-spinal cord preparations in situ were also analyzed by adding 9-phenanthrol (20 -50 M) and Pyr3 (50 M) to the perfusion solution. Perturbations of the inspiratory PN motor output as well as VN inspiratory and post-inspiratory activity were analyzed. Throughout these experiments, the perfusion pressure was maintained with vasopressin added to the perfusion solution as required or by adjusting the perfusion pump speed to avoid possible effects of perfusion pressure changes on respiratory activity, since 9-phenanthrol and Pyr3 caused reductions (10 -20 mmHg) in perfusion pressure, consistent with the proposed role of TRPM4 and TRPC3 channels in the control of vascular smooth muscle tone (Brayden et al., 2008).
Signal analyses of respiratory parameters
All digitized electrophysiological signals were analyzed by automated procedures to extract respiratory parameters from integrated nerve or neuronal population activities, performed with IDL (Exelis VIS) and Matlab (R2016a, Matlab, RRID: SCR_001622) software using the NIH high-performance computing Biowulf cluster. Inspiratory events were detected from the smoothed integrated XII (in vitro) or PN (in situ) signals via a 300-ms window moving average and peak detection algorithm that calculated a threshold-based zero derivative (positive peak) point. After peak detection, inspiratory activity time (T I ), expiratory interval time (T E ), respiratory period (T TOT ), and frequency (f R ϭ 1/T TOT ) were computed. T I was measured as the original integrated burst width at 20% of the peak height above baseline; T E was calculated as T TOT -T I . Inspiratory amplitude was calculated by subtracting the local baseline value from the peak value of the integrated signals. The endpoint of the parameter quantification was defined when inspiratory amplitude declined to either a quasisteady state value as assessed by inspection, or to noise level with the disappearance of inspiratory activity. Representative time courses of these parameters were extracted by a 300-s-window, time-based moving median. Data were then pooled per experimental condition, and summary time courses were computed with the parameter values normalized to mean values during the control period (300 -0 s before drug application).
We also quantified effects of the pharmacological manipulations on the regularity of the inspiratory rhythm by analyzing Poincaré plots of periods for 80 inspiratory bursts before and after drug application as the integrated inspiratory amplitude reached the defined endpoint. Short-and long-term period variability was quantified by plotting each T TOT as a function of the preceding T TOT and fitting a Gaussian distribution to these points projected onto the line perpendicular to the y ϭ x line (with standard deviation SD1) and the points projected onto the y ϭ x line (with standard deviation SD2). SD1 represents total burstto-burst period variability, and SD2 represents total variability minus burst-to-burst variability, serving as measurements of short-and long-term rhythm regularity, respectively (Tulppo et al., 1996;Fishman et al., 2012). Values of SD1 and SD2 during periods of drug application were normalized to control values. Coefficients of variation of inspiratory burst periods were also calculated to measure mean normalized variability of T TOT over the same time intervals used to determine SD1 and SD2.
For the in situ experiments, in addition to quantifying the respiratory parameters indicated above (T TOT , T I , T E , f R ), we analyzed perturbations of amplitudes and durations of individual phases of the respiratory cycle. Amplitudes and durations of inspiratory activity in PN and VN recordings and post-inspiratory activity in VN recordings were analyzed from integrated, cycle phase-triggered neurograms aligned at the onset of inspiration defined by PN activity. Successive cycle-triggered traces were either overlaid (PN and VN separately) or represented as a dynamic raster plot to depict temporal profiles of activity before, during, and after drug application periods.
For statistical analysis (Table 1), the endpoint values, as described above, of each experiment within a group were compared with the control values with a two-sided Wilcoxon signed-rank test. Correlation analyses on data from imaging experiments were performed by computing either Pearson's (r) or Spearman's rank (r s ) correlation coefficient. In all tests, significance level was set at p Ͻ 0.05.
Results
Immunohistochemical labeling of TRPM4 and TRPC3 channels in pre-BötC neurons, regions of the ventral respiratory column adjacent to the pre-BötC, and motoneurons TRPM4 and TRPC3 channel antibodies labeled neurons bilaterally within the pre-BötC region ( Fig. 1) in medullary slices from neonatal and mature rats/mice (n ϭ 3 each). These channels were also labeled by antibody in (1) motoneurons defined by ChAT immunolabeling within nucleus ambiguus (NA) and the XII motor nucleus containing subpopulations of respiratory motoneurons; (2) neurons within the medullary reticular formation zone dorsal to pre-BötC where inspiratory XII premotor neurons are distributed (Koizumi et al., 2008;Revill et al., 2015); (3) neurons within the rostral ventral respiratory group (rVRG) region, adjacent and caudal to the pre-BötC, where bulbospinal respiratory neurons are localized; and (4) neurons in the BötC region containing respiratory neurons rostral to the pre-BötC. TRPM4 and TRPC3 channels are not exclusively expressed in these regions, but as indicated by antibody labeling are widely expressed in neurons throughout the medullary reticular formation at these levels of the medulla.
TRPM4 and TRPC3 channel mRNA in glutamatergic, glycinergic/GABAergic pre-BötC inspiratory neurons and cranial motoneurons detected by single-cell multiplex RT-PCR
To confirm that respiratory neurons express TRP channels, we probed for TRPM4 and TRPC3 channel mRNA in single functionally identified pre-BötC inspiratory neurons in rhythmically active in vitro medullary slice preparations from WT neonatal rats and mice. Pre-BötC inspiratory neurons were identified by imaging neuronal Ca 2ϩ dynamics with OGB at depths up to 150 m in these slices, in which inspiratory neurons exhibit rhythmic Ca 2ϩ fluorescence transients in phase with the inspiratory XII nerve activity (Koizumi et al., 2013). Under current-clamp recording, all optically identified pre-BötC inspiratory neurons exhibited spike discharge synchronized with rhythmic XII activity. In the cytoplasm harvested from these neurons (n ϭ 41 neurons in total; n ϭ 33 from 8 rat slices and n ϭ 8 from 3 mouse slices) during whole-cell recording, we probed for TRPM4 and TRPC3 channel mRNA as well as VgluT2, GlyT2, and/or GAD67 mRNA to identify neuronal transmitter phenotype (Fig. 3). Only neurons with clean negative controls from "mock harvests" in the slice and appropriate positive controls (see Methods) were used for the analysis. In this sample, we identified 32 excitatory pre-BötC inspiratory neurons expressing only VgluT2 mRNA (28 neurons from rat slices; 4 neurons from mouse slices) and 9 inhibitory neurons expressing either GlyT2 mRNA only (n ϭ 1 each from rat and mouse slices), GAD67 mRNA only (n ϭ 1 each from rat and mouse), or coexpression of GlyT2 and GAD67 mRNA (n ϭ 3 from rat and n ϭ 2 from mouse slices), a phenotype previously documented for pre-BötC inhibitory interneurons (Koizumi et al., 2013). No VgluT2 mRNA was detected in these inhibitory neurons. Most of the TRP channel mRNApositive pre-BötC inspiratory neurons in this sample were glutamatergic (n ϭ 32/41, 78%), and almost half of these excitatory neurons (n ϭ 15/32, 47%) coexpressed both TRPM4 and TRPC3 mRNA, while other excitatory neurons expressed either TRPM4 mRNA only (n ϭ 5) or TRPC3 mRNA only (n ϭ 12). Inhibitory pre-BötC inspiratory neurons also expressed TRPM4 mRNA only (n ϭ 2), TRPC3 mRNA only (n ϭ 5), or both TRPM4 and TRPC3 mRNA (n ϭ 2).
We also analyzed expression of TRPM4 and TRPC3 channel mRNA in NA and XII motoneurons identified electrophysiologically as inspiratory motoneurons from whole-cell recording in rhythmically active slice preparations from both neonatal rats (n ϭ 4) and mice (n ϭ 2). We identified coexpression of TRPM4 and TRPC3 channel mRNA in all NA (n ϭ 5 motoneurons in total; n ϭ 2 from rats and n ϭ 3 from mice) and XII (n ϭ 10 total; n ϭ 7 from rats and n ϭ 3 from mice) inspiratory motoneurons sampled. Thus, these mRNA expression patterns are consistent with our results from immunolabeling demonstrating prominent TRPM4 and TRPC3 channel antibody labeling in all NA and XII motoneurons, and are consistent with previous results showing TRPM4 channel mRNA in lasercaptured XII motoneurons (Alvares et al., 2014).
Perturbations of inspiratory motor output in vitro by pharmacological inhibitors of TRPM4 and TRPC3 channels
The expression of TRPM4 and TRPC3 channel mRNA in identified pre-BötC inspiratory neurons and inspiratory cranial motoneurons, and the extensive antibody labeling of these channels in the pre-BötC region and adjacent respiratory-related regions as well as motor nuclei, suggest possible functional roles of these channels in rhythm and motor pattern generation. To test for functional endogenous activity of these channels in the rhythmically active neonatal rat and mouse slice preparations in vitro, we initially analyzed perturbations of the inspiratory rhythm and burst amplitude/duration of integrated XII inspiratory motor output after bath-application of the TRPM4 channel inhibitor 9-phenanthrol, the TRPC3 chan-nel inhibitor Pyr3, and the I CAN blocker FFA. In preliminary experiments, we determined that 9-phenanthrol (10 -50 M), Pyr3 (10 -50 M), and FFA (20 -75 M) progressively reduced the amplitude of XII inspiratory activity and, in Figure 3. Expression of TRPM4 and TRPC3 channel mRNA in glutamatergic and glycinergic/GABAergic pre-BötC inspiratory neurons. A, Overview of experimental in vitro neonatal rat rhythmic slice preparation showing whole-cell patch-clamp recording from the pre-BötC inspiratory neurons and suction electrode recordings from hypoglossal (XII) nerves to monitor inspiratory activity. NAsc, semicompact division of nucleus ambiguus; V4, fourth ventricle. B, Two-photon single optical plane images of pre-BötC inspiratory neuron (arrow) targeted for whole-cell recording and subsequent harvesting of cytoplasm, showing Ca 2ϩ -sensitive dye (OGB) labeling (B1) and Dodt structural image (B2). B3, Identification of inspiratory neuron by verifying that the Ca 2ϩ fluorescence signals in real time are synchronized with integrated inspiratory XII nerve activity (͐ XII). C1, Current-clamp recording (upper traces) from excitatory pre-BötC inspiratory neuron in B illustrates inspiratory bursts synchronized with ͐ XII. Under voltage-clamp (lower traces), the same neuron exhibited rhythmic inward synaptic currents synchronized with ͐ XII. This neuron was shown to be excitatory (VgluT2expressing) by post hoc single-cell RT-PCR (below). C2, Current-clamp recording (upper traces) and voltage-clamp recording (lower traces) from inhibitory pre-BötC inspiratory neuron illustrating inspiratory bursts and rhythmic inward synaptic currents synchronized with ͐ XII. This neuron was shown to be inhibitory (co-expression of GlyT2 and GAD67 mRNA) by post hoc single-cell RT-PCR (see below). D, Representative electrophoresis gel generated by single-cell multiplex RT-PCR from mRNA in cytoplasm harvested during whole-cell recording from two electrophysiologically identified pre-BötC inspiratory neurons (C1 and C2) in neonatal rat slices. In addition to cDNA probes for TRPM4 and TRPC3 channel mRNA, probes for vesicular glutamate transporter type 2 (VgluT2), glycine transporter type 2 (GlyT2), and glutamatic acid decarboxylase 67 (GAD67) mRNA were used to identify excitatory or inhibitory neuronal phenotypes, examples of which are shown. Expected numbers of base pairs (bp) for reaction products are indicated. Assays for both of these neurons had clean negative controls from "mock harvests" in the slice and appropriate positive controls from 100 pg total rat brain RNA run as RT template (not shown, see Materials and Methods). some cases, could completely eliminate XII inspiratory motor output at 50 M in our rat and mouse slice preparations. We therefore routinely used a single application of 50 M for these drugs as a near upper bound for circuit activity perturbations.
With 50 M 9-phenanthrol, for both rat and mouse slices, we consistently found large perturbations of integrated XII burst amplitude without significant perturbations of inspiratory burst frequency (f R ) relative to control values. Fig. 4A shows an example from an individual experiment, as well as the averaged, normalized time course of the reduction in XII amplitude and nonsignificant perturbation of normalized f R as burst amplitude reached a quasi-steady state value from a set of rat slices (n ϭ 6). The reduction of peak inspiratory burst amplitude for the group of experiments (57 Ϯ 7% reduction in mean amplitude; p ϭ 0.03) was accompanied by a significant reduction of inspiratory burst duration (T I , 29 Ϯ 8% reduction; p ϭ 0.03) at the defined endpoint of the time series at 32.6 Ϯ 8.1 min. This change in amplitude and T I was accompanied by a nonsignificant change of f R (reduction by 6 Ϯ 9%, p ϭ 0.31) due to a small, insignificant increase in T E (15 Ϯ 12% increase; p ϭ 0.44). Similarly, in mouse slices (n ϭ 6, Fig. 5A) the significant reduction in peak integrated XII amplitude and T I from control values was 60 Ϯ 8% (p ϭ 0.03) and 28 Ϯ 3% (p ϭ 0.03), respectively, without a significant change in f R (8 Ϯ 9% increase, p ϭ 0.44) or T E (2 Ϯ 10% decrease, p ϭ 0.69) at 20.28 Ϯ 3.4 min after bath-application of 50 M 9-phenanthrol. In these pharmacological experiments, as well as those described below, we obtained only partial recovery of XII burst amplitude after up to 1 h of continuous drug washout.
Comparable data sets for perturbations of XII amplitude and T I after bath application of the I CAN blocker FFA (50 M) are shown, respectively, for rat and mouse slices in Figs. 4C and 5C. FFA reduced the group (n ϭ 6) mean XII amplitude and T I , respectively, in rat slices (Fig. 4C) by 57 Ϯ 5% (p ϭ 0.03) and 25 Ϯ 6% (p ϭ 0.03), while the mean f R was unchanged (0 Ϯ 10% change from control, p ϭ 1.00) at the 28.3 Ϯ 4.1-min endpoint. FFA significantly reduced the group mean (n ϭ 6) XII amplitude in mouse slices (Fig. 5C) by 45 Ϯ 12% (p ϭ 0.03), although T I was not significantly reduced (5 Ϯ 1% reduction, p ϭ 0.06) and f R was nonsignificantly increased (16 Ϯ 12%, p ϭ 0.31) from control values at the quasi-steady state We also performed control experiments (n ϭ 6 each in rats and mice), in which the XII nerve activity was recorded for 60 min without application of any pharmacological agents, and found no significant changes in inspiratory burst amplitude of XII nerve activity (100 Ϯ 3% in rats and 99 Ϯ 4% in mice compared with the control value at 60 min; p ϭ 0.36 in rats and p ϭ 0.36 in mice).
Simultaneous perturbations within pre-BötC excitatory circuits and hypoglossal motor output in vitro with bath-applied channel inhibitors
Because TRPM4 and TRPC3 channels were expressed in pre-BötC and XII inspiratory neurons, reductions in XII activity amplitude could potentially reflect reduced excitability of XII motoneurons. To establish contributions of pre-BötC neurons, we analyzed correlations between the observed perturbations in motor output amplitude and perturbations of pre-BötC excitatory neuron population activity. For these experiments, we used in vitro rhythmic slices from VgluT2-GCaMP6 transgenic mice expressing the fluorescent Ca 2ϩ -sensor GCaMP6f in glutamatergic neurons to image neuronal and population activity within the pre-BötC (see Methods) during simultaneous recording of XII motor output (Fig. 7). Application of 50 M 9-phenanthrol, Pyr3, or FFA to the transgenic mouse slice preparations significantly decreased the amplitude of the field ⌬F (i.e., F -F 0 ), indicating reduced excitatory neuron population activity, which typically reached quasi-steady state by 20 min after drug application (e.g., see Fig. 7D) and was accompanied by a significant reduction in amplitude of integrated XII activity. The time-dependent reductions of ⌬F and ͐ XII amplitudes (XII Amp) were linearly correlated (see regression lines and Pearson correlation coefficients for data in Figs. 7D and 8C). The reductions in amplitude of field ⌬F normalized to control values with 9-phenanthrol (n ϭ 5), Pyr3 (n ϭ 5), and FFA (n ϭ 4) were 49 Ϯ 7%, 52 Ϯ 6%, and 39 Ϯ 8%, respectively, at 20 min after drug administration (Fig. 8B). These amplitude re-continued stantaneous f R and T I ; solid lines: running median). Right panels show group summary of mean time course (solid lines) and SEM (light-color bands) of normalized integrated XII amplitude (XII Amp), f R , and T I after drug administration (n ϭ 6 slice preparations each). Time of drug administration is indicated by vertical dashed lines. ductions were significant over time in all cases: for 9-phenanthrol, the Spearman correlation coefficient r s ϭ -0.88 (p ϭ 0.017), for Pyr3 r s ϭ -0.63 (p ϭ 0.0028), and for FFA r s ϭ -0.72 (p ϭ 0.031). The ⌬F amplitude perturbations were accompanied by nonsignificant changes in normalized f R for the imaged population (Fig. 8B) of 14 Ϯ 6%, -17 Ϯ 18%, and 5 Ϯ 6% at 20 min with 9-phenanthrol (r s ϭ 0.14, p ϭ 0.52), Pyr3 (r s ϭ -0.11, p ϭ 0.60), and FFA (r s ϭ -0.30, p ϭ 0.22), respectively. We performed control experiments (Fig. 8A, n ϭ 5 mice) to test for possible photobleaching or time-dependent changes in population activity, in which calcium imaging was performed without any drug application with exactly the same protocol of image acquisition as the pharmacological experiments. The results showed that there were no significant changes in the pre-BötC field ⌬F (102 Ϯ 3% of control at 20 min, Spearman correlation coefficient r s ϭ 0.11; p ϭ 0.62), integrated XII amplitude (99 Ϯ 3%, r s ϭ -0.078; p ϭ 0.74), and respiratory frequency (100 Ϯ 2%, r s ϭ 0.092; p ϭ 0.70).
We also tracked perturbations of Ca 2ϩ transients of individual pre-BötC glutamatergic neurons (cell ⌬F) in relation to population-level (field ⌬F) perturbations (Figs. 9 and 10). The reduction in mean amplitude of cell ⌬F (normalized to control amplitudes) for sets of imaged neurons with inspiratory Ca 2ϩ transients was correlated with the reduction in normalized field ⌬F over time after drug application. We note that some inspiratory neurons in these experiments exhibited normalized ⌬F values that were unaffected or increased during drug application (outlier points in Fig. 10). Regardless, the mean cell ⌬F for the entire group of inspiratory neurons analyzed was strongly correlated (see identity lines in Fig. 10) with the mean field ⌬F.
Perturbations of pre-BötC activity and motor output by TRPM4 and TRPC3 channel inhibitors in perfused brainstem-spinal cord preparations in situ
We analyzed contributions of endogenously active TRPM4 and TRPC3 channels to rhythm and motor pattern generation in mature rat and mouse arterially perfused brainstem-spinal cord preparations in situ to assess functional roles in more intact respiratory circuits generating a three-phase rhythmic activity pattern similar to that in vivo. We analyzed perturbations of extracellularly recorded pre-BötC and PN nerve inspiratory activities as well as VN inspiratory and post-inspiratory (post-I) activities after systemic application of 9-phenanthrol and Pyr3 in the brainstem-spinal cord via perfusion solution.
Discussion
Biophysical mechanisms generating and transmitting rhythmic activity within excitatory pre-BötC circuits remain undefined despite extensive experimental and modeling studies investigating possible Na ϩ -and Ca 2ϩ -based mechanisms of rhythmic pre-BötC cellular and population-level bursting activity . Ca 2ϩ signaling-based theories incorporating I CAN , postulated to be mediated by Ca 2ϩ -activated TRPM4 channels (Mironov, 2008;Del Negro et al., 2010), have been proposed. However, TRPM4 and other potentially important TRP channels mediating nonselective cationic currents involved in Ca 2ϩ -related signaling such as TRPC3, although proposed, have not been identified in pre-BötC and other respiratory neurons. Furthermore, their functional roles in respiratory circuits have not been clearly defined.
We obtained evidence for TRPM4 and TRPC3 channel mRNA in pre-BötC inspiratory neurons as well as medullary respiratory motoneurons. The pharmacologically induced perturbations of circuit activity by their putative selective channel inhibitors indicate endogenous activity of these channels with major functional roles in formation of inspiratory and post-inspiratory respiratory activity. However, our results indicate that these channels do not contribute to the generation and stability of inspiratory rhythm in pre-BötC circuits. These results therefore do not support previous Ca 2ϩ signaling-based hypotheses incorporating TRPM4/I CAN in pre-BötC neurons as a fundamental mechanism for rhythm generation.
TRPM4 and TRPC3 channels in respiratory neurons
Our initial survey of neuronal expression of TRPM4 and TRPC3 channels by antibody labeling in neonatal/adult rats and mice, including in genetically specified excitatory and inhibitory neurons in mice, established that these channels are present in neurons in the pre-BötC region and adjacent medullary respiratory-related regions as well as in motor nuclei known to contain respiratory neurons. We assayed for TRPM4 and TRPC3 channel mRNA in identified inspiratory pre-BötC neurons and motoneurons, which has previously not been performed, although the presence of these channels has been suggested by TRPM4/5 mRNA or TRPC3/C7 channel protein detection in bulk tissue obtained from the pre-BötC region (Crowder et al., 2007;Ben-Mabrouk and Tryba, 2010). Similarly, TRPM4 channel mRNA has been detected in lasercaptured XII rat motoneurons (Alvares et al., 2014), and immunolabeling of TRPM4 channel protein in mouse NA has been reported (Del Negro et al., 2010). These approaches have not specifically established channel expression in respiratory neurons.
Our scmRT-PCR and immunohistological analyses identified TRPM4 and/or TRPC3 mRNA in both excitatory and inhibitory pre-BötC inspiratory neurons in slices from neonatal rats and mice. Coexpression of mRNA for these two channels in single pre-BötC inspiratory neurons was Figure 8. Time-dependent changes in the amplitude of integrated XII inspiratory burst activities (XII Amp) and the field GCaMP6f fluorescence transients (⌬F) of the pre-BötC glutamatergic population after TRPM4, TRPC3, and I CAN inhibitors. A, Control experiments (n ϭ 5 mice) to test for possible photobleaching and time-dependent changes in population activity, in which calcium imaging was performed without any drug application with exactly the same protocol of image acquisition as the pharmacological experiments. The results (mean normalized values Ϯ SEM) show that there were no significant changes in the pre-BötC field ⌬F amplitude, XII Amp (normalized to control values), and normalized inspiratory burst frequency (f R ). B, Group summary data (mean normalized values Ϯ SEM) for 9-phenanthrol, Pyr3, and FFA (n ϭ 5, 5, and 4, respectively) shows reduction in both XII Amp and the pre-BötC field ⌬F amplitude, while f R changed nonsignificantly after applying channel inhibitors in all cases. C, Time-dependent reductions of XII Amp and field ⌬F amplitudes after drug application were positively correlated (solid lines: linear regression; Pearson linear correlation coefficient for 9-phenanthrol, Pyr3, and FFA: r ϭ 0.864, 0.845, and 0.749, respectively). The linear regression on mean amplitude reduction between XII Amp and field ⌬F for 9-phenanthrol, Pyr3, and FFA yielded corresponding linear models with slopes m ϭ 0.859, 0.463, and 0.676, and intercepts b ϭ 0.103, 0.499, and 0.277, respectively. Dashed lines represent the identity line.
found in nearly half of the excitatory neurons. Our sample of inspiratory pre-BötC inhibitory neurons was not sufficient to allow conclusions about channel coexpression in these neurons. Although TRPM5 mRNA (Crowder et al., 2007) and TRPC7 channel protein (Ben-Mabrouk and Tryba, 2010) in the pre-BötC region has been reported, we did not systematically probe for other TRPM/C channels in our sample of pre-BötC inspiratory neurons. Consistent with our immunolabeling results, we also found mRNA for TRPM4 and TRPC3 channels in functionally identified XII and NA inspiratory motoneurons. In general, our findings imply that TRPM4/C3 channels may be func- . Effects of TRPM4, TRPC3, and I CAN channel inhibitors on inspiratory Ca 2ϩ activity of the pre-BötC field and glutamatergic neurons expressing GCaMP6f. A, Example of relationship between normalized pre-BötC peak field GCaMP6f ⌬F and normalized individual inspiratory cell ⌬F, 10 and 20 min after bath-applied 50 M 9-phenanthrol. Eight neurons (shown in Fig. 9) were tracked through time (connected green dots) within the two-photon optical section using automated ROI detection. Group mean values Ϯ SEM of the normalized fluorescence transients are plotted (green filled circles with error bars) and the identity line (dashed) is indicated. Note that two of the neurons showed augmented fluorescence transients in this example, but the mean group cellular ⌬F nevertheless followed the field ⌬F. B, Group summary of effects of TRPM4, TRPC3, and I CAN inhibitors on the inspiratory pre-BötC field ⌬F and cellular ⌬F. Left: mean values of cellular ⌬F (red, n ϭ 6 neurons; green, n ϭ 8, same as A) Ϯ SEM during control period, 10 min, and 20 min after bath-applied 9-phenanthrol from two slices are indicated (diamonds and error crosses). Inspiratory neurons with unaffected or increased ⌬F amplitude included in the group statistics are plotted individually at the top. Middle: three-experiment summary for Pyr3 (red, n ϭ 6 neurons; green, n ϭ 13; blue, n ϭ 4). Right: two-experiment summary for FFA (red, n ϭ 12 neurons; green, n ϭ 6). Identity line (dashed) is indicated. tionally involved at multiple levels within medullary respiratory circuits.
Role of TRPM4 and TRPC3 channels in respiratory pattern generation in vitro
We analyzed functional contributions of TRPM4 and TRPC3 channels with the selective pharmacological inhibitors 9-phenanthrol and Pyr3, respectively, initially within rhythmically active in vitro slices from neonatal rats and mice. Perturbations of circuit activity were compared with those caused by FFA, a blocker of I CAN and TRPM4 (Guinamard et al., 2013), that has been used previously to evaluate roles of this current in generating rhythmic bursting activity of respiratory neurons and circuits in vitro (e.g., Peña et al., 2004;Del Negro et al., 2005). We empirically determined from initial in vitro studies and subsequently used concentrations (maximally 50 M for the data presented) of 9-phenanthrol, Pyr3, and FFA that produced large or near-maximal perturbations of the amplitude of inspiratory motor outputs in vitro, and are also expected to be effective and relatively selective for TRPM4, TRPC3, and I CAN in physiologic preparations (Kiyonaka et al., 2009;Guinamard et al., 2013Guinamard et al., , 2014. The three channel inhibitors similarly reduced the amplitude of XII inspiratory motoneuronal activity in vitro without significant perturbations of inspiratory frequency and regularity of the rhythm. These amplitude perturbations indicate that currents inhibited by 9-phenanthrol, Pyr3, or FFA are endogenously active in respiratory neurons in vitro. The similar perturbations with 9-phenanthrol and FFA are consistent with the proposal from pharmacological studies in other cells and tissues that FFA blocks TRPM4 channels (Guinamard et al., 2014), as well as the proposal that TRPM4, known to be directly activated by increases in intracellular Ca 2ϩ , mediates an I CAN (Launay et al., 2002;Hofmann et al., 2003) active in respiratory neurons.
In previous pharmacological studies investigating the role of I CAN in active respiratory circuits in neonatal mouse slices in vitro (Peña et al., 2004), high concentrations of FFA (500 M) reduced the amplitude/area of integrated inspiratory population activity but caused a relatively small (ϳ20%) reduction of inspiratory burst frequency without affecting regularity of the rhythm. Although these high concentrations of FFA can also depress voltagegated Na ϩ currents (at Ͼ100 M in hippocampal neurons; Yau et al., 2010) and voltage-gated Ca 2ϩ currents (Shimamura et al., 2002), as well as cause other nonselective perturbations of neuronal excitability (Guinamard et al., 2013), these observations are generally consistent with our results with FFA in both neonatal rat and mouse in vitro slices showing relatively large perturbations of the amplitude of pre-BötC and XII motoneuronal inspiratory activity and only small perturbations of inspiratory frequency without affecting stability of the inspiratory rhythm. In other earlier in vitro mouse slice experiments, 100 M FFA reduced inspiratory drive potentials of individual pre-BötC neurons, but without reducing the amplitude or altering the frequency of XII motor discharge (Del Negro et al., 2005;Pace et al., 2007). The lack of perturbations of XII inspiratory activity at this concentration of FFA is difficult to reconcile with our electrophysiological and Ca 2ϩ imaging results showing correlations between the amplitude of pre-BötC inspiratory activity, as assessed by intracellular Ca 2ϩ dynamics (below), and the reduction of XII inspiratory discharge amplitude. In those earlier studies, however, higher concentrations (300 -350 M) of FFA that are considered nonselective for I CAN reduced the XII inspiratory activity amplitude and could have eliminated rhythmic inspiratory XII motor output.
Consistent with our scmRT-PCR results indicating TRPC3 channel mRNA expression in identified inspiratory neurons, the amplitude perturbations produced by Pyr3 suggest that TRPC3 channels are also functionally activated during respiratory circuit activity in vitro. These channels are not directly Ca 2ϩ activated, but mediate Na ϩ /Ca 2ϩ currents, and may be involved in regulating neuronal Ca 2ϩ -related signaling (Talavera et al., 2008;Birnbaumer, 2009;Guinamard et al., 2013) in respiratory neurons. This TRPC3 channel-mediated Ca 2ϩ flux/intracellular Ca 2ϩ regulation can potentially also affect TRPM4/I CAN in respiratory neurons. We found coexpression of mRNA for TRPC3 and TRPM4 channels in approximately half of the excitatory pre-BötC inspiratory neurons and all of the respiratory motoneurons assayed, suggesting that such a functional interaction may be possible. The roles of TRPC3-mediated cationic currents/Ca 2ϩ -related signaling in generating respiratory neuron activity have not been previously investigated. Our results suggest an important functional role of these channels in activity amplitude modulation but not in rhythmogenesis, like TRPM4/I CAN channels. Whether the similar amplitude perturbations by the TRPC3 and TRPM4 channel inhibitors reflects involvement with TRPM4/ I CAN activation by Ca 2ϩ -related functions of TRPC3 channels remains to be determined.
In general, the optimal pharmacological strategy for probing the roles of TRPM4/I CAN or TRPC3 channels active in respiratory circuit neurons has not been definitively established. The major problem is to identify and analyze the neuronal currents attenuated by the channel inhibitors in respiratory neurons at any applied concentrations. Measurements of whole-cell currents mediated by TRPM4/I CAN or TRPC3 channels in respiratory neurons have not yet been performed, so that 9-phenanthrol and Pyr3 concentrations likely to be effective/selective have been inferred in part from pharmacological analyses performed in other (typically nonneuronal) cell types (Kiyonaka et al., 2009;Guinamard et al., 2014). The problem is particularly complicated for resolving the Ca 2ϩ -activated TRPM4/I CANmediated currents, because the sources of Ca 2ϩ flux activating these currents in respiratory neurons need to be preserved and taken into account in a detailed pharmacological analysis of currents activated endogenously during respiratory neuronal activity. Although Ca 2ϩ flux through voltage-gated Ca 2ϩ channels (Peña et al., 2004;Morgado-Valle et al., 2008), and/or synaptically activated Ca 2ϩ -fluxes, including through ionotropic glutamatergic receptors and/or activation of metabotropic glutamatergic receptors to induce ER Ca 2ϩ release, have been postulated to activate I CAN Mironov, 2008;Del Negro et al., 2010), these mechanisms have not been established.
Correlated perturbations of pre-BötC excitatory neuronal population activity and hypoglossal motor output in vitro
We initially evaluated roles of TRPM4 and TRPC3 channels by analyzing perturbations of XII inspiratory motor output in slices, but this approach does not necessary allow assessment of the contributions of these channels in pre-BötC neurons to perturbations of the motor output, since TRPM4 and TRPC3 channels are also expressed in XII inspiratory neurons. We also note that TRPM4 or TRPC3 channel antibody labeling was identified in regions of the reticular formation dorsal to the pre-BötC region that contains inspiratory XII premotoneurons (Koizumi et al., 2008;Revill et al., 2015). Accordingly, reductions in XII activity amplitude by channel inhibitors in our in vitro slice preparations potentially reflect reduced activity of XII motoneurons and possibly other neurons within inspiratory drive transmission circuits. We therefore more directly established involvement of pre-BötC neurons by dynamic Ca 2ϩ imaging of inspiratory pre-BötC neuronal activity in slices from transgenic mice expressing GCaMP6f in glutamatergic neurons during bath application of the channel inhibitors. This allowed us to assess activity perturbations of the critical populations of pre-BötC excitatory neurons generating inspiratory rhythm and synaptic drive in transmission circuits to XII motoneurons, and to compare simultaneous perturbations of activity of these neurons and XII motor output. All of the channel inhibitors caused reductions in the amplitude of spatially averaged GCaMP6f fluorescence transients (field ⌬F) and the fluorescence transients of individual glutamatergic inspiratory pre-BötC neurons (cell ⌬F), confirming inspiratory activity perturbations at the level of pre-BötC excitatory neurons. Furthermore, we established that the field ⌬F reflects the mean cell ⌬F of sets of individual excitatory inspiratory neurons. We also found significantly correlated, linear relationships (Fig. 8) between the amplitude of field ⌬F and the amplitude of integrated XII inspiratory activity. The slopes of these linear relationships for 9-phenanthrol, FFA, and Pyr3 (0.86, 0.68, and 0.46, respectively), particularly with Pyr3, reflect that initially the activity amplitude perturbations of our imaged sample of inspiratory glutamatergic neurons tended to occur more rapidly than the reduction in integrated XII activity, but the group mean amplitude perturbations tended to converge toward the identity line of the field ⌬F versus integrated XII amplitude relationships as the quasi-steady-state perturbations were reached. Our results suggest that the number of active pre-BötC inspiratory cells and the burst amplitude of each active pre-BötC neuron after application of the channel inhibitors are important factors contributing to the overall reduction of pre-BötC field ⌬F and decrease of XII burst amplitude.
We conclude that the perturbations of pre-BötC inspiratory activity correlate with the perturbation of XII inspiratory activity amplitude. The ⌬F amplitude perturbations occurred without significant changes in the frequency of inspiratory-related activity within the pre-BötC and simultaneously recorded XII inspiratory activity. The extent to which activity perturbations of XII inspiratory motoneurons or neurons within the rhythmic inspiratory drive transmission premotor circuits contribute to the reduction of XII motor output remains to be determined. We also note that there is a subpopulation of pre-BötC inspiratory neurons with axonal projections to XII motoneurons (Koizumi et al., 2008(Koizumi et al., , 2013, and reduced activity of these neurons may also contribute to the overall reduction of inspiratory activity in the transmission circuits without perturbing rhythm generation.
Contributions of TRPM4 and TRPC3 channels to respiratory pattern generation in mature rodent brainstem circuits in situ
We also analyzed functional contributions of TRPM4 and TRPC3 channels with 9-phenanthrol and Pyr3, respectively, in mature rat and mouse arterially perfused in situ brainstem-spinal cord preparations to evaluate roles of these channels in more intact circuits generating a eupneic-like three-phase respiratory motor output pattern. Moreover, extending our analysis to mature animals was necessary, since I CAN -dependent neuronal bursting, and accordingly potential involvement of TRPM4, has been proposed to contribute to inspiratory rhythm generation predominantly in mice older than P5 (Peña et al., 2004;Del Negro et al., 2005), although I CAN is postulated to contribute to formation of drive potentials generating inspiratory bursts throughout development (Del Negro et al., 2005). In agreement with our results obtained in vitro, presumptive inhibition of TRPM4 or TRPC3 channels in the more intact rat and mouse circuits in situ significantly reduced the amplitude of pre-BötC inspiratory activity, accompanied by reduced amplitudes of inspiratory motor outputs as evaluated from integrated vagal and phrenic nerve inspiratory activities. In addition, the channel inhibitors, especially 9-phenanthrol, caused large reductions in vagal post-I activity (e.g., Fig. 11), indicating an important contribution of endogenous channel activation to inspiratory-expiratory pattern generation in more intact respiratory circuits. The increase of respiratory frequency, primarily with TRPM4 channel inhibition, occurred with the reduction of expiratory phase duration as post-I activity was reduced , although rhythm generation was not disrupted.
The reduction of post-I vagal activity could reflect contributions of TRPM4 and TRPC3 channel activation at the level of vagal motoneurons, or at the interneuronal level in excitatory/inhibitory circuits in ventral medullary respiratory-related regions, including within the BötC, that generate post-I activity Richter and Smith, 2014) and where these channels may be present in excitatory/inhibitory neurons as suggested by our immunolabeling results. According to the respiratory central pattern generation (CPG) network model based on experimental analyses with in situ preparations (Rubin et al., 2009b), different types (e.g., neurotransmitter phenotypes, active phase, bursting pattern) of respiratory interneurons in the pre-BötC and BötC are functionally interacting to generate a normal three-phase pattern of respiratory neural activity. Our experimental results of different effects of TRPM4 or TRPC3 channel inhibition on f R in the more intact in situ preparations (Fig. 12) suggest different sensitivity to TRPM4 or TRPC3 channel inhibitors among different types of respiratory neurons in the CPG circuits. Contributions of TRPM4 and TRPC3 channel activation in different types of excitatory and/or inhibitory respiratory neurons remain to be clarified.
Our results suggest that inhibiting TRPM4/I CAN or TRPC3 in excitatory pre-BötC inspiratory neurons primarily contributes to the amplitude decrease of inspiratory motor outputs in vitro and in situ, whereas inhibiting TRPM4 or TRPC3 channels in inhibitory neurons, possibly BötC expiratory neurons, in the more intact in situ circuits causes perturbations of post-I activity (e.g., Marchenko et al., 2016) and therefore respiratory frequency. In summary, we suggest that endogenous activation of TRPM4/ I CAN or TRPC3 plays an important role in regulating activity of excitatory and inhibitory respiratory neurons, the latter particularly in the intact in situ CPG circuits for inspiratory-expiratory pattern generation.
Implications for proposed I CAN -dependent and other mechanisms of respiratory rhythm generation in pre-BötC circuits
Based on previous experimental studies in neonatal mouse rhythmic medullary slices in vitro (Crowder et al., 2007;Pace et al., 2007;Pace and Del Negro, 2008) and organotypic pre-BötC cultures (Mironov, 2008(Mironov, , 2013, and also computational modeling studies (Rubin et al., 2009a;Dunmyre et al., 2011), an emergent I CAN -dependent mechanism in pre-BötC excitatory circuits was postulated to play a major role in respiratory rhythmogenesis according to the "group pacemaker" hypothesis (Feldman and Del Negro, 2006;Del Negro et al., 2010). In this model, synapticallyactivated Ca 2ϩ fluxes, especially mediated by metabotropic glutamate receptors (mGluRs), were proposed to trigger I CAN activation through intracellular Ca 2ϩ signaling involving inositol triphosphate (IP 3 )-mediated Ca 2ϩ release from ER stores. Activation of I CAN is proposed to generate depolarization of excitatory inspiratory pre-BötC neurons to primarily produce synaptically mediated (i.e., network-dependent) inspiratory drive potentials underlying inspiratory bursts (Crowder et al., 2007;Pace et al., 2007;Pace and Del Negro, 2008). During population-level inspiratory bursts, the I CAN -dependent depolarization has been suggested to cause partial voltage-dependent inactivation of neuronal spikegenerating transient Na ϩ channels, associated with transient depression of recurrent excitation and circuit-generated excitatory synaptic drive to deactivate I CAN and terminate inspiratory bursts (Rubin et al., 2009a;Del Negro et al., 2010). In other more complex models with multiple sources of neuronal Ca 2ϩ flux (Jasinski et al., 2013;Rybak et al., 2014), including voltage-gated Ca 2ϩ currents, it has been theoretically shown that I CAN -induced bursting, and subsequent burst termination sufficient for rhythmogenesis, can occur by dynamic Ca 2ϩ -dependent activation-inactivation of IP 3 receptor-mediated Ca 2ϩ release, without or with involvement of other burst-terminating mechanisms such as Na ϩ /K ϩ pump currents. The Na ϩ /K ϩ pump currents can hypothetically contribute importantly to inspiratory burst termination and may be regulated by I CAN -mediated Na ϩ flux, linking I CAN activation to another mechanism for inspiratory burst termination critical for rhythm generation. Interfering with ER Ca 2ϩ release mechanisms does not disturb inspiratory rhythm generation in the pre-BötC in vitro, however, indicating that normally this source of Ca 2ϩ flux is not critically involved in rhythm generation or control of inspiratory amplitude in vitro (Beltran-Parrazal et al., 2012), so other Ca 2ϩ sources explored in these models seem to be involved in activating I CAN .
Another important hypothesis for inspiratory rhythm generation long proposed in the field is that I CAN -dependent, FFA-sensitive pre-BötC inspiratory pacemaker neurons (i.e., inspiratory neurons with intrinsic oscillatory bursting properties when isolated from synaptic inputs), with I CAN activation driven by voltage-gated, Cd ϩ2 -sensitive Ca 2ϩ currents, have a critical rhythmogenic role (Peña et al., 2004) in pre-BötC circuits together with other populations of neurons with oscillatory bursting properties mediated by persistent Na ϩ current (I NaP ; dual pacemaker hypothesis; Thoby-Brisson and Ramirez, 2001;Peña et al., 2004;Ramirez et al., 2011), following the proposal of I NaP -dependent cellular and excitatory population rhythm generation mechanisms in pre-BötC excitatory circuits (Butera et al., 1999a, b).
In general, our results do not support the concept that populations of pre-BötC neurons with I CAN -mediated bursting properties are critically involved in generating inspiratory rhythm. However, they support the proposal that TPRM4/I CAN -mediated currents are functionally active in respiratory neurons and importantly contribute to inspiratory burst generation determining the amplitude of pre-BötC neuronal population activity. TRPC3 channels also have this fundamental role, possibly by providing Ca 2ϩ flux activating TRPM4/I CAN . Remarkably, this amplitude control is essentially independent of the inspiratory rhythm generation mechanism and indicates there is a rhythmogenic kernel subpopulation of neurons within the pre-BötC excitatory network that rely on a fundamentally different oscillatory mechanism. Previous studies have proposed (Butera et al., 1999a, b) and presented evidence (Koizumi and Smith, 2008) that I NaP -dependent mechanisms are sufficient to account for a number of features of inspiratory rhythm generation when neonatal pre-BötC circuits are isolated in vitro, as well as in reduced in situ preparations from mature rats . In the more intact mature system, this I NaP -dependent oscillatory mechanism may not be sufficient to explain rhythm generation, which involves more complex sets of inhibitory circuit interactions with the pre-BötC excitatory circuits Rubin et al., 2009b). The present studies indicate that although TPRM4/I CAN -mediated currents are functionally active in the more intact system and have a basic role in inspiratory-expiratory respiratory pattern generation, they are also not essential for rhythm generation. | v3-fos-license |
2016-05-04T20:20:58.661Z | 2012-01-21T00:00:00.000 | 208925953 | {
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} | pes2o/s2orc | 2,4-Diphenyl-6-trifluoromethyl-2,3-dihydro-1H,5H-pyrrolo[3,4-c]pyrrole-1,3-dione
The asymmetric unit of the title compound, C19H11F3N2O2, contains two crystallographically unique molecules which differ in the rotation of a phenyl ring and a –CF3 substituent. The dihedral angles involving the pyrrole ring and the attached phenyl ring are 62.82 (8) and 71.54 (7)° in the two molecules. The difference in the rotation of the CF3 groups with respect to the pyrrolo rings to which they are attached is 23.5(1)°. For one molecule, there is a close contact between an H atom and the centroid of the phenyl ring of an adjacent molecule (2.572 Å). A similar contact is lacking in the second molecule. In the crystal, N—H⋯O interactions connect adjacent molecules into a chain normal to (01). Crystallographically unique molecules alternate along the hydrogen-bonded chains.
The asymmetric unit of the title compound, C 19 H 11 F 3 N 2 O 2 , contains two crystallographically unique molecules which differ in the rotation of a phenyl ring and a -CF 3 substituent. The dihedral angles involving the pyrrole ring and the attached phenyl ring are 62.82 (8) and 71.54 (7) in the two molecules. The difference in the rotation of the CF 3 groups with respect to the pyrrolo rings to which they are attached is 23.5(1) . For one molecule, there is a close contact between an H atom and the centroid of the phenyl ring of an adjacent molecule (2.572 Å ). A similar contact is lacking in the second molecule. In the crystal, N-HÁ Á ÁO interactions connect adjacent molecules into a chain normal to (011). Crystallographically unique molecules alternate along the hydrogenbonded chains.
Comment
The biological activity of compounds with pyrrol-3,4-dicarboximide scaffolds includes analgesic, central nervous system depressive action, and antiproliferative activities (Malinka et al. 2005;Malinka et al. 1999;Shen et al. 2010). Furthermore, pyrrol-3,4-dicarboximides are very interesting compounds because of their structural similarity to lamellarins (Yu et al. 2011). The title compound was synthesized and its crystal structure is reported herein.
The asymmetric unit contains two molecules of the title compound (see Figure 1 for a view of the molecular structure).
After overlapping the central fused ring of the independent molecules using OLEX2 (see Figure 2; Dolomanov et al., 2009), it is clear that the molecules differ only in rotation of the phenyl ring and CF 3 substituents. The phenyl ring planes differ by approximately a 41° rotation about the N-C bond. The CF 3 substituent is rotated by 16.5°. The r.m.s. deviation of atomic positions between the two molecules is 0.56 Å (all atoms), 0.065 Å for matched atoms. The center of one of the phenyl rings that differ in orientation (C7-C12) has a close contact (2.572 Å) to the hydrogen atom bonded to C14 on a symmetry related molecule. This contact is lacking for the other molecule.
Intermolecular hydrogen bonds connect molecules into a ribbon throughout the crystal. Figure 3 shows molecular packing and hydrogen bonds in the crystal. Hydrogen bonds exist between O1 and N3 (2.8395 Å) and O4 and N1 (2.8757 Å) and connect molecules into a chain normal to (0 1 -1). Crystallographically unique molecules alternate along the hydrogen bonded chains. The graph set description is C2,2(12)>a>b (determined using Mercury (Macrae et al., 2008)).
Refinement
All hydrogen atoms were visible in a difference Fourier map and were added at calculated positions. Bonds distances are set to 0.95 Å for carbon-hydrogen bonds, and 0.88 Å for nitrogen-hydrogen bonds. | v3-fos-license |
2019-06-11T18:22:56.070Z | 2019-06-09T00:00:00.000 | 189921540 | {
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} | pes2o/s2orc | The Role of Rhodomyrtus tomentosa (Aiton) Hassk. Fruits in Downregulation of Mast Cells-Mediated Allergic Responses
Rhodomyrtus tomentosa, a flowering plant of Myrtaceae family from southern and southeastern Asia, was known to possess a rich source of structurally diverse and various biological activities. In this study, the inhibitory effect of R. tomentosa fruit extract (RFE) on allergic responses in calcium ionophore A23187-activated RBL-2H3 mast cells was investigated. The result showed that RFE was able to inhibit mast cell degranulation via decreasing β-hexosaminidase release and intracellular Ca2+ elevation at the concentration of 400 μg/ml. Moreover, the suppressive effects of RFE on the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evidenced. In addition, RFE effectively scavenged DPPH radical and suppressed the reactive oxygen species generation in a dose-dependent manner. Notably, the pretreatment of RFE caused the downregulation of tyrosine kinase Fyn phospholipid enzyme phospholipase Cγ (PLCγ), extracellular-signal-regulated kinase (ERK), and nuclear factor kappa B (NF-κB) phosphorylation. These results indicated that RFE could be a promising inhibitor of allergic responses and may be developed as bioactive ingredient for prevention or treatment of allergic diseases.
Introduction
Allergy is a hypersensitivity disorder that relates to exaggerated reaction of the immune system to harmless environmental substances such as animal dander, house dust mites, foods, pollen, insects, and chemical agents [1]. Allergic rhinitis, asthma, and atopic eczema are among the most common allergic diseases [2]. The prevalence, severity, and complexity of these diseases in the population are rapidly rising. So far, various medicines such as antihistamine, mast cell stabilizers, and immune suppressors have been applied for ameliorating allergic symptoms and reducing the suffering of anaphylaxis [3]. However, these medicines were not available to cure allergic diseases completely and exhibited several side effects [4]. Therefore, the discovery of safe and efficient therapeutics derived from natural products for prevention and treatment of allergic diseases is necessary.
Rhodomyrtus tomentosa is a flowering plant that belongs to the family Myrtaceae and is native to southern and southeastern Asia. It has been used in traditional Vietnamese, Chinese, and Malaysian medicine for a long time for treatment of diarrhea, dysentery, gynecopathy, stomachache, and wound-healing [5]. Moreover, R. tomentosa has been known to contain a rich source of structurally diverse and biologically active metabolites such as triterpenes, steroids, and phenolic compounds [6,7]. In particular, various biological activities of R. tomentosa have been evaluated and reported recently [8]. Hence, it is considered as a potential source for exploring novel therapeutic agents. In the present study, the biological activity of R. tomentosa was further evaluated via investigating its inhibitory capacity on allergic responses in vitro.
Rat basophilic leukemia (RBL-2H3) cells display properties of mucosal-type mast cells. The activation of these cells leads to the release and generation of several inflammatory mediators [9]. Thus, RBL-2H3 cells have been commonly and successfully used in in vitro studies for screening antiallergic agents. Herein, RBL-2H3 cells were used as an in vitro model 2 BioMed Research International for evaluation of antiallergic activity of R. tomentosa fruit extract.
Materials and Methods
2.1. Materials. R. tomentosa fruits were purchased from Duong Dong Town, Phu Quoc district, Kien Giang province. ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). The antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2.
Extraction. R. tomentosa fruits were air-dried under shade and powdered using a grinder. The powder was soaked with ethanol 80% under the extract conditions of ratio (1/4, w/v), time (4 h), and temperature (60 ∘ C). The R. tomentosa fruit extract (RFE) was kept at 4 ∘ C for further investigation.
Cell Viability.
The cytotoxic effect of the extract on RBL-2H3 cells was examined by MTT assay as previously described [10]. The cells (1x10 5 cells/ml) were treated with the extract (100, 200, or 400 g/ml) for 24 h before incubation with MTT solution (1 mg/ml, final concentration) for 4 h. The supernatant was then removed, and DMSO (100 l) was added to solubilize the formed formazan salt. The absorbance was measured at 540 nm using a microplate reader (GENios5 Tecan Austria GmbH, Austria). The cell viability was shown as a percentage compared to blank.
Degranulation
Assay. RBL-2H3 cells (2 × 10 5 cells/ml) were pretreated with the extract (100, 200, or 400 g/ml) for 24 h. The culture medium was replaced by Tyrode buffer before stimulation with calcium ionophore A23187 (1 M) for 30 min at 37 ∘ C. The level of -hexosaminidase release was measured as previously described [11]. The -hexosaminidase releases were calculated as a percentage compared to control: release ratio (%) = (T -B)/(C -B) × 100, where B is blank group, C is control group, and T is the tested group.
Measurement of Cytokine
Production. Different concentration of the extract (100, 200, or 400 g/ml) was added to the culture medium of RBL-2H3 cells (2 × 10 5 cells/ml) for 24 h. The culture medium was then removed and Tyrode buffer was replaced before stimulation with calcium ionophore A23187 (1 M) for 1 h at 37 ∘ C. The supernatants were collected, and the amount of IL-1 or TNF-was measured by ELISA kit.
Measurement of Reactive Oxygen Species Production (ROS).
The extract (400 g/ml) was added to the culture medium of RBL-2H3 cells (1 × 10 3 cells/ml) for 24 h before incubation with dihydroethidium (2 M) for 60 min at 37 ∘ C. The culture medium was then removed and Tyrode buffer was replaced before stimulation with calcium ionophore A23187 (1 M) for 30 min at 37 ∘ C. Paraformaldehyde 3% was used to fix the cells and the fluorescence intensity was conducted under a fluorescence microscope (CTR 6000, Leica, Wetzlar, Germany).
Western Blot Analysis.
The protein expression level was measured by Western blot method. Various doses of the extract (100, 200, or 400 g/ml) were introduced to RBL-2H3 cells for 24 h prior to stimulation with calcium ionophore A23187 (1 M) for 30 min at 37 ∘ C. The procedure of protein detection was performed as previously described [12]. The protein band was visualized using LAS30005 Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan).
Statistical
Analysis. Statistical analysis was performed by using the analysis of variance (ANOVA) test of Statistical Package for the Social Sciences (SPSS). The statistical significance of differences among groups was analyzed using Duncan's multiple range tests, wherein p < 0.05 was considered significant.
Effect of RFE on Mast Cell Degranulation.
Mast cells play an important role in the development of allergic diseases and inflammatory processes [14]. Activation of mast cells triggers a cascade of intracellular events, especially degranulation [15]. Mast cell degranulation is considered to be one of the critical steps in allergic responses, causing the elevation of intracellular Ca 2+ level and the subsequent release of various preformed mediators. These mediators are the origination of various pathophysiologic events in acute allergic responses [16]. Therefore, various antiallergic drugs have been developed so far, which are able to inhibit degranulation of mast cells. In this study, the inhibitory effect of RFE on mast degranulation was evaluated via measuring -hexosaminidase release and intracellular Ca 2+ elevation in the activated RBL-2H3 cells (Figure 1). It was observed that RFE was able to reduce -hexosaminidase release to 79, 63, and 37% at the concentrations of 100, 200, and 400 g/ml (Figure 1(a)). Moreover, the increase in intracellular Ca 2+ level induced by calcium ionophore A23187 was remarkably alleviated by RFE at concentration of 400 g/ml (Figure 1(b)).
In particular, the inhibitory effect of RFE on mast cell degranulation was not due to cytotoxicity (Figure 1(c)). Notably, RFE possessed the similar inhibitory activity as compared with Smilax glabra [17], Morinda citrifolia [18], and grapeseed extract [19]. Evidently, calcium ionophore A23187 induces mast cell degranulation by increasing cellmembrane permeability [20,21]. Thus, antiallergic agents having a membrane-stabilizing action may be desirable such as disodium cromoglycate or sodium hydroxypropylcromate. As a result, RFE may be suggested to stabilize the lipid bilayer membrane, thus reducing the degranulation in RBL-2H3 mast cells. Indeed, various phytochemical components of medicinal plants such as saponins, glycosides, flavonoids, Figure 2: The suppressive effect of RFE on cytokine productions in calcium ionophore A23187-activated RBL-2H3 mast cells. Cells were pretreated with various concentrations of RFE for 24 h before exposure to A23187 for 1 h. The production level of IL-1 (a) and TNF-(b) was quantified in culture media using commercial ELISA kits. Each determination was made in three independent experiments, and the data are shown as means ± SD. Different letters A-D indicate significant difference among groups (p < 0.05) by Duncan's multiple-range test. The same letter indicates that the difference between the means is not statistically significant. If two groups have different letters, they are significantly different from each other at p < 0.05. If a group is similar to A and B or B and C, it was addressed by letters "AB" or "BC," respectively. tannins, and phenolic compounds have been evidenced as mast cell stabilizing agents [22]. Meanwhile, RFE has been reported to contain various tannins and phenolic compounds that may contribute to stabilizing mast cell membrane and preventing degranulation [23].
Effect of RFE on Cytokine
Production. The allergic reactions are also characterized by the production of various cytokines. The activated mast cells are well established as an important source of several cytokines such as tumor necrosis factor-(TNF-), interleukin-1 (IL-1 ), IL-4, IL-6, IL-8, and IL-13 [24]. The excessive production of these cytokines leads to the recruitment of inflammatory cells such as neutrophils and eosinophils, which increase the inflammatory responses [25]. Therefore, the decrease in cytokine production from the activated mast cells is one of the key indicators of the ameliorated allergic symptoms. Herein, the suppressive effect of RFE on IL-1 and TNFproductions was investigated in RBL-2H3 mast cells. It was shown that the production levels of IL-1 and TNFwere increased in the culture supernatants of A23187exposed RBL-2H3 cells (Figure 2). The amount of IL-1 and TNF-from the exposed cells was 167 ± 4.9 and 216 ± 6.4 pg/ml, respectively. Conversely, this increase was significantly reduced in a concentration-dependent manner by RFE pretreatment. At the concentration of 400 g/ml, RFE reduced IL-1 and TNF-levels to 93 ± 5.7 and 80 ± 6 pg/ml, respectively.
Radical Scavenging Activity of RFE.
A free radical is considered as a molecule that contains one or more unpaired electrons in its outermost atomic or molecular orbital. It is generated from endogenous sources such as intracellular autooxidation and inactivation of small molecules or from exogenous sources such as tobacco smoke, certain pollutants, organic solvents, and pesticides [26]. The free radicals are recognized as agents involved in the pathogenesis of allergic diseases [27]. Meanwhile, the antioxidant agents from natural products have been proposed as an approach to reduce the allergic diseases [28]. Herein, RFE was determined to be effective in scavenging radicals (Figure 3). RFE was able to scavenge 85% DPPH radical at the concentration of 400 g/ml (Figure 3(a)). Moreover, the result from light microscope assay showed that the fluorescence density of ROS was markedly decreased in RFE-pretreated group as compared to A23187-stimulated group. It evidenced radicals as an inducer of Ca 2+ elevation and subsequent degranulation in the mast cells [29]. Meanwhile, the blockade of ROS production by DPI or antioxidants such as (-)epigallocatechin gallate suppressed mast cell degranulation [30,31]. These results suggested that the antioxidant activity of RFE may contribute to the depression of mast cell degranulation.
Effect of RFE on the Intracellular Signaling Molecules in the Activated RBL-2H3 Mast Cells.
Evidently, the allergic responses were also indicated by the activation of a cascade of intracellular signaling molecules such as Fyn, PLC , MAPKs, and NF-B [15,32]. It was reported that the activation of Fyn and PLC leads to microtubule polymerization, intracellular Ca 2+ elevation, and subsequent mast cell degranulation [33,34]. Meanwhile, the activation of NF-B triggers gene expression and production [32]. Thus, the inhibition of mast cell degranulation and cytokine production may relate to the inactivation of these intracellular signaling molecules. As shown in Figure 4, the phosphorylation of Fyn, PLC , ERK MAPK, and NF-B was increased in the control group exposed to A23187 alone. Conversely, the RFE pretreatment caused significant suppression on Fyn, PLC , ERK MAPK, and NF-B phosphorylation at the concentration of 400 g/ml. As a result, this suppressive effect of RFE may contribute to the inhibition of mast cell degranulation.
Conclusion
In conclusion, this study has evidenced the inhibitory effect of R. tomentosa fruit extract on allergic responses in A23187-activated RBL-2H3 mast cells. The inhibitory effect has been found due to decreasing -hexosaminidase release and cytokine productions, suppressing ROS production, and downregulating the phosphorylation of tyrosine kinase Fyn and phospholipid enzyme phospholipase C . Therefore, R. tomentosa fruits could offer an attractive strategy for the control of allergic diseases. However, further studies related to safety and efficacy need to be evaluated.
6
BioMed Research International
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that there are no conflicts of interest. | v3-fos-license |
2021-10-22T15:52:01.280Z | 2021-09-01T00:00:00.000 | 239626325 | {
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} | pes2o/s2orc | Emergence of cationic polyamine dendrimersomes: design, stimuli sensitivity and potential biomedical applications
For decades, self-assembled lipid vesicles have been widely used in clinics as nanoscale delivery systems for various biomedical applications, including treatment of various diseases. Due to their core–shell architecture and versatile nature, they have been successfully used as carriers for the delivery of a wide range of therapeutic cargos, including drugs and nucleic acids, in cancer treatment. Recently, surface-modified polyamine dendrimer-based vesicles, or dendrimersomes, have emerged as promising alternatives to lipid vesicles for various biomedical applications, due to their ease of synthesis, non-immunogenicity, stability in circulation and lower size polydispersity. This mini-review provides an overview of the recent advances resulting from the use of biomimetic hydrophobically-modified polyamine-based dendrimersomes towards biomedical applications, focusing mainly on the two most widely used polyamine dendrimers, namely polyamidoamine (PAMAM) and poly(propylene imine) (PPI) dendrimers.
Introduction
Dendrimers are emerging as potential non-viral vectors for efficiently delivering drugs and nucleic acids to the brain and cancer cells. They are polymeric, nanometer-sized synthetic molecules with perfectly branched multiple monomers that emerge radially from a central core similar to a tree (dendron in Greek). [1][2][3][4] Dendrimers consist of three main architectural components: a reactant core molecule, which is considered the origin of the dendrimer, highly branched polymeric layers (or generations) that bind to the core in a specic way to form a uniformly branched spherical macromolecule, and a multivalent periphery. 5,6 Their modiable surface functionalities, low polydispersity and available internal cavities make them particularly attractive as delivery systems for drug and gene delivery applications. 1,2,[4][5][6][7][8] Recently, the ability of surface-modied amphiphilic polyamine dendrimers to form selfassembled nanostructures in water was the object of much scrutiny. These amphiphilic dendrimers were synthesized by conjugating various hydrophobic groups (i.e. aromatic, long hydrophobic, uorinated chains) to the hydrophilic dendrimers. 5,[9][10][11][12][13] The polyamine dendrimer-based vesicles, or dendrimersomes, resulting from their self-assembly, have emerged as promising delivery systems for various biomedical applications.
The self-assembly of amphiphilic molecules in water is a natural phenomenon resulting in various ordered thermodynamically stable nano or microstructures, which is mostly driven by various forces, such as hydrophobic interactions, hydrogen bonding and metal-ligand interactions. [14][15][16][17] Hydrophobic interactions are important factors causing the selfassembly of amphiphilic molecules in aqueous solution, which actually led to the rational design and synthesis of various low-and high-molecular weight amphiphiles with various hydrophobes towards nano or micro assemblies in the last decades. [18][19][20][21] Despite many potential applications, most articial nanostructures constituted of low-and high-molecular weight amphiphiles showed inherent drawbacks due to their in vivo instability and inefficient delivery of cargos, which led this eld evolving to develop enhanced nano-carriers for biomedical applications, especially for the delivery of therapeutic agents in cancer therapy.
Among various self-assembled nanostructures, vesicles, three-dimensional entities with an aqueous lumen separated from the outside aqueous medium by one or more hydrophobic bilayer, are widely studied delivery systems as they resemble the structure of naturally occurring cells, viral capsids or the recently developed exosomes. [22][23][24][25][26] They are advantageous over other delivery systems for biomedical applications as they can carry hydrophobic as well as hydrophilic cargos to the target site of action. Several synthetic vesicles such as liposomes (lipidbased vesicles), polymersomes (polymer-based vesicles) and dendrimersomes (dendrimer-based vesicles) can mimic natural carriers and are currently under investigation for various biomedical investigations, such as therapy, diagnostic, analytical science and synthetic biology.
Over the years, the evaluation of natural and synthetic vesicles for biomedical use appeared to follow the following timeline: viral capsids (natural carriers, but immunogenic), chronologically followed by liposomes (no immunogenicity, but low stability), stealth liposomes (improved stability, but additional complexities due to the presence of several additives such as cholesterol and poly(ethylene glycol) (PEG)), polymersomes (high stability and longer circulation time, but high polydispersity and poor control over size), and nally dendrimersomes (high stability with low polydispersity, excellent physical properties in the bulk state, high degree of branching and a multiple number of end groups at the periphery) ( Fig. 1 and 2). 27-31 The formation of dendrimersomes has mainly been reported from Janus type of dendrimers for various applications over the years, 30,32-34 which is synthetically challenging and involves more time, organic solvents and synthetic skills. On the other hand, a new generation of dendrimersomes made from cationic polyamine dendrimers is less synthetically challenging, less time consuming, requires less organic solvents and less synthetic skills. As a result, the evaluation of these vesicles was the focus of much research, including the recent development of such dendrimersomes towards anti-cancer therapy in our laboratory.
Much work has been devoted so far to the studies of polyamidoamine (PAMAM)-based amphiphilic dendrimers bearing Surface-modified amphiphilic dendrimers forming stimuli-responsive and non-stimuli-responsive dendrimer-based vesicles, or dendrimersomes, presented in this review (color code for surface-modifying groups: lipids or aliphatic chains (blue), aromatic groups (green); "double colored bar": conjugated amphiphiles or hydrophobe-dendrimer conjugate, "/)": complexed amphiphiles or hydrophobe-dendrimer complex). long, so chains at their periphery. 5,[9][10][11][12][13]35 By contrast, it is only recently that the aggregation behavior of another polyamine hydrophilic dendrimer, poly(propylene imine) (PPI), was investigated. 36,37 This mini-review provides an overview of the development of the self-assembled amphiphilic polyamine dendrimer-based vesicles (dendrimersomes) towards potential biomedical applications, including cancer therapy. As there is already a vast amount of literature available on the formation of selfassembled nanostructures derived from dendritic amphiphiles made from Janus type of dendrimers, 30,32-34,38-41 our discussion will be restricted to the formation of vesicular selfaggregated structures from surface-modied dendritic polyamine amphiphilic molecules, which corresponds to a more recent development, focusing on PAMAM and PPI dendrimers, that are the most promising polyamine cationic dendrimers currently in use ( Fig. 3 and 4).
PPI-based dendrimersomes: design, stimuli-sensitivity and biomedical applications
Recent developments of spontaneously formed dendrimersomes from amphiphilic PPI dendrimer aer hydrophobic surface modication were reported in this part, which was subdivided based on the type of hydrophobic modication on the surface of PPI polyamine dendrimers, using various lipids and aromatic groups. Additional stimuli-responsive behavior, redox-sensitivity in particular, and gene transfection capabilities of these dendrimersomes towards their successful biomedical implication were further discussed in each of these particular sections. Generation 3 (G3)-PPI dendrimer was previously reported to be safer and more efficacious than its higher generation counterparts for gene transfection in in vitro and in vivo conditions. 42-45 A polyethylene glycol (PEG)-based heterobifunctional cross-linker (orthopyridyl disulde polyethylene glycol succinimidyl carboxymethyl ester OPSS-PEG-SCM) was used to conjugate the hydrophobic groups to G3-PPI dendrimer, as PEG is a biocompatible material well known to increase the water solubility of delivery systems and a stealth material to reduce non-specic interactions with circulatory proteins, thereby leading to an enhanced circulatory lifetime of drug and gene delivery systems. 26,[46][47][48][49] Our recent work also demonstrated that the conjugation of low molecular weight PEG (2 kDa) to generation 3-and generation 4-PPI dendrimers signicantly decreased their cytotoxicity as compared to unmodied dendrimers, leading to increased gene expression on B16F10-Luc, A431, DU145 and PC3-Luc cells lines. 50 2.1. Lipid-modied PPI dendrimers 2.1.1. Octadecyl chain-modied PPI dendrimer. We reported the spontaneous formation of dendrimersomes by octadecyl (C18) lipid chain-modied PEGylated G3-PPI dendrimer for drug and gene delivery. Being essential structural and functional hydrophobic constituents of animal cell membrane, 51-53 long fatty acid chains have always been a preferred lipid choice for the conjugation to hydrophilic substances towards the formation of self-assembled nanostructures, as hydrophobic interactions are one of the main driving forces for any amphiphilic low molecular weight surfactants and high molecular weight polymers to self-aggregate. [54][55][56] Additionally, the presence of lipid chains conjugated to the dendrimer backbone proved to enhance the overall transfection capability of cationic polymers, including polyamine dendrimers, due to the strong fusogenic activity of lipids and the formation of more compact, smaller-sized dendriplexes. 57-60 Furthermore, transfection activity was reported to be enhanced for the octadecyl lipid-modied generation 3-PAMAM dendrimer. 61 In line with that, we successfully synthesized disulphide-linked octadecyl (C18 alkyl) or stearyl chain-bearing PEGylated (MW 2 kDa) generation 3-PPI dendrimer, or DAB-PEG-SS-ODT (Fig. 5) through one-pot in situ twostep reactions, where the lipid : dendrimer ratio was nearly 1 : 1. 62 We were unable however to synthesize octadecyl-bearing PEGylated generation 3-PPI dendrimer at other ratios of lipid : dendrimer such as 2 : 1 or 1 : 2 due to freeze thawing issues, which is a common problem for various cationic lipids and emulsion-based foods. 63,64 Stable, cationic, nanosized vesicles were formed spontaneously from this dendrimer as a result of molecular self-assembly above its critical aggregation concentration value ($50 mg mL À1 ) ( Fig. 6A and B). The hydrodynamic diameter (d H ) of these lipid-dendrimer-based dendrimersomes showed a gradual decrease in average size from low to high concentrations, due to the formation of more compact and stable nanostructures as a result of increased hydrophobic interactions among lipid parts with increasing concentration. Thus, the sizes of DAB-PEG-SS-ODT-based vesicles at various concentrations (260.8 nm AE 36.5 nm at 100 mg mL À1 , 221.4 nm AE 39.4 nm at 200 mg mL À1 and 162.1 nm AE 8.1 nm at 400 mg mL À1 ) were below the cut-off size for extravasation to most tumor cells (400 nm) (Fig. 6C). 65 DAB-PEG-SS-ODT-based vesicles were found to bear a positive zeta potential at all the tested concentrations, which increased with increasing concentrations, reaching a maximum value of 2.43 mV AE 0.54 mV at a concentration of 400 mg mL À1 (Fig. 6C). The positive charge on the surface of the vesicles helped to enhance the non-specic uptake of these nanostructured cationic vesicles by the cells, because of favorable electrostatic interactions between the negatively charged cell membrane and positively charged nanoparticles. 66 The C18 lipid-bearing dendrimersome showed a crystallization exotherm at 0.2-0.3 C and an adsorption tendency on mica surface ( Fig. 6D and E). These features were more pronounced with increasing concentration of lipid-dendrimer, indicating a linear relationship of such properties with the content of conjugated lipid ( Fig. 6D and E). The steady-state uorescence intensity of emission maximum of coumarin-153 (C153) increased with increasing concentrations of dendrimersomes, suggesting an increasing hydrophobicity of the vesicular bilayer membrane with increasing lipid-dendrimer concentration resulting from a more favorable packing of amphiphiles. Overall a small blue shi ($4 nm) of the emission maximum of C153 was observed in presence of the dendrimersomes than that in absence of dendrimersomes, indicating a comparatively polar microenvironment of this vesicular bilayer. Furthermore, the calculated micropolarity (p*-value ¼ 0.83) of the hydrophobic microenvironment of bilayer at 1000 mg mL À1 lipid-dendrimer solution was found to be similar to the polarity of the polar aprotic solvent propylene carbonate (0.83). 67 The comparatively polar microenvironment of the bilayer membrane made it less favorable for encapsulation of a substantial amount of hydrophobic drugs. Because of the presence of a disulphide linkage (-S-S-) in the backbone, this lipid-dendrimer successfully showed a redox-responsive disintegration of dendrimersomes in presence of a glutathione (GSH) concentration similar to that of the cytosolic reducing environment (10 mM GSH) in comparison to that of extracellular reducing environment (10 mM GSH) or non-reductive environment. In addition, this lipid-dendrimer was found to condense more than 70-75% of the DNA at dendrimer : DNA weight ratios of 5 : 1 and higher. Even aer the dendriplex formation, the lipid-dendrimer had the capability to retain its vesicular forming properties. Dendriplexes showed less adsorption tendency on mica surface, leading to less deformation in comparison to that observed with the naked dendrimer. The cationic, stable, nanometer-sized dendrimersomes-based dendriplexes helped to enhance the cellular uptake of DNA both at dendrimer : DNA weight ratios of 10 : 1 and 20 : 1, respectively by up to 15.3-fold and 16.3-fold compared with naked DNA on PC3 prostate cancer cell line. Similarly, in DU145 prostate cancer cell line, the cellular uptake of DNA increased following treatment of the cells with this lipid-dendriplex by up to 28.6-fold and 27.6-fold compared with naked DNA. The dendriplexes showed an increased gene transfection on PC3 cell line at both dendrimer : DNA weight ratios of 10 : 1 and 20 : 1 compared with naked DNA, whereas in DU145 cells transfection was only enhanced at dendrimer : DNA weight ratio of 20 : 1. Octadecyl chain-bearing PEGylated PPI dendrimer-based (DAB-PEG-SS-ODT) dendrimersomes therefore were shown to be promising as redox-sensitive drug and gene delivery systems for potential applications in combination cancer therapy.
2.1.2. Cholesterol-modied PPI dendrimer. Since hydrophobic interactions are one of the main driving forces for any aggregated structure, we have chosen to conjugate PEGylated PPI dendrimer with a more lipophilic cholesterol moiety (instead of comparatively less hydrophobic lipidic alkyl chains) to facilitate comparatively more robust and efficient self-assembly and to form more compact, smaller-sized dendriplexes. 68 Due to its higher hydrophobicity than that of the hydrocarbon chains of fatty acids, cholesterol has been extensively utilized for the synthesis of amphiphilic low molecular weight surfactant and high molecular weight polymer-based self-assembled nanostructures. [69][70][71] Literature data suggested that cholesterol not only helps to enhance the stability of the hydrophobic rigid microenvironment of nanostructures and facilitate any drug delivery systems to cross the cellular membrane, but also increase the transfection efficacy of gene vectors due to its strong fusogenic activity. 6 In this study, we have reported the development of redox-sensitive dendrimersomes as drug and gene delivery systems comprising of disulde-linked cholesterol-bearing PEGylated (MW 2 kDa) generation 3-PPI (or DAB-PEG-S-S-CHOL) dendrimers ( Fig. 5) via an in situ two-step reaction. In this work, we have synthesized two cholesterol-dendrimers where the ratio of cholesterol (and PEG) and PPI were 2 : 1 for high cholesterol-dendrimer (12.5% conjugated cholesterol per dendrimer) and 1 : 1 for low cholesterol-dendrimer (9.5% conjugated cholesterol per dendrimer). Both of them were able to spontaneously self-assemble into stable, cationic, nanosized vesicles ( Fig. 7A and B) above their critical aggregation concentrations (CAC: $21 mg mL À1 for low cholesterol dendrimer and $10 mg mL À1 for high cholesterol dendrimer). A lower CAC value for high cholesterol-dendrimer vesicles in comparison to the low cholesterol-dendrimer vesicle indicated that the overall more hydrophobic content actually facilitates the dendrimersome formation at lower lipid-dendrimer concentration. These cholesterol-dendrimers showed comparatively lower CAC values (and therefore self-assembly formation at lower lipiddendrimer concentration) than that of the C18 alkyl chain modied dendrimers (mentioned in the previous section), due to the higher hydrophobicity of cholesterol than the alkyl chain. Both low-and high-cholesterol dendrimer-based dendrimersomes displayed an average size less than 230 nm with low polydispersity index (PDI) values at all the tested concentrations, with a gradual decreasing trend from low to high concentrations. They showed the smallest sizes of 91.3 nm AE 11.3 nm and 133.4 nm AE 48.8 nm for low-and high-cholesterol dendrimerbased dendrimersomes at 400 mg mL À1 (among tested concentrations) respectively. The formation of more compact and stable dendrimersomes at higher dendrimer concentrations due to increased hydrophobic interactions among cholesterol was the reason behind such a decrease in size. Both the dendrimersomes showed positive zeta potential between 4 mV and 7 mV with an increasing trend with increasing concentrations. The maximum zeta potential values of 5.7 mV AE 0.5 mV and 6.5 mV AE 0.3 mV was observed at 400 mg mL À1 (among tested concentrations) for low-and high-cholesterol dendrimer-based dendrimersomes, respectively. The size and zeta potential of low-and highcholesterol dendrimersomes were not statistically different at all the tested concentrations with the change of the amount of cholesterol within the dendrimers. TEM images of the dried samples on carbon coated copper grid revealed the formation of spherical unilamellar vesicles of sizes less than 50 nm. These dendrimersomes showed a successful entrapment of hydrophilic and hydrophobic dyes. Both of them showed no lower critical solution temperature (LCST) or cloud point (CT) in the temperature ranging from 20 to 90 C, facilitating their no phase separation of vesicles at body temperature (37 C), and therefore no premature release of any entrapped pharmaceutically active agent at body temperature. In DSC study, both the dendrimersomes showed an endothermic melting peak (T m ) due to the phase change of the vesicle membrane from the "gel" state to the "liquid crystalline" or "uid" state at nearly 0 C (À0.6 C and À0.8 C for low-and high-cholesterol dendrimer-based vesicles, respectively) during the rst heating phase and second heating cycle. Such a low T m values for these dendrimersomes might be due to the presence of unsaturation in cholesterol and/or in the disulde bond. AFM images showed that both dendrimer-based vesicles adsorbed on a freshly cleaved mica surface aer airdrying, leading to the diffusion of the vesicles. Due to the presence of disulde-linked hydrophobic cholesterol in the bilayer of dendrimer-based vesicles, they showed redox-sensitive properties in presence of intracellular reducing environment (10 mM GSH). It was found that both the dendrimersomes showed a gradual increase in size over time in presence of a higher concentration (10 mM) of GSH in comparison to that in the presence of 10 mM or without GSH, which can be attributed to the cleavage of the disulde bonds and disintegration of vesicles, forming larger, unstable and less compact aggregates in intracellular redox condition. Both dendrimersomes showed a higher amount of cumulative release of entrapped hydrophobic Nile red over time from the vesicles in in the presence of a glutathione concentration similar to that of a cytosolic reducing environment (up to 40% and $25% Nile red release from low-and high-cholesterol dendrimersomes respectively in the presence of 10 mM GSH) rather than in extracellular redox condition. Thus the disassembly of the vesicles and concomitant release of the model drug in the intracellular or cytosolic reducing environment (corresponding to 10 mM GSH) proved the possibility of future use of these dendrimer-based vesicles as redox-responsive drug delivery systems for cancer treatment. The high-cholesterol dendrimersomes showed higher melting enthalpy (in DSC study), increased adsorption tendency on mica surface (in AFM study), have higher entrapment ability for hydrophobic drugs and displayed an increased resistance to redox-responsive environments in comparison with their low-cholesterol counterpart due to a progressive "dilution" of the lipids (cholesterol) in the bilayer membrane from high-to low-cholesterol dendrimer-based vesicles. Both dendrimersomes were able to condense plasmid DNA quite efficiently along with the retention of their vesicular structures aer complexation with DNA ( Fig. 7C-F). Lowcholesterol vesicles were able to condense more than 85% of the DNA at all the tested dendrimer : DNA weight ratios, whereas high-cholesterol vesicles having fewer primary amine at the periphery, showed condensation ability over 85% at dendrimer : DNA weight ratios of 1 : 1 and higher. Due to the presence of highly hydrophobic cholesterol in the backbone, these dendriplexes showed an overall smaller hydrodynamic diameter, leading to the formation of more compact dendriplexes in comparison to not only DAB-PEG-S-S-ODT-based dendrimersomes (described in the previous sections), but also the unmodied DAB dendriplex. Both dendrimers showed positive surface charge ranging between 21.4 AE 0.9 and 41.1 AE 0.6 mV for most dendrimer : DNA weight ratios from 1 : 1 and above. Further, both dendriplexes retained the vesicular morphology aer complexation with DNA, as evidenced from the TEM images of dendriplexes at dendrimer : DNA weight ratios of 5 : 1 and 10 : 1. Thus, positively charged and smaller-sized compact dendrimersome-based dendriplexes enhanced the cellular uptake of DNA through internalization mechanisms by up to 8-12-fold and 10-15-fold when compared with unmodied G3-PPI dendriplex and naked DNA respectively. Overall, these dendriplexes helped to increase the gene transfection in the PC-3 prostate cancer cell line at various dendrimer : DNA weight ratios, by about 5-fold compared with the gene expression observed following treatment of the cells with naked DNA. These cholesterol-bearing PEGylated dendrimersomes were more promising as redox-sensitive drugs and gene delivery systems than the C18 alkyl-bearing PEGylated dendrimersomes.
Aromatic group-modied PPI dendrimers
2.2.1. Camptothecin (quinoline alkaloid)-modied PPI dendrimer. In spite of their advantages regarding the combination delivery of drugs and genes, cholesterol-based dendrimersomes (mentioned in Section 2.1.2.) may face the undesired release of the entrapped drugs before reaching their target site of action. To overcome this potential issue associated with the physical entrapment of drugs in the delivery systems, we have designed and synthesized a pro-drug dendrimer by conjugating the anti-cancer drug camptothecin (CPT) with PEGylated generation 3-diaminobutyric polypropylenimine dendrimer (or DAB-PEG-S-S-CPT) through a redox-sensitive disulphide linkage (Fig. 5) in a similar fashion as DAB-PEG-S-S-ODT and DAB-PEG-S-S-CHOL. 72 The ratio of CPT (and PEG) and PPI was found to be nearly equal to 1 : 1 for the synthesized CPT-dendrimer, DAB-PEG-S-S-CPT. This PEGylated pro-drug dendrimer was found to spontaneously self-assemble into cationic vesicles at pH 7.4. It formed the dendrimersomes above a comparatively higher critical aggregation concentration ($200 mg mL À1 ) than that of lipid-dendrimer-based dendrimersomes due to the lower hydrophobicity of CPT than that of lipid. The hydrodynamic size of dendrimersomes measured at various tested concentrations (0.5, 1 and 2 mg mL À1 ) showed a sharp decrease with time and increasing concentrations. The hydrodynamic sizes became steady aer 12 h at 37 C and remained stable at around 250-300 nm (for 0.5 mg mL À1 ) and 150-200 nm (for 1 and 2 mg mL À1 ), leading to the formation of more stable and compact nanostructures at higher concentrations due to increased hydrophobic interactions among CPT molecules. The size of dendrimersomes in presence or absence of complete medium containing 10% FBS (size $ 100 nm) was below the cutoff size for extravasation. The formation of stable cationic dendrimersomes at various tested concentrations (0.5-2 mg mL À1 ) was evidenced from the positive zeta potential ($3-5 mV) for up to 7 days. TEM images revealed the unilamellar, spherical-shaped vesicles (dendrimersomes) with a size lower than 100 nm (Fig. 8A-C). The micropolarity (p*-value ¼ 0.278) value obtained using coumarin-153 (C153) probe study suggested that the polarity of hydrophobic bilayer microenvironment was similar to the polarity of hexauorobenzene (0.27), conrming that the vesicular bilayer was composed of the aromatic benzene rings of CPT. The appearance of the shoulder band in the uorescence spectrum of C153 indicated a distribution of probe molecules at multiple locations in vesicular assemblies. Circular dichroism (CD) spectra of dendrimersomes (2 mg mL À1 ) in phosphate buffer (pH 7.4) suggested a chiral packing of the CPT moieties in the vesicular bilayer with negative chirality and le-handed helical arrangement. The CPT release from dendrimersomes (1 mg mL À1 in phosphate buffer, pH 7.4) was found to be around 10 to 15% in non-redox and extracellular redox condition in normal cells (in presence of 10 mM GSH). It sharply increased with time to about 30-35% within 7 days in presence of 10 mM GSH (equivalent to the intracellular concentration in normal cells) and 70% in presence of 50 mM glutathione (equivalent to the intracellular environment of tumor tissue) due to the disintegration of dendrimersomes. Additionally, the pro-drug dendrimer could instantly condense more than 85% of the DNA at dendrimer : DNA weight ratios of 5 : 1 and higher, forming stable vesicular aggregates over time. Such stable and cationic dendrimersome-DNA complexes at dendrimer : DNA weight ratios of 5 : 1 and higher led to an enhanced cellular uptake of DNA (by up to 1.6-fold) and increased gene transfection (by up to 2.4-fold) in prostate cancer cells in comparison with the unmodied G3-PPI dendrimer (Fig. 8D). These redoxresponsive camptothecin-bearing cationic dendrimersomes are therefore promising carrier-based delivery systems towards combination cancer therapy involving chemotherapy along with gene therapy. Further, such a strategy could also be used for synergistic combination of drugs and nucleic acids of interest against any particular cancer. Moreover, due to the capability of these drug-conjugated dendrimersomes to entrap various hydrophobic and hydrophilic guests in their hydrophilic aqueous core or hydrophobic bilayer, such dendrimersomes could be used for theranostic applications by delivering imaging agents in addition to therapeutic drugs and nucleic acids to the disease site. [73][74][75] 2.2.2. Azobenzene-and palmitoyl-modied PPI dendrimers. Tsuda and colleagues reported in 2000 the formation of giant micrometer-sized spherical vesicles with a multilamellar "onion-like" structure in aqueous solution below pH 8 from generation 5-PPI dendrimer functionalized with aliphatic chains. 37 Their study described the synthesis and characterization of three surface-modied generation 5-PPI dendrimers 1, 2, and 3, decorated with side chains containing 64 palmitoyl, 32 palmitoyl and 32 azobenzene, and 64 azobenzene groups respectively (Fig. 9).
Transmission electron microcopy and confocal uorescence microscopy experiments demonstrated the formation of dendrimer-based giant vesicles in aqueous dispersion. Additionally, they demonstrated that the azobenzene-functionalized vesicles (2 and 3) were able to uoresce, with a maximum at l max 600 nm, due to the dense and ordered arrangement of the azobenzene chromophore in the hydrophobic bilayer of the vesicular aggregates. Hydrogen bonds between protonated cores of the dendrimers (at pH < 8) and the p-p stacking of the azobenzene moieties inside the bilayer (as evidenced by the emission of the vesicles) were the main factors involved in the stabilization of such micrometer-sized vesicles. The vesicular structure and size distribution were found to be dependent on the end groups at the periphery of the dendrimer and the pH of the solution. Giant vesicles 2 and 3 have their morphology altered along with the refractive index of the solution, upon exposure to laser irradiation with 1064 and/or 420 nm light, as (i) infrared light induced a structural rearrangement, and (ii) 420 nm light induced a cis-trans isomerization of azobenzene units, leading to structural changes accompanied by an increase in the emission intensity. Further, vesicles in an aqueous solution at pH 1 showed an increase in uorescence intensity with a blue shi of the emission maximum to 540 nm (due to higher degree protonation of the vesicles only aer illumination), whereas this blue shi was not observed for vesicle solutions in Milli-Q® water at pH 5.5. The enhanced uorescence intensity due to the reorganization of the chromophores within the giant vesicles was attributed to the fact that the kinetically formed giant vesicular systems in which the packing of the liquid-crystalline azobenzene units were not optimal, changed to a thermodynamically more relaxed state aer lightinduced isomerization.
2.2.3. Polystyrene-modied PPI dendrimer. In 1995, Meijer group reported the formation of novel amphiphilic macromolecules resulting from the conjugation of well-dened polystyrene (PS) moiety to PPI dendrimers of 5 generations (Fig. 10). 76 PS-dendr-(NH 2 ) 32 , PS-dendr-(NH 2 ) 16 and PS-dendr-(NH 2 ) 8 respectively formed spherical micelles, micellar rods and vesicular structures in aqueous phase, as demonstrated by dynamic light scattering, conductivity measurements and transmission electron microscopic studies. The lower generations of this class of amphiphilic macromolecules showed inverted micellar behavior in toluene, which was predominantly determined by the apolar PS chain. Conductivity measurements were used to assess the amphiphilic properties of PS-dendr-(NH 2 ) x in toluene-water mixtures as a function of the toluene/ water ratio, which actually helped to identify the point of phase inversion from water to toluene as the continuous phase. All lower generation amphiphiles displayed a strong tendency to stabilize toluene as the continuous phase. The highest inversion point was observed for PS-dendr-(NH 2 ) 16 , which was well beyond 50% toluene in volume. Further, the inversion point of PS-dendr-(NH 2 ) 32 could not be determined, due to the dendrimer insolubility in toluene. Based on the differences in phase inversion from water to toluene, the authors suggested various types of aggregation pattern by these amphiphiles due to their different amphiphilic behavior. The DLS measurements was not very conrmatory, especially for higher generation of dendrimer, due to the clustering between the aggregates. Finally, TEM study using three different techniques (negative Fig. 9 Chemical structure of the surface-modified dendritic amphiphiles made of generation 5-poly(propylenimine) dendrimers 1, 2, and 3, with the dendrimers decorated with side chains containing 64 palmitoyl, 32 palmitoyl, 32 azobenzene and 64 azobenzene groups, respectively. staining with uranyl acetate, Pt shadowing, and freeze fracture) showed a clear change in aggregation pattern from PS-dendr-(NH 2 ) 8 to PS-dendr-(NH 2 ) 32 , from vesicles [for PS-dendr-(NH 2 ) 8 ] to micellar rods [for PS-dendr-(NH 2 ) 16 , 12 nm in diameter], to spherical micelles [for PS-dendr-(NH 2 ) 32 , 10 to 20 nm in diameter], which is in qualitative agreement with the theory of Israelachvili and colleagues on the relation between the type of aggregation and the amphiphile molecular shape. 77 Thus, the size of the amphiphilic dendrimer head group played an important role to form such various aggregates, from spherical to inverted micelles. The vesicular structures formed by PSdendr-(NH 2 ) 8 showed a resemblance to the structure proposed by Kunitake and colleagues. 78 Their diameters (10 to 20 nm) were in the same order of magnitude as the estimated bilayers of the PS-dendrimer block copolymers. These amphiphiles were reported to be closer in shape and in size as compared with traditional surfactants and traditional block copolymers respectively.
PAMAM-based dendrimersomes: design, stimuli-sensitivity and biomedical applications
In this part, we have reported recent developments of spontaneously and non-spontaneously (stimuli-sensitive) formed dendrimersomes from amphiphilic PAMAM dendrimer In 2015, Doura and colleagues reported the synthesis and characterization of pHresponsive lipid-bearing PAMAM dendron-based vesicular assemblies, formed following conjugation of two octadecyl hydrophobic chains to amine-terminated generation 1-(G1) polyamidoamine dendron (Fig. 11). 11 The presence of two primary amines and two tertiary amines in the lipid-dendron moiety made this dendrimer particularly promising due to its dynamic molecular shape at various pHs. It was able to form vesicle assemblies at neutral and alkaline pHs, but its structure changed to a micelle-like structure below pH 6.4. DSC analysis of this lipid-dendrimer showed a sharp endothermic peak (around 43 C) at pH 10 and 7.4, but a broader endothermic peak with a gradual shi to a lower temperature at pH below 6.0, indicating the formation of a bilayer structure with a gel-to-liquid crystalline transition around 43 C under neutral and alkaline conditions but not in acidic condition. The pH-responsive property of the lipiddendrimer-based vesicles was conrmed by DLS study. The hydrodynamic size of the vesicles was around 75 nm at pH 7.4, whereas their diameter decreased to around 20 nm below pH 6.5 aer a structural shi from vesicles to micelles. This was conrmed further by TEM imaging, which also showed the formation of vesicles with a diameter around 100 nm at pH 7.4, and the formation of micelles with diameter less than 20 nm at pH 6.5 and pH 4.0. Furthermore, the lipid-dendron amphiphile also produced sharp pH-responsive vesicles with high colloidal stability in presence of PEG-lipid, which eventually transformed to micelle-like structure at around pH 6.5. The PEGincorporated lipid-dendron vesicles were able to encapsulate FITC-labeled ovalbumin (a water-soluble protein found in egg white, with a molecular weight of 45 kDa) in their internal aqueous space, and also acid-triggered its almost complete release at pH 6.0. An efficient ovalbumin delivery from PEGincorporated dendron-lipid vesicles was also observed into the cytosol of DC2.4 mouse dendritic cells aer internalization through endocytosis. These pH-responsive dendrimersomes showed a promising efficacy for cytoplasmic delivery of bioactive molecules, such as proteins, indicating their future potential towards pH-responsive cancer therapy.
3.1.1.2. Temperature-responsive dendrimersomes. Kono and colleagues reported, in 2011, the formation of temperaturesensitive vesicles by a series of alkyl amide-PAMAM dendrons with varying chain terminal moiety that undergoes a temperature-dependent transition through a change in hydration of the vesicle surface. 35 They synthesized PAMAM-based lipiddendrimers by conjugating amine groups of generation 2-(G2) and generation 3-(G3) PAMAM dendrons with two octadecyl chains, where the dendrimer and the lipid chains were used as head group and hydrophobic tails, respectively (Fig. 12). They also made these lipid-dendrimers temperature-sensitive by modifying the thermosensitive isobutyramide (IBAM) groups at every chain terminal of the dendron moiety to produce two IBAM-based thermos-sensitive dendrimers, IBAM-G2-DL and IBAM-G3-DL. To correlate their assumption on the impact of the terminal groups with the change of temperature, they also prepared lipid-PAMAM dendrimer with acetamide (ACAM) groups at every chain terminal of the dendron moiety, ACAM-G2-DL and ACAM-G3-DL.
The authors successfully demonstrated the temperature sensitization of lipid-bearing dendrimer-based vesicles. Temperature-sensitive IBAM-terminated lipid-dendrimers showed an increase in the cloud point with decreasing pHs of the dispersions, due to the protonation of the tertiary amines of the PAMAM dendrimer leading to the enhanced hydration of the conjugated lipid moiety at lower pH values. Transmission electron microscopy conrmed the vesicular morphology of IBAM-G2-DL assemblies in aqueous solution, with a diameter of 50-100 nm at 15 C. However, these vesicular assemblies were changed to brous structures about 8-10 nm in thickness aer 10 min incubation at 50 C (higher than the cloud point of IBAM-G2-DL). By contrast, the IBAM-G3-DL molecules not only formed spherical vesicles with diameters of 100-200 nm at 15 C, but also were able to retain their spherical vesicular morphology aer 10 min incubation above the cloud point, indicating a fusion of vesicles during the incubation. Such a morphology transition phenomenon of these lipiddendrimer-based vesicular assemblies below and above the cloud point was also supported by DLS, AFM and SAXS proles at various temperatures. In DSC measurements, ACAM-G2-DL and ACAM-G3-DL displayed no endotherm, whereas the Fig. 11 Chemical structure of amine-terminated, generation 1-polyamidoamine dendron bearing two octadecyl hydrophobic chains.
IBAM-G2-DL and IBAM-G2-DL dendrimers showed clear endothermic peaks in the same temperature region. These DSC results demonstrated the importance of the dehydration of the IBAM groups on the vesicle surface for the generation of such endothermic peaks. Additionally, a concomitant increase of cloud point with increasing ACAM content proved the enhanced hydration of the surface of the assemblies due to the inclusion of hydrophilic ACAM groups. Thus, in contrast to IBAM-G2-DL, ACAM-G2-DL dispersion was found to be stable under the experimental conditions.
The amphiphilic AD could self-assemble in water into spherical unilamellar vesicles of about 200 nm in diameter, as demonstrated by DLS and TEM studies. Additionally, TEM images showed a bilayer structure (corona of the vesicles) of 7 nm thickness, which is approximately twice the theoretical length of AD ($3.5 nm). Furthermore, the formation of Langmuir monolayer lm at the air/water interface indicated a lipidlike behavior and vesicle-forming properties of amphiphilic AD. The authors observed an adaptive supramolecular assemblies formed by AD upon interaction with siRNA, where ADdendrimersomes underwent structural rearrangement to form nanosized siRNA/AD complexes. Such siRNA/AD complex-based spherical substructures of 6-8 nm in diameter (twice the size of AD) indicated the formation of micellar nanostructures. This structural transition from the larger-sized vesicles into smaller-sized micelles upon complexation with siRNA helped to expose the positively charged dendrimer surface area, therefore providing better stabilization of complexes through electrostatic interactions with the negatively charged siRNA. Additional mesoscale computational studies using dissipative particle dynamics (DPD) supported that AD self-assembled into large supramolecular structures, and planar section demonstrated the formation of double-layered dendrimersomes. The presence of siRNA actually destabilized the balance between the two opposing forces, hydrophobic interactions among conjugated alkyl chains (favorable for cooperative construction of the Fig. 13 Chemical structure of lipid-modified amphiphilic PAMAM dendrimer (AD) obtained by conjugating two precursors (azide-functionalized hydrophilic dendrimer moiety and alkyne-functionalized C-18 alkyl bearing hydrophobic moiety) through click chemistry. supramolecular assemblies), and repulsive interactions/steric effects among the positively charged head groups of amphiphilic dendrimers, leading to structural changes. These stable siRNA-AD nano-assemblies effectively delivered siRNAs to various cell lines in comparison to the commercially available vector Lipofectamine RNAiMAX® (lipo), including PBMC-CD4+ human primary peripheral blood mononuclear cells, HSC-CD34+ hematopoietic stem cells and glioblastoma stem cells (GSCs) towards the development of successful non-viral vectors for delivery of RNAi therapeutics. These siRNA-AD nanocomplexes were resulted in 50% reduction of viral infection in both PBMC-CD4+ and HSC-CD34+ cells. Furthermore, the signicant reduction of gene silencing ability of AD/siRNA complex in the presence of balomycin A1 (a proton pump inhibitor) proved that the proton sponge effect (protonation of tertiary amines in the dendrimer interior leading to endosomal disruption and subsequent cargo release in the cytoplasm) was highly important for successful AD-mediated siRNA delivery.
3.1.2. Hexadecyl or C16 (fatty acid) chain-modied PAMAM dendrimer 3.1.2.1. Balance of hydrophobic and hydrophilic groups. In 2015, Zhang and colleagues reported the self-aggregation of a series of amphiphilic G n QPAMC m (where n ¼ 1 (dendrimer generation) and m ¼ 8, 12, 16 (alkyl chain length aer the modication of the end group)) lipid-modied PAMAM dendrimers, formed by the conjugation of the same hydrophilic group (generation-1 PAMAM) to alkyl groups of various chain length in aqueous solution (Fig. 14). 13 The authors observed signicant differences in the morphology of self-aggregates of amphiphiles with their chemical structures or with the change of hydrophobic and hydrophilic balance of amphiphiles, more specically with the chain length of alkyl groups attached to the dendrimer surface. Cryo-TEM images conrmed the formation of spherical aggregates with a particle size of 50-100 nm for all G 1 QPAMC m , which was in agreement with DLS results. The aggregates of G 1 QPAMC 8 were irregularly spherical, unlike the regular spherical shape observed for those made of G 1 QPAMC 12 and G 1 QPAMC 16 . Additionally, only G 1 QPAMC 16 (with an alkyl chain length of 16) was able to form 50-100 nm vesicles with clear boundaries in comparison to the other two amphiphiles G 1 QPAMC 8 and G 1 QPAMC 12 . Critical micelle concentration was found to decrease with the increase in chain length, though not linearly indicating a differentiation in the morphology. With the help of uorescence studies using two hydrophobic uorescent probes pyrene and DPH, the authors revealed (i) a lower polarity, (ii) higher solubilization ability of microdomains of such self-aggregates and (iii) enhancement of the hydrophobic microenvironment with the increase in alkyl chain length from C8 to C12. In contrast, for G 1 QPAMC 16 amphiphile, they observed an increase not only in the polarity of the microdomain of aggregates (in comparison to that of G 1 QPAMC 12 ) but also a sharp hydrophobic probe solubilization ability, indicating a boundary structure (bilayer or multilayer) within the vesicular aggregates. The thermodynamic properties and related parameters also helped to understand the formation of aggregates with various morphologies. The enthalpies of micelle formation (or DH mic values) for G 1 QPAMC 8 , G 1 QPAMC 12 , and G 1 QPAMC 12 were À2.3, À8.5, and À5.2 kJ mol À1 , respectively, indicating that the enthalpy and entropy jointly contributed in the self-assembly process of the amphiphilic G 1 QPAMC m . An enhancement of absolute value of the enthalpy, from À2.3 kJ mol À1 (G 1 QPAMC 8 ) to À8.5 kJ mol À1 (G 1 QPAMC 12 ), proved the dominating impact of enhanced hydrophobic interactions with the increase in alkyl chain length, whereas for G 1 QPAMC 16 , the decrease in the absolute value of the enthalpy indicated an opposite feature. It was found that the ratios of theoretical alkyl chain length/radius are less than one for G 1 QPAMC 8 (10.16/19.77) and G 1 QPAMC 12 (15.24/ 19.77), and higher than one for G 1 QPAMC 16 (20.32/19.77), indicating that the head group of such amphiphiles also played an important role along with the hydrophobic interactions in the self-aggregation process and thereby enthalpy changes. Such an alkyl chain length/radius value (higher than 1) for G 1 QPAMC 16 led to the conclusion that the intra-cohesion of the alkyl chain (C16) was involved in the process of selfassembly of the lipid-modied dendrimer, which resulted in the weakening of the hydrophobic interactions and thereby in the increase in ÀDH mic .
Aromatic group-modied dendrimers
3.2.1. Aniline pentamer (AP) modied-dendrimer. Hung and colleagues reported the formation of bilayer vesicular assembly from a series of PAMAM dendrimer-based amphiphiles, synthesized by conjugating various generations (G2-G5) of hydrophilic PAMAM dendrimer with a hydrophobic shell of aniline pentamer (AP) (Fig. 15). 10 The CAC of such AP-modied dendrimers self-assembled to vesicular aggregates in water were not only very low (10 À6 -10 À7 M), but also decreased with increasing generation of PAMAM. This trend suggested that the formation of vesicles is favored in the order G5 > G4 > G3 > G2, as the higher-generation (G4 and G5) amphiphilic PAMAM-AP dendrimers holds stronger selforganization tendency due to the p-p interactions among AP units and the H-bonding interactions between the dendrimer branches. The TEM images of PAMAM-AP G2-5 demonstrated the formation of 100-200 nm spherical vesicular aggregates, with gradually decreasing size of the bilayer vesicles as the dendrimer generation increased from G2 to G5, due to the loosely held nanostructures for the lower-generation dendrimers. These vesicles were very adhesive due to the presence of exible hydrophilic dendritic branches in the outer layer, leading to a stronger self-adhesion via H-bonding interactions of the branches for the higher-generation dendrimers than for the lower-generation counterparts. Moreover, due to the strong p-p interactions of the long hydrophobic rigid AP units in the self-assembled nanoarchitectures, the average thickness of the hydrophobic interphase was reduced to 1.7-2.1 nm, which is well below the head-to-head theoretical distances (4.8 nm) of the AP double layer. Stained TEM images with uranyl acetate showed exactly reversed images in comparison to the unstained images, as the outer and inner layers of the vesicles appear in black with negative staining leading to the conclusion that the hydrophilic dendritic branches are localized in this region. TEM images also conrmed the presence of multiple aggregates containing two vesicles (twins for G2-PAMAM-AP and G3-PAMAM-AP) and multi-vesicles (large number of multiple aggregates for G4-PAMAM-AP and G5-PAMAM-AP), due to the increased number of hydrophobic AP units in the outer periphery group of the PAMAM dendrimers. Moreover, SEM and AFM images of G2-and G4-PAMAM-AP supported the selfassembly formation of vesicles of various diameters. The packing parameters of G2-G5-PAMAM-AP were found to be in the range of 0.5-1.0 (as evaluated from EDX O-mapping images), supporting the formation of bilayer vesicular structures. Comparatively larger-sized vesicles were observed under acidic and alkaline conditions, in comparison to that in neutral medium for each generation of amphiphilic dendrimers. At low pH, the generation of ammonium salts from the tertiary amine groups of PAMAM-AP helped to form the polarons from AP units by doping with strong acids, which eventually increased the stability of vesicular aggregates at low pH than that in double distilled water. At high pH, the deprotonation of PAMAM-AP amphiphiles enhanced p-p interactions, leading to the formation of twins or multilayered vesicles. X-ray diffraction (XRD) analyses of the vesicles helped to elucidate the possible molecular packing patterns of G2-G5-PAMAM-AP along with self-assembly driving force analysis. XRD patterns conrmed the presence of a more ordered structure for these higher generation amphiphilic dendrimers, due to the crystallization induced by the result of enhanced p-p interactions (compacted effect) of the strongly emeraldine APs.
AFM and TEM imaging demonstrated the presence of spherical vesicular aggregates of PAMAM-NPD amphiphiles in water, with an average diameter about 100 nm and a bilayer thickness of about 3.2-5.0 nm. DLS study clearly depicted an increase in hydrodynamic radius with increasing generations for PAMAM-NPD-based vesicles (78.4 nm for G0, 57.7 nm for G1, 75.7 nm for G2, and 113.0 nm for G3), whereas no ordered aggregates were obtained for dendrimers with generations higher than G3. Furthermore, PAMAM-DNS also formed vesicular structures with an average diameter of about 135 nm and a bilayer thickness of about 3.2 nm in water. A p-p interaction due to the presence of rigid and short aromatic groups at the periphery of amphiphilic PAMAM dendrimers played an important role, in addition to the amphiphilic nature of modied dendrimers, in the self-assembly process towards vesicle aggregates. This p-p interaction, along with vesicles formed by interactions between two non-uorescent dendrimer building blocks and Ar27, successfully served as hosts for a zwitterionic uorescent dye, FRM 480. This dye did not cause any aggregation with either of the cationic and anionic building blocks because of its zwitterionic nature. The size of these vesicles (R H $ 140 nm) did not appear to be impacted by such dye encapsulation, as evidenced from DLS study, but the vesicles became visible through CLSM study due to the encapsulation of this uorescent dye in their microenvironment. Following imaging of dye-loaded vesicles on mica surface, the formation of un-sharp and ''diffuse'' particles was observed due to the movement of the particles on mica surface as a result of weak immobilization between negatively changed mica surface and positively charged vesicles. Also, the uorescence correlation spectroscopy (FCS) of the vesicles showed a radius of 1 and 150 nm for only dye and the dye-vesicle system respectively supporting DLS results. Additionally, the vesicles have been shown to be able to encapsulate uorescently labeled 10 kDa peptide, showing their future potential use in drug delivery.
Conclusion and perspectives
Polyamine dendrimer-based vesicles, or dendrimersomes, have demonstrated to be highly promising delivery systems of therapeutic genes and drugs for various biomedical applications. This mini-review focused on dendrimersomes prepared from hydrophobically surface-modied hydrophilic PPI and PAMAM dendrimers, as these two dendrimers are currently the most widely used and promising polyamine dendrimers. Additional cationic charges on the surface, stimuli-sensitivity (such as pH, redox, light and temperature) of such dendrimersomes made from various amphiphilic dendrimers helped them to be used as nano-carriers for drugs and genes and to release their cargo in cancer cell-specic environment. The lipid-modied PPI dendrimers were found to form more stable dendrimersomes (with lower CAC) than the drug-modied PPI dendrimers. In addition, cholesterol-modied PPI dendrimersomes showed more efficacy than the fatty acid chain-modied PPI dendrimersomes as drug and gene delivery systems. These lipid-modied, stimuli-responsive polyamine dendrimersomes could be used for drug-gene combination cancer therapy, due to their ability to encapsulate drugs, to form stable complexes with nucleic acids and to enhance overall transfection. They could also be used in future theranostic applications, as a result of their potential entrapment of a therapeutic agent together with an imaging agent.
Although the therapeutic, diagnostic, biosensor efficacies and safety of the dendrimersomes are yet to be conrmed in vivo, the early ndings of the described studies show that such platforms hold a promising future in cancer management. A further analysis of the structure-property correlation of these newly developed polyamine dendrimersomes should facilitate their development into even more efficacious delivery systems. Overall, because of their ease of preparation, low immunogenicity, embedded stimuli-sensitivities and multifunctional architecture, these polyamine dendrimersomes are likely to be used in therapeutic strategies combining targeting, imaging, diagnostics and therapy. Continued research in this area should therefore enable the preparation of highly specic, highly efficacious dendrimersomes towards the treatment of cancer and other biomedical applications.
Conflicts of interest
There are no conicts to declare. | v3-fos-license |
2019-09-12T13:06:38.015Z | 2019-10-22T00:00:00.000 | 202554253 | {
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} | pes2o/s2orc | Tetranuclear oxido-bridged thorium ( IV ) clusters obtained using tridentate Schi ff bases † ‡
Thorium(IV) complexes are currently attracting intense attention from inorganic chemists due to the development of liquid-fluoride thorium reactors and the fact that thorium(IV) is often used as a model system for the study of the more radioactive Np(IV) and Pu(IV). Schiff-base complexes of tetravalent actinides are useful for the development of new separation strategies in nuclear fuel processing and nuclear waste management. Thorium(IV)–Schiff base complexes find applications in the colorimetric detection of this toxic metal ion and the construction of fluorescent on/off sensors for Th(IV) exploiting the ligandbased light emission of its complexes. Clusters of Th(IV) with hydroxide, oxide or peroxide bridges are also relevant to the environmental and geological chemistry of this metal ion. The reactions between Th (NO3)4·5H2O and N-salicylidene-o-aminophenol (LH2) and N-salicylidene-o-amino-4-methylphenol (L’H2) in MeCN have provided access to complexes [Th4O(NO3)2(LH)2(L)5] (1) and [Th4O(NO3)2(L’H)2(L’)5] (2) in moderate yields. The structures of 1·4MeCN and 2·2.4 MeCN have been determined by singlecrystal X-ray crystallography. The complexes have similar molecular structures possessing the {Th4(μ4-O) (μ-OR’)8} core that contains the extremely rare {Th4(μ4-O)} unit. The four Th atoms are arranged at the vertexes of a distorted tetrahedron with a central μ4-O ion bonded to each metal ion. The H atom of one of the acidic –OH groups of each 3.21 LH or L’H ligand is located on the imine nitrogen atom, thus blocking its coordination. The Th centres are also held together by one 3.221 L or (L’) group and four 2.211 L or (L’) ligands. The metal ions adopt three different coordination numbers (8, 9, and 10) with a total of four coordination geometries (triangular dodecahedral, muffin, biaugmented trigonal prismatic, and sphenocorona). A variety of H-bonding interactions create 1D chains and 2D layers in the crystal structures of 1·4 MeCN and 2·2.4 MeCN, respectively. The structures of the complexes are compared with those of the uranyl complexes with the same or similar ligands. Solid-state and IR data are discussed in terms of the coordination mode of the organic ligands and the nitrato groups. H NMR data suggest that solid-state structures are not retained in DMSO. The solid complexes emit green light at room temperature upon excitation at 400 nm, the emission being ligand-centered.
Introduction
The ions of early actinide (An) elements are unique in inorganic chemistry in that they combine large radii and lanthanide (Ln) ions' Lewis acidity with the potential for remarkable covalency (due to greater f-orbital radial extension for An vs. Ln ions), and due to the fact that they can achieve a broad range of oxidation states (mainly for U, Np and Pu). [1][2][3] These characteristics provide An complexes with some special reactivity profiles. Thorium was discovered in 1829 by the famous chemist Jöns Jacob Berzelius and owes its name to Thor, the Scandinavian god of thunder and war. 4 Natural Th is radioactive, but many of its current uses exploit its chemical, rather than its nuclear, features. Thorium is the first true An element and has an empty 5f orbital, its outer electronic con-figuration being 6d 2 5f 2 ; therefore, Th(IV) is the most stable and almost exclusive oxidation state. 5 There has been a renaissance in the coordination and material chemistry of Th(IV) for the last 5 years or so. 1,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] There are several reasons for this. The main reason is that Th is widely considered to be the next generation nuclear fuel as the development of Liquid-Fluoride Thorium Reactors (LFTRs) comes closer to commercialization. 5,6,22,23 India is keen on using thorium as the nuclear fuel (this country has approximately 30% of the world's thorium reserves) and plans to produce one third of its electricity from thorium by 2050. 5 The production of thorium is usually achieved by the separation of Th 4+ from Ln 3+ ions, since the main thorium production mineral is monazite which contains the ions of 4f metals; mining is based on liquid-liquid extraction with organic ligands, 24,25 hence the importance of the Th(IV) coordination chemistry. There is also intense interest for the development of new efficient solid absorbents for the selective extraction of Th 4+ ions from 4f-metal ions. 16 Other research areas of Th(IV) chemistry that are currently attracting attention from inorganic chemists include the incorporation of this diamagnetic ion into coordination clusters instead of the paramagnetic U(IV) to elucidate the magnetic exchange interactions between the d-metal ions in d/U IV complexes, 26 its interaction with ionic liquids to model the aspects of the extraction of An ions from radioactive feeds (a process of great relevance in nuclear fuel cycle activities), 13 the understanding of Th(IV) peroxide chemistry, 8,14 the study of the solution and solid-state structural chemistry of Th(IV) hydrolysis and condensation products, 9,11 the stabilization of novel secondary building units in Th(IV)-based Metal-Organic Frameworks (MOFs), 15,19 the characterization of complexes with very high coordination numbers (up to 14) and unusual coordination geometry, 7 the in-depth investigation of the thermodynamic and electronic properties of heterometallic An z /Th IV (z = various) and An/Th IV /3d-metal ion MOFs with "structural memory" 20 and the progress in compounds with Th(IV)-ligand multiple bonds. 12 Schiff bases have been used extensively as ligands with metals across the Periodic table. 27 These molecules are attractive for their easy and high-yield preparation, their solubility in a variety of organic solvents, and their ability to stabilize metals in various oxidation states and control behaviour of metal ions during catalysis; [28][29][30][31][32][33] in addition, their steric and electronic properties are highly tunable. The coordination chemistry of the trans-UO 2 2+ ion (uranyl ion) with Schiff bases is a well explored area. [34][35][36][37][38] However, tetravalent actinides, An(IV), have been little studied. Schiff-base complexes of An(IV) are of great utility for the development of new An separation strategies in nuclear fuel processing and nuclear waste management. 39 Restricting the discussion to Th(IV), its Schiff-base complexes (which are scarce in the literature [40][41][42] are closely related to the development of colorimetric detection of this metal ion and applications in real-time samples, 43 to the construction of on/off sensors for Th(IV) exploiting the ligandbased light emission of its complexes, 44 and to the study of Th(IV) reactions with the derivatives of vitamin B 6 as a means to understand the assimilation of this metal ion and other heavy nuclei by living organisms and its acute toxicological effects. 45 A drawback associated with tetradentate dianionic O, N,N,O ligands of the salen 2− -type is their planarity and charge. The valence completion of An(IV), e.g. Th(IV), requires often two ligands per metal ion and this may result in hindered reactivity due to steric effects. Once the bis-Schiff-base complex is isolated, no further ligand-substitution is possible and the redox chemistry of redox-active An IV centres, e.g. U VI , is rather poor. 41 Therefore, the interest in this area has shifted to potentially dianionic, tridentate Schiff bases. Two such ligands are The number 4 in the first (empirical) name of L′H 2 (which is in disagreement with the numbering code in Scheme 1) is used because its origin is from the name of the starting material, i.e., 4-methyl-2-aminophenol used for the synthesis of L′H 2 . 46 Upon deprotonation of one or both -OH groups of LH 2 and L′H 2 , each negatively charged phenoxido atom can bridge two or three metal ions favouring cluster formation, while the position of the imino N atom simultaneously assures the formation of chelating rings that can lead to thermodynamic stability of the products in solution. The anions of the ligands have been widely used in main group-, 47 3d-, 48-51 4f-52,53 and mixed 3d/4f-metal 54 chemistry by our group and other scientists, with emphasis on the magnetic properties of the complexes. The use of LH 2 and L′H 2 in 5f-metal chemistry is negligible (vide infra). No Th(IV) complexes have been prepared with these ligands.
Having all the above mentioned in mind, and given our interest in the chemistry of An ions [55][56][57] and in the coordination chemistry of LH 2 and its derivatives, 46,48-52 we report herein the reactions of Th(NO 3 ) 4 ·5H 2 O with LH 2 and L′H 2 , and the full characterization of the interesting products.
Experimental section
Materials, physical and spectroscopic measurements All manipulations were performed under aerobic conditions using materials (reagent grade) and solvents as received. The free ligands LH 2 and L′H 2 were synthesized by the reactions of salicylaldehyde, and 2-aminophenol and 4-methyl-2-amino- phenol, respectively, in refluxing MeOH or EtOH using published procedures; 58-60 the yields were in the 75-80% range. Their purity was checked by microanalyses and 1 H NMR spectra. Caution! Natural thorium ( primary isotope 232 Th) is a weak α-emitter (4.012 MeV) with a half-life of 1.41 × 10 10 years. Manipulations and reactions should be carried out in monitored fume hoods in a radiation laboratory which has αand β-counting equipment; the laboratory atmosphere is inspected monthly for contamination. Elemental analyses (C, H, and N) were performed by the University of Patras microanalytical service. FT-IR spectra (4000-400 cm −1 ) were recorded using a PerkinElmer 16PC spectrometer with samples prepared as KBr pellets and as Nujol or hexachlorobutadiene mulls between CsI disks. Solid-state emission and excitation spectra were recorded using a Cary Eclipse fluorescence spectrophotometer. 1 H NMR spectra in DMSO-d 6 were recorded using a 400 MHz Bruker Avance DPX spectrometer with TMS as an internal standard. FT-Raman spectra were recorded using a Bruker (D) FRA-106/S component attached to an Equinox 55 spectrometer. An R510 diode-pumped Nd:YAG laser at 1064 nm was used for Raman excitation with a laser power of 250 mW on the sample, utilizing an average of 100 scans at 4 cm −1 resolution. Conductivity measurements were carried out at 25°C with a Metrohm-Herisau E-527 bridge and a cell of standard constant.
Synthetic details
To a colourless solution of Th(NO 3 ) 4 ·5H 2 O (0.057 g, 0.10 mmol) in MeCN (16 mL) was added solid orange LH 2 (0.043 g, 0.20 mmol). The solid soon dissolved and the colour of the solution turned yellow. The reaction solution was stirred for a further 10 min, filtered and stored in a closed flask at room temperature. X-ray quality, yellowish crystals of the product were obtained in a period of 3 d. The crystals were collected by filtration, washed with cold MeCN (2 × 2 mL) and Et 2 O (3 × 2 mL), and dried in vacuo overnight. The yield was ∼30% (based on the metal available). The product was analyzed satisfactorily as lattice MeCN-free, i.e., 1. Analytical data, calcd for C 91 [Th 4 O(NO 3 ) 2 (L′H) 2 (L′) 5 ]·2.4MeCN (2·2.4MeCN). To a red solution containing L′H 2 (0.045 g, 0.20 mmol) in MeCN (16 mL) and Bu n 4 NOH (25% solution in MeOH, 57 μL, 0.20 mmol) was added solid Th(NO 3 ) 4 ·5H 2 O (0.057 g, 0.10 mmol). The solid soon dissolved and the resulting light orange solution was vigorously stirred for 10 min, filtered and stored in a closed flask at room temperature. X-ray quality orange crystals of the product were precipitated over a period of 24 h. The crystals were collected by filtration, washed with cold MeCN (2 mL) and Et 2 O (3 × 2 mL), and dried in vacuo overnight. Typical yields were in the 35-45% range (based on the metal available). The product was analyzed satisfactorily as lattice MeCN-free, i.e., 2.
Single-crystal X-ray crystallography
Yellowish orange (0.06 × 0.11 × 0.43 mm) and orange (0.17 × 0.17 × 0.36 mm) crystals of 1·4MeCN and 2·2.4MecN, respectively, were taken from the mother liquor and immediately cooled to −103°C (1·4MeCN) and −93°C (2·2.4MecN). X-ray diffraction data were collected using a Rigaku R-AXIS SPIDER Image Plate diffractometer using graphite-monochromated Mo Kα radiation. Data collection (ω-scans) and processing (cell refinement, data reduction and empirical absorption correction) were performed using the CrystalClear program package. 61 The structures were solved by direct methods using SHELXS-97 62 and refined by full-matrix least-squares techniques on F 2 with the latest version of SHELXL (2014/7). 63 The H atoms were either located by difference maps and refined isotropically or were introduced at calculated positions as riding on their respective bonded atoms. All non-H atoms were refined anisotropically. Most structural plots were drawn using the Diamond 3 program package. 64 Important crystallographic data are listed in Table 1. Full details can be found in the CIF files.
Synthetic comments
In the present study, we have investigated the reactions between the tridentate Schiff bases LH 2 and L′H 2 and thorium 5 ]·4MeCN (1·4MeCN) in rather low yields (∼30%), eqn (1). Efforts to prepare a compound with all the ligands in their doubly deprotonated form (L 2− ), e.g., [Th 4 O(L) 7 ] were not successful, even when we used an external base (Et 3 N, Bu n 4 NOH) to LH 2 ratios up to 2 : 1. The obtained product was again 1; somewhat to our surprise the yield of the reaction increased only by 10%, reaching 40-45% (the procedure with the addition of the base is not described in the Experimental section).
A completely analogous reaction between Th(NO 3 ) 4 ·5H 2 O and L′H 2 in MeCN, using the same concentrations of the reactants as in the case of 1, resulted only in a few orange crystals (yield lower than 10%) of [Th 4 O(NO 3 ) 2 (L′H) 2 (L′) 5 ]·2.4MeCN (2·2.4MeCN). The addition of an external base was deemed necessary to increase the yield. Thus, the Th(NO 3 ) 4 ·5H 2 O/L′H 2 / Bu n 4 NOH (1 : 2 : 2) reaction system in MeCN gave a light orange solution, from which the product was isolated in a ca. 40% yield, eqn (2). Increase of the L′H 2 to OH − ratio from 2 : 2 to 2 : 4, to further improve the yield, resulted in the precipitate of amorphous hydroxide thorium(IV) species. the vertexes of a distorted tetrahedron, with a central μ 4 -O 2− ion (O1) bonded to each metal ion. The Th IV centres are also held together by two 3.21 (Harris notation 65 ) LH − groups, one 3.221 L 2− and four 2.211 L 2− ligands (Scheme 2). Two slightly anisobidentate chelating nitrato groups (donor atoms O16/O18 and O19/O20) complete the coordination spheres of Th2 and Th4. The core of the molecule is {Th IV 4 (μ 4 -O)(μ-OR′) 8 } (Fig. 2). Of interest is the fact that the H atom of one of the acidic -OH groups of each LH − ligand is clearly located on the imine nitrogen atom (N2 and N6), thus blocking the coordination of the latter. )-Th IV angles are in the 103.7(1)-112.6(1)°range, in agreement with the distorted tetrahedral arrangement of the metal centres. Th1 is 8 coordinate; it is coordinated by seven oxygen atoms, with the Th1-O bond lengths ranging from 2.205(3) to 2.486(3) Å and one nitrogen atom. Two oxygen atoms are terminal from one LH − (O13) and one L 2− (O3) ligands, four are bridging from a LH − (O4) and three L 2− (O2, O7, and O15) ligands and the seventh oxygen atom is the oxido group. Th2 is 9-coordinate; it is surrounded by eight oxygen atoms, with the Th2-O bond lengths ranging from 2.235(3) to 2.612(3) Å, and by one nitrogen atom. One oxygen atom is bridging from a LH − (O4) ligand, four oxygen atoms (one terminal, O6; three bridging, O7/O8/O10) belong to three L 2− ligands, two oxygen atoms are from a chelating nitrato group (O16 and O18) and the eighth oxygen is the oxido group. Th3 is 8-coordinate; it is bonded to seven oxygen atoms, with the Th3-O bond length ranging from 2.228(3) to 2.574(3) Å, and to one nitrogen atom. Two oxygen atoms are terminal from one LH − (O5) and one L 2− (O9) ligands, four are bridging from a LH − (O12) and three L 2− (O2, O8, and O11) ligands and the seventh oxygen atom is the oxido group. Finally, Th4 is surrounded by eight oxygen and two nitrogen atoms being 10-coordinate. The Th4-O bond lengths range from 2.213(3) to 2.652(3) Å. One oxygen atom is bridging from a LH − (O12) ligand, four oxygen atoms (one terminal, O14; three bridging, O10/O11/O15) belong to two L 2− ligands, two oxygen atoms are from a chelating nitrato group (O19 and O20) and the eighth oxygen atom is the oxido group. The Th-N bond distances are in the range of 2.601(4)-2.741(4) Å and are typical for 8-, 9-and 10-coordinate species with Schiff bases as ligands. [40][41][42][43][44][45] To estimate the closer coordination polyhedra defined by the donor atoms around the Th IV centres in 1·4MeCN, a comparison of the experimental structural data with the theoretical values for the most common polyhedral shapes with 8, 9 and 10 vertices was performed using the SHAPE program. 66 The best fit was obtained for the triangular dodecahedron (Th1), the muffin (Th2), the biaugmented trigonal prism (Th3) and sphenocorona (Th4) (Fig. 3 and Tables S3-S5 ‡).
There are two classical, relatively strong, intraligand H bonds within the tetranuclear molecule (Fig. S1, Table S1 ‡), with the protonated nitrogen atoms (N2 and N6) of the LH − ligands as donors and their terminal, formerly salicylaldehyde, phenolato oxygen atoms (O5 and O13) as acceptors. A variety of intermolecular H-bonding interactions are present in the crystal structure. Three lattice MeCN molecules, three coordinated nitrato oxygen atoms (O16, O19, and O20) and one non-coordinated nitrato oxygen atom (O21) act as acceptors, while the donors are aromatic and aliphatic carbon atoms from the ligands and MeCN. The overall result is the formation of chains parallel to the α axis (Fig. 4).
Molecule 2 has a very similar structure ( Fig. 5 and S2 ‡) to that of 1 (Fig. 1). The core, the metal topology, the coordination polyhedra (Fig. S3, Tables S3-S5 ‡) and the coordination modes of the organic and nitrato ligands are almost identical in the two structures. In the numbering schemes of the metal centres, Th1, Th2, Th3 and Th4 in 2 are equivalent to Th4, 1·4MeCN and 2·2.4MeCN, and the Harris notation that describes these modes. 67 where EO4 is the tetraethylene glycolate(−2) ligand, and [Th 4 OCl 2 I 6 {O(CH 2 ) 2 OCH 3 } 6 ]. 68 Interestingly, the authors note that the oxido bridge in the former complex is the result of an oxide impurity from the ThCl 4 starting material. 67 In the latter complex, the source of the central μ 4 -O 2− unit was verified to be 68 dimethoxyethane (DME) present in the starting [Th 4 Cl 4−x I x (DME) 2 ] material, and not adventitious H 2 O (the reaction and the manipulation were performed in a strictly inert atmosphere (N 2 ) under vacuum). In the case of 1 and 2, the source of the oxido group seems to be H 2 O from the solvent (MeCN) and the hydrated thorium(IV) nitrate. Such μ 4 -O 2− groups are extremely rare in An(IV) cluster chemistry despite their occurrence in the fluorite-type structures. 8 Generally, the tetravalent actinides Th-Pu represent some of the hardest cations of the Periodic table; because of their high charge density and acidity, these metal ions are particularly prone to hydrolysis and condensation. 9,18 Compounds 1 and 2 are new members of a large family of complexes of 3d-, 4f-and mixed 3d/4f-metal ions (ref. [47][48][49][50][51][52][53][54] are only representative) containing anionic forms of LH 2 and L′H 2 . However, they join a handful of structurally characterized An complexes which are listed in Table 4. Unfortunately, no U(IV), Np(IV) and Pu(IV) complexes with any forms of LH 2 and L′ H 2 have been reported, so no direct comparison of An complexes with the metal in the IV oxidation state are possible. The previous An examples were mononuclear (neutral 69 and anionic 34,70 ) and dinuclear 55 uranyl (UO 2 2+ ) complexes. The completely different structures between the uranyl and thorium(IV) complexes (Table 4) are attributed to the completely different chemical nature of the two metal species. In a general sense, as compared to the hexavalent actinides, which almost invariably form the well-established actinyl cation (An VI O 2 2+ ), the tetravalent actinides adopt more spherical or isotropic coordination geometries, thus leading to structural units and types that are quite distinct from those of the hexavalent An ions. 18 Restricting this discussion to Th(IV) and U VI O 2 2+ , Th(IV) exhibits an effective charge that matches its formal oxidation state, whereas U(VI), in the form of the uranyl species, behaves as a metal ion with an effective charge of +3.3. 10 A final interesting point is the fact that the 3.210 ligation mode of LH − and L′H − established in the structures of 1·4MeCN and 2·2.4MeCN, respectively, is observed for the first time in the coordination chemistry of LH 2 and L′H 2 .
Spectroscopic studies
In the IR spectra of 1 and 2 ( Fig. S4 and S5 ‡), the mediumintensity and broad band at ∼3450 cm −1 is attributed to the ν(NH + ) vibration of the LH − or L′H − ligands (vide supra); 71 the broadness of the band is indicative of H bonding. The spectrum of 1 exhibits a medium-intensity and a strong band at 1622 and 1604 cm −1 , respectively, which is assigned 46,55,71 to the CvN stretching vibration, ν(CvN), of the Schiff base linkage. These bands have been shifted to lower wavenumbers compared with the corresponding band in the spectrum of the free LH 2 ligand (1630 cm −1 , Fig. S6 ‡), in agreement with the protonation or coordination of the imine nitrogen. The appearance of the two bands reflects the presence of two types ( protonated and coordinated) of CvN groups in the complex. The two bands appear at 1628 and 1602 cm −1 in the spectrum of 2. The band at 1602 cm −1 has been shifted to a lower wavenumber compared with the band in the spectrum of the free L′H 2 ligand (1628 cm −1 , Fig. S7 ‡), whereas the band at 1628 cm −1 has remained at the same wavenumber. The latter experimental fact is not unusual. 72,73 Extensive experimental and theoretical studies on complexes with Schiff bases containing a CvN bond (with the carbon atom attached to an aromatic ring) have revealed that a change in the s character of the N lone pair occurs upon coordination or protonation and the s character of the N orbital involved in the CvN bond increases; this change in hybridization leads to a greater CvN stretching constant relative to the free neutral ligands, thus shifting the ν(CvN) vibrational mode to slightly higher wavenumbers. The IR bands at ∼1470, ∼1270 and ∼1030 cm −1 in the spectra of both complexes are assigned 74 to the ν 1 (A 1 )[ν(NvO)], ν 5 (B 2 )[ν as (NO 2 )] and ν 2 (A 1 )[ν s (NO 2 )] vibrations of the bidentate chelating (C 2v ) nitrato group; the separation of the two highestwavenumber stretching bands is ∼200 cm −1 , in agreement with the bidentate character of the coordinated nitrato groups. 52,74 The Raman spectra of the well dried samples of complexes 2 and 1 are shown in Fig. 7 and S8, ‡ respectively. The characteristic ν(C aromatic -H) peak appears at ∼3050 cm −1 in both spectra; 11,46,72,75,76 extra peaks at 2918 and 2860 cm −1 in the spectrum of 2 are due to the ν as (CH 3 ) and ν s (CH 3 ) modes, respectively. 46,75 The peak at ∼1620 cm −1 in both spectra is due to the ν(CvN) mode of the Schiff-base linkage; 46,76 the clear splitting of this peak reflects the presence of two types of imine linkages ( protonated and coordinated) in the tetranuclear complexes. The two highest-frequency stretching nitrogen-oxygen vibrations of the bidentate chelating nitrato groups are located 74,77 at 1477 and 1266 cm −1 for 1, and at 1500 and 1284 cm −1 for 2. The 1 H NMR spectra of 1 and 2 in DMSO-d 6 ( Fig. S9 and S10 ‡) are extremely complicated, suggesting the presence of several different species in solution and indicating that the structures of the complexes are not retained in solution. For example, the spectrum of 2 shows four signals between δ 2.00 and 2.30 ppm, with different integration ratios, attributed 11,46 to the protons of the methyl groups. The imine protons appear around δ 9 ppm in the form of multiple signals. 43 Of particular interest is the appearance of signals at δ values in the region 14.08-13.76 ppm and at ∼9.7 ppm. The former can be assigned to the very acidic protons of -CvNH + groups from the LH − and L′H − ligands; the latter most probably indicates the presence of -OH groups arising from protonation of the deprotonated phenolato oxygen atoms, the most possible proton sources being some of the -CvNH + groups and/or hydrolysis of [Th(H 2 O) x ] 4+ species; a strong evidence of hydrolytic processes comes from the appearance of signals in the δ 4.8-6.2 ppm region assigned 11 to coordinated OH − groups. Our proposal for the decomposition of the complexes in solution is reinforced by the molar conductivity values, Λ M (10 −3 M, 25°C), in DMSO, which are 86 (1) and 69 (2) S cm 2 mol −1 ; these values indicate the presence of ionic species in this solvent. 78 Solid-state, room-temperature emission spectra of the free ligands LH 2 and L′H 2 ( Fig. S11 and S12, ‡ respectively), and complexes 1 and 2 ( Fig. 8 and S13 ‡) were recorded. The optical behavior of the two complexes is similar. Upon maximum excitation at 400 nm, the solid complexes show a rather sharp emission band with a maximum at ∼ 540 nm (emission of green light). As Th(IV) is non-emissive, the emission can be assigned to a charge transfer state within the coordinated organic ligands. 44,[79][80][81][82][83][84] Tetravalent thorium has a 5f 0 configuration. All of the electrons are spin paired in this electronic state and emission is not expected. 81 It is also hardly oxidizing and consequently low-energy metal-centered charge-transfer excited states do not exist. 80 Additional evidence for the intraligand character of the green emission in the complexes comes from the study of the emission properties of the free ligands. Upon maximum excitation at 497 nm (LH 2 ) or 465 nm (L′H 2 ), the ligands emit at 540 nm (LH 2 ) and 610 nm (L′H 2 ). The development of "turn-on" or "turn-off" fluorescent sensors for Th(IV) is an area of intense interest due to their potential use for thorium analysis/identification in nuclear waste investigation. 44,[82][83][84] Conclusions and perspectives Even at the time of submission of the present work, thorium chemistry continues to surprise the inorganic chemistry community. [85][86][87][88][89][90][91] For example, the number of the crystallographically characterised Th(III) complexes (mainly with cyclopentadienyl ancillary ligands) increases and the first example of a square planar thorium complex has been just reported. 89 Many scientists believe that with the harmful effects of global climate change becoming more evident with every passing year, more research activity and allocated resources will help the Z = 90 element realize its great potential and become a truly valuable source of materials in our energy economy. 5 As far as the present work is concerned, it is rather difficult to conclude on a research topic, i.e., Th(IV) complexes with Schiff-base ligands, which is still at its infancy. In this report, we believe that we have contributed to some extent into the chemistry of thorium(IV) clusters, and into the coordination chemistry of LH 2 and L′H 2 and related tridentate Schiff bases. Complexes 1 and 2 have interesting molecular structures and contain the extremely rare {Th IV 4 (μ 4 -O)} 14+ core. They also join a handful of fluorescent Th(IV) complexes. The observed ligandbased green emission might indicate that LH 2 and L′H 2 and related tridentate Schiff bases containing naptholate (instead of phenolate) aromatic rings, can be considered to be fluorescent sensors for toxic Th(IV) analysis. The development of such a fluorescence detection in solution might provide a simple, rapid, selective and low-cost method for thorium ion determination. Several fluorogenic optodes for Th(IV), with a variety of organic molecules as sensing materials, have been developed in the last 10 years or so. However, some limitations such as a poor sensitivity, a limited working pH range and minor or major interference from other metal ions (e.g. Al 3+ , UO 2 2+ , Ti 4+ , Zr 4+ and trivalent lanthanides), due to their similar chemical properties, need to be addressed. Very recently Kumar and Kumar designed, synthesized, fabricated and used an excellent 8-aminoquinoline-based fluorescent optode for the quantification of Th 4+ in various aqueous, monazite sand and gas mantle samples. 92 In the present case, the ligand L′H 2 could be, in principle, used as the sensor of Th(IV) in the presence of UO 2 2+ , since the complex of the latter with (L′) 2− does not emit light at room temperature under several excitation wavelengths. 55 Future work will be focused on the study of the coordination chemistry of tetradentate Schiff bases containing one -OH and one -COOH group (instead of two -OH groups) on different aromatic rings towards Th(IV) and the investigation of the emission properties of Th(IV) complexes in solution. Work is also in progress to develop the Th(IV)-oxime/oximate chemistry (oximes also contain a CvN group) and synthesize the almost elusive thorium η 2 -(O,N)-oximate complexes. 93 These efforts, already well advanced, will be reported in due course.
Conflicts of interest
There are no conflicts to declare. | v3-fos-license |
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} | pes2o/s2orc | Evaluation of continuous water vapor δ D and δ 18 O measurements by off-axis integrated cavity output spectroscopy
Recent commercially available laser spectroscopy systems enabled us to continuously and reliably measure the δD and δ18O of atmospheric water vapor. The use of this new technology is becoming popular because of its advantages over the conventional approach based on cold trap collection. These advantages include much higher temporal resolution/continuous monitoring and the ability to make direct measurements of both isotopes in the field. Here, we evaluate the accuracy and precision of the laser based water vapor isotope instrument through a comparison of measurements with those found using the conventional cold trap method. A commercially available water vapor isotope analyzer (WVIA) with the vaporization system of a liquid water standard (Water Vapor Isotope Standard Source, WVISS) from Los Gatos Research (LGR) Inc. was used for this study. We found that the WVIA instrument can provide accurate results if (1) correction is applied for time-dependent isotope drift, (2) normalization to the VSMOW/SLAP scale is implemented, and (3) the water vapor concentration dependence of the isotopic ratio is also corrected. In addition, since the isotopic value of water vapor generated by the WVISS is also dependent on the concentration of water vapor, this effect must be considered to determine the true water vapor concentration effect on the resulting isotope measurement. To test our calibration procedure, continuous water vapor isotope measurements using both a laser instrument and a cold trap system were carried out at the IAEA Isotope Hydrology Laboratory in Vienna from August to December 2011. The calibrated isotopic values measured using the WVIA agree well with those obtained via the cold trap method. The standard deviation of the isotopic difference between both methods is about 1.4 ‰ for δD and 0.28 ‰ for δ18O. This precision allowed us to obtain reliable values for d-excess. The day-to-day variation of d-excess measured by WVIA also agrees well with that found using the cold trap method. These results demonstrate that a coupled system, using commercially available WVIA and WVISS instruments can provide continuous and accurate isotope data, with results achieved similar to those obtained using the conventional method, but with drastically improved temporal resolution.
between both methods is about 1.4 ‰ for δD and 0.28 ‰ for δ 18 O.This precision allowed us to obtain reliable values for d-excess.The day-to-day variation of d-excess measured by WVIA also agrees well with that found using the cold trap method.These results demonstrate that a coupled system, using commercially available WVIA and WVISS instruments can provide continuous and accurate isotope data, with results achieved similar to those obtained using the conventional method, but with drastically improved temporal resolution.
Motivation
Global monitoring of oxygen and hydrogen isotope contents in precipitation has been carried out by the International Atomic Energy Agency (IAEA) in cooperation with the World Meteorological Organization (WMO) for more than 40 yr through the IAEA/WMO Global Network of Isotopes in Precipitation (GNIP).GNIP has led to a great deal of study on the temporal and spatial variations of environmental stable water isotopes (δ 18 O and δD) and is widely used in various scientific disciplines, such as hydrology, climatology, ecology and biology (e.g.Araguas-Araguas et al., 2000;Hoffmann et al., 2000;Bowen et al., 2005).On the other hand, although water vapor is ubiquitous in the atmosphere, the availability of isotopic data on atmospheric water vapor is quite limited.One of the main reasons for this scarcity is that water vapor sampling presents more difficulties than precipitation sampling.Traditionally, atmospheric water vapor samples for isotope analysis were trapped in a cold bath filled with a cooling agent, such as liquid N 2 or dry ice/ethanol.Next, a collected water sample was extracted from the cold trap and analyzed using isotope ratio mass spectrometry (IRMS) or laser spectroscopy methods from the liquid water samples.Because incomplete trapping of water vapor results in isotope fractionation, self-designed trap system (Schoch-Fischer et al., 1984;Yakir and Wang, 1996;He and Smith, 1999;Uemura et al., 2008) have been developed to meet required trapping efficiency.In addition, extensive laboratory work is necessary to extract a vapor sample from a cold trap.More recently, alternative moisture trapping techniques have been reported, resulting in more sophisticated methods (Helliker et al., 2002;Han et al., 2006;Peters and Yakir, 2010).However, these methods are still labor intensive and require samples to be prepared in a laboratory.
A more recent alternative utilizes laser based instruments (e.g.Kerstel et al., 2006) and instruments based on optical spectroscopy for water vapor isotope analysis (e.g.Griffith et al., 2006).These instruments can directly measure atmospheric water vapor isotopes in real-time with high temporal resolution and without the time-consuming physical collection of condensed moisture.In addition, the latest instruments are of a reasonably small size and power consumption is low, allowing for the application of this technique to in-situ measurements (Lee et al., 2005;Gupta et al., 2009;Sturm and Knohl, 2010;Aemisegger et al., 2012).Notably, Lee et al. (2006) have reported continuous δD water vapor records for New England, USA, for a period of one year, using a tunable diode laser (TDL) spectroscopy system.However, because of instrumental nonlinearity, drifting signal, dependence on water vapor concentration, temperature sensitivity, and pressure variations in the absorption cell, regular and frequent calibration with a water vapor standard of known isotope content is required for longterm field observations.In addition, a required water vapor isotope standard of known isotopic content is not commercially available.Therefore, custom-made vaporizer systems, which can provide a frequent supply of water vapor of known isotope content to an in-situ instrument, had to be developed before such an instrument could be deployed in the field (Lee et al., 2005;Iannone et al., 2009;Schmidt et al., 2010;Sturm and Knohl, 2010;Gkinis et al., 2010).This represented a major limitation for the widespread use of this technique.However, a recently developed robust vaporizer system, coupled with the laser instrument, has become commercially available.Tremoy et al. (2011) have tested the performance of the calibration system (Standard Delivery Module, SDM) produced by Picarro, Inc. coupled with their laser instrument (Wavelength-Scanned Cavity Ring-Down Spectroscopy, WS-CRDS).
In this study, we use another commercially available instrument based on off-axis integrated cavity output spectroscopy, manufactured by Los Gatos Research, Inc. (LGR) (Water Vapor Isotope Analyzer, WVIA) coupled with an accessory device for the vaporization of a liquid water standard (Water Vapor Isotope Standard Source, WVISS).The WVISS is programmable from the WVIA and thus this coupled system is capable of automatically conducting a calibration routine at specific intervals.A detailed assessment of the WVIA has already been undertaken in previous studies (Wang et al., 2009;Sturm and Knohl, 2010;Rambo et al., 2011).Sturm and Knohl (2010) have reported a strong concentration dependence and temperature sensitivity of the WVIA.Rambo et al. (2011) have used the same coupled system with the WVISS and proposed a routine calibration procedure to correct for both concentration dependence and temperature sensitivity.Recently, Aemisegger et al. (2012) completed an evaluation of this coupled system through comparison with the WS-CRDS, in conjunction with the SDM.These studies have highlighted the usefulness of this system to the field monitoring, however long-term water vapor isotope monitoring testing across several seasons has not been reported yet.In particular, the performance and reliability of the calibration system (WVISS) must be stringently examined before it can be applied in field conditions.Here, we detail the results of extensive laboratory experiments to develop a calibration procedure for routine field application.Then, the calibration procedure developed from the laboratory experiment was evaluated through a long-term monitoring program of water vapor isotopes, using both the laser instrument and the conventional cryogenic moisture trapping method.
Laser spectroscopy system
The laser spectroscopy system used to measure water vapor isotopes (δ 18 O and δD) was the model DLT-100 water vapor isotope analyzer (WVIA, LGR Inc., Mountain View, CA, USA) manufactured in June 2009 (model 908-0004).To optimize performance of the WVIA, the operating software was updated to the latest version in May 2011.This analyzer is based on an off-axis integrated cavity output spectroscopy system (OA-ICOS) using a semiconductor diode laser with a wavelength of around 1.39 µm (e.g.Bear et al., 2002).Here we describe the OA-ICOS measurement technique briefly.In OA-ICOS, the laser beam is directed off-axis with respect to the optical cavity.The laser wavelength is swept through the selected absorption line of each species (H 2 O, HDO, and H 18 2 O) and the transmitted laser intensity through a cavity is recorded.In addition, to determine effective optical path length in the cavity at each wavelength, ring down-time measurement is made by rapidly switching off the laser diode, which is measuring decay of the light intensity over time.The typical ring-down time is around 10 µs at 10 000 ppm H 2 O concentration and it corresponds to the effective path length of 3 km in a 0.59 m-long cell.This path length is more than 10 times longer than that of a multi-pass cell and results in a high signal to noise ratio for isotope measurement.The mixing ratio χ of each water isotope species is determined through integration of the absorption spectrum as follows: where I ν is the transmitted laser intensity at wavelength ν, I o is the initial intensity, P is gas pressure in the cell, S is absorption line strength, L eff is effective optical path length.The subscript x represents each water isotope species.Calculated mixing ratios of water isotopologues are converted into a δ value with respect to the international standard Vienna Standard Mean Ocean Water (V-SMOW) (Coplen, 1996).
where R is the atomic ratio, D/H or 18 O/ 16 O, respectively.
Experimental setup for water vapor isotope measurement
A sample of air is introduced into an absorption cell (optical cavity) of the WVIA via an external pump (KNF, N920AP.29.18) downstream of the instrument at a constant flow rate of 0.5 l min −1 .The volume of an absorption cell is around 830 ml, while inside pressure is controlled at 37 ± 0.007 hPa via a pressure controller and temperature is maintained at 49 • C to avoid the condensation of water vapor from ambient air in the cell.Based on these figures, the exchange time is estimated to be approximately 4 s.The data output frequency for water isotopologue measurements is 1 Hz.
To introduce water vapor standards into the WVIA, we used an LGR Water Vapor Isotope Standard Source (WVISS) device, manufactured in April 2010 (see Fig. 1a).It consists of a two-stage air dryer unit and a heated evaporation jar (1 l) for accepting water from a 0.5 l container via a nebulizer (part no.800-1-005-01-00; Savillex Ltd., Eden Prairie, MN, USA).Compressed room air is passed through a desiccant dryer (part no.MDH5-FLE-S37, Twin tower engineering, Broomfield, CU, USA) and a column of granular Drierite (size 8-20 mesh), providing dry air into the system (H 2 O concentration < 10 ppm).The compressed dry air stream from the drying unit is then split into two lines: one goes into the evaporation jar and the other supplies air to the nebulizer.The nebulizer aspirates liquid water from a water reservoir at a rate of 50 µl min −1 and injects tiny water droplets into the evaporation jar.The jar is heated to about 80 • C so that droplets are immediately evaporated; the resulting vapor should have the same isotopic composition as that of the source water.The humidity of the output air can be varied from 2500 to 25 000 ppm by adjusting the dry air mixing ratio in the evaporation jar.The flow of dry air is controlled by a mass flow controller (part no.FMA5420, OMEGA Engineering, Stamford, CT, USA, 0-5 l min −1 ).
The output tubing from the jar is connected to a T-shape splitter via 3/8 inch (9.52 mm) Teflon tubing.One outlet constitutes an exhaust to room air, whereas the other outlet is directed to the analyzer through a 3-way valve, which controls airflow by switching between the ambient air inlet and WVISS-generated standard gas.The WVISS-generated standard gas is drawn by an external pump downstream of the analyzer (see above).The tubing joining the WVISS and the analyzer is made of 1/4 inch (6.35 mm) Teflon tubing (with a length of around 1 m).The dilution rate with dry air and valve operation is controlled by the WVIA.Even though standard gas is released from the heated chamber (80 • C), there is no clear temperature shift in the optical cavity when the valve is switched from the ambient air inlet to the standard gas line.Raw data from the analyzer is stored on the internal hard disk, and is transferred via ethernet cable.Calibration is undertaken as a post-processing step using the external computer system.
Performance test of the WVISS
To test the assumption that no fractionation occurs in the WVISS, air generated in the unit was physically collected using a cold trap (see Fig. 1a).A water reservoir was filled with an in-house laboratory water standard (δD = −78 ‰ and δ 18 O = −11.3‰), then the WVISS was run continuously during vapor trapping (2-6 h) to obtain a sufficient amount of water for isotope analysis.WVISS air exhaust was drawn by an external pump at a flow rate of 1.5 l min −1 , then water vapor was collected with a glass trap submerged into a dry-ice/isopropanol mixture at −78 • C.This experiment was repeated more than 10 times at 6 levels of water vapor concentration (3000 ppm, 4000 ppm, 5500 ppm, 6000 ppm, 8000 ppm, 10 000 ppm).In this study, we used a custom manufactured glass trap for vapor collection.This trap has been used for water vapor sample collection in various field projects (Kurita and Yamada, 2007;Kurita, 2011;Kurita et al., 2011).Along with this, we undertook a trapping test using the WVISS/WVIA system before starting the laboratory experiment.A glass trap was inserted between WVISS and WVIA and output humidity from a cold trap was monitored by the WVIA.This pretest confirmed complete trapping of water vapor because the reported humidity from the WVIA is less than 10 ppm during such a trapping period.The collected amount of water was less than 1.5 g.Sampling time varied from two to six hours, depending on the concentration.After sample collection, water in the trap was subsequently thawed and poured into a 9-ml glass vial.Isotopic analysis of the collected water was undertaken at the IAEA Isotope Hydrology Laboratory using IRMS (Delta plus, Thermo Scientific, Bremen, Germany) with a locally developed equilibration device and through cavity ring-down spectroscopy isotopic water analysis (model L1102-i; Picarro Inc., Sunnyvale, CA, USA) with a CTC Analytics autosampler (model HTC-PAL; Leap Technologies, Carrboro, NC, USA).Measurement precision, including internal and external variation, is better than ± 0.15 ‰ for δ 18 O and ± 2 ‰ for δD.
During vapor trapping, the WVISS generated vapor was introduced into the WVIA for 10 min in every hour.Regarding comparison with the values of water vapor samples collected by cold trap method, the time-averaged isotopic values of WVIA measurements were calculated from the last 5 min of each 10 min continuous measurement run every hour.
Accuracy and precision of the WVIA measurement
Isotope measurements of nine in-house water standards with different δ 18 O and δD contents (Table 1) were performed to help determine a routine calibration procedure for the coupled WVIA and WVISS system.The water standards ranged in isotope values from −397.9 to −0.1 ‰ for δD and from −50.87 to −0.08 ‰ for δ 18 O.Each water standard was stored in a 9-ml glass vial.The nebulizer feed tube was inserted into a vial of standard, and the mixing ratio of the WVISS-generated vapor was set to 10 000 ppm. Vapor was continuously introduced into the WVIA and the isotope data for the last 10 min of the 20 min measuring periods was averaged.After a 20 min measurement period, the water bottle with the isotope standard was replaced.To reduce memory effects from residual water, water in the tubing was flushed away with dry air before the tube was inserted into a next standard.As described in Sturm and Knohl (2010), the WVIA response time is short, thus we did not observe any memory effect in the isotopic data during the final 10 min period, even though the most negative water standard (STD10) was measured just after the least negative water standard (STD11).Measurements of the nine water standards were performed during the same day (the measurement sequence was random) and the experiment was repeated more than 15 times.
Ambient air analysis
To evaluate the overall calibration procedure, we measured water vapor isotopes in ambient air using the WVIA-WVISS coupled system from August to December 2011.A sampling tube for outdoor air (5 m length of BEV-A-Line V tubing, 1/4 inch O.D.) was connected to a 3-way valve attached to the WVISS (see Fig. 1b).The outdoor air tube was used to sample the ambient water vapor present in Vienna, Austria outside our laboratory (tubing was routed through a window).A 1.0 µm filter with PTFE membrane (part no.10463523, Whatman, Dassel, German) was placed where the tube enters the WVISS.Air was drawn via the WVIA external pump at a flow rate of 0.5 l min −1 .Every 50 min, the 3-way valve automatically switched from ambient/outdoor inlet to WVISS standard air, whereupon standard air with a H 2 O concentration of 10 000 ppm was introduced to the WVIA for 10 min.After finishing the reference gas measurement, the valve switches back and ambient air sampling is resumed.
Atmospheric water vapor sampling using the cold trap method was also carried out to evaluate the validity of laser based water isotope measurements.Air sampling tubing (10 m length of PVC tubing, 14 mm O.D.) with an inlet at the same location as the WVISS ambient inlet was attached to a glass trap, which is the same as that used in the WVISS performance test (Fig. 1b).Air was drawn at a rate of 1.5 l min −1 through the trap immersed into a dry-ice/isopropanol bath for 6 to 15 h.The amount of water vapor trapped ranged from 1.6 to 13.2 g.We collected 154 samples from August to December 2011.The isotopic analysis of these samples was carried out using the procedure described above.
Performance test of the WVISS
The mean isotopic values and the statistical distribution of the WVISS-generated air collected by cold trap, at several water vapor concentrations are shown in Fig. 2.There was a large spread in isotopic values, greater than analytical error, arising from water isotope measurement at each vapor concentration (see Sect. 2.3).To examine whether a large spread in isotopic values in WVISS-generated air is related to WVISS vapor production, the values of water vapor samples using the cold trap (δ TRAP ) technique are compared with WVIA-reported raw isotopic values (δ WVIA ) in Fig. 3.For δD, the spread of values from the cold trap samples is almost twice as big as that of the WVIA reported values.The variation in δD WVIA values is less than ±2 ‰ from the mean value.The δD TRAP values include analytical error, which take place during liquid water isotope measurement (around 2 ‰).In addition, decanting small amounts of trap water (less than 1.5 g) into vials leads to additional scatter.The variation in δD TRAP is similar or less than the analytical uncertainty of cold trap samples.This uncertainty may result in a large spread and thus data points plot under a 1 : 1 line.However, there is a positive correlation between δD WVIA and δD TRAP (R = 0.369) and a similar feature that the δD WVIA values at 3000 ppm showed slightly enriched values, similar to the δD TRAP (see purple dots in Fig. 3a).Although the amplitude of a spread may be overestimated due to analytical uncertainty of cold trapped samples, the variation of δD TRAP values may reflect some isotopic shift in WVISS-generated air.
For δ 18 O, the relationship between δ 18 O WVIA and δ 18 O TRAP is scattered across the entire water vapor concentration range (3000 to 10 000 ppm) because of the timedependent isotope variation (see next section).However, a linear correlation between δ 18 O WVIA and δ 18 O TRAP was observed in the measurements at 10 000 ppm when the timedependent isotopic variation was relatively small.The points at 10 000 ppm measured between 27-29 November 2011 (red circles) are clearly distributed along a 1:1 line and the spread is significantly larger than the analytical error (0.15 ‰) (Fig. 3b).This suggests that a large spread of 10 000 ppm may reflect isotopic changes in WVISS-generated air.This is supported by the fact that δ vapor trapping varied, ranging from 9500 ppm to 10 500 ppm within each experiment.The maximum (minimum) value of δ 18 O TRAP corresponds to the lowest (highest) vapor concentration.From these results, we can infer that the large spread in δ 18 O values of WVISS-generated air at each concentration may be primarily related to WVISS vapor production.The average standard deviation in the range of 3000 to 10 000 ppm is about 0.25 ‰ for δ 18 O.In addition to a large spread in isotopic values, Fig. 2 shows that the mean isotopic value of WVISS-generated air varied in response to changing water vapor concentration.Measured δ 18 O at higher concentrations (6000 to 10 000 ppm) is higher than the known value, and at lower water vapor concentrations (3000 to 5500 ppm), δ 18 O values tend to be more negative than for higher concentrations.The mean value shifts from −11.1 ‰ at higher concentrations to −11.4 ‰ at lower concentrations.The difference between them is statistically significant (p < 0.05).Interestingly, this tendency runs opposite to the gradual decrease in δ 18 O TRAP with water vapor concentrations of 10 000 ppm.This suggests that factors resulting in a large δ 18 O spread are not primary drivers of concentration dependence; several effects may contribute to changing the isotopic values of WVISS-generated air.However, identification of the source of this bias and explaining the large spread in isotopic values of WVISS-generated air are beyond the scope of this paper.On the other hand, δD data do not suggest the same behavior.A positive bias is observed at all concentrations.There is also no systematic difference in the isotopic contents of water vapor for concentrations ranging from 3000 to 10 000 ppm.
Long-term stability
The long-term stability of the WVIA was evaluated via repeated measurements of a water standard over a period of four months.The uncalibrated hourly measurement time series of a water standard (δD = −78 ‰ and δ 18 O = −11.3‰) at a constant concentration (10 000 ppm) is shown in Fig. 4.Each plot represents the last 5 min average value of a 10 min measurement.Measured isotopic values range from −77 ‰ to −82 ‰ for δD and from −8.0 ‰ to −12.5 ‰ for δ 18 O.The spread in δ 18 O is three times larger than the difference between the maximum and minimum of WVISS-generated air at the same concentration (Fig. 2), indicating the variation stems mainly from the WVIA.Because an analyzer was installed in an air-conditioned laboratory, the observed large isotopic variation is too large to be explained by temperature variations (Sturm and Knohl, 2010).It is not clear yet what is causing this large δ 18 O variation, however it is clear that the system does have significant non-linear drift.Thus, frequent measurement of a water standard is crucial to correct for time-dependent isotope variation.Next, we examined the similarity between the timedependent variability in water standards with different isotopic values.The temporal variations in δD and δ 18 O of the six water standards (see Table 1) are shown in Fig. 5.The data are represented as a normalized anomaly from the longterm mean value.The temporal trends of each standard water matched each other well for both isotopes, and they do not depend on the isotopic content of water.Thus, repeated measurements of a single reference water sample should be enough to remove time-dependent drift.To move the runto-run variability, the WVIA-reported isotopic value of the water standard was converted to the VSMOW scale using a water standard (STD-11, see Table 1).
where δ sample/raw and δ STD11/raw are the raw δD or δ 18 O values of the sample and STD-11, respectively, and δ STD11/VSMOW is the VSMOW scale value determined by IRMS measurements.
Linearity
To test the linearity of the WVIA-WVISS coupled system, the WVIA-measured isotopic values of several water standards were compared with those obtained via IRMS measurements.The differences between values from the WVISS/WVIA and known values of each standard are shown in Fig. 6.For δD, except STDX3 (δD = −332.7 ‰), the calculated VSMOW scale value of each water standard agreed well with the known value.However, the WVISS/WVIAδ 18 O values significantly underestimated the true values, and the amplitude of this underestimation decreased linearly with increasing δ values.A two-point calibration method was applied to normalize WVIA-reported data (δ WVIA ) to the international VSMOW/SLAP scale.The calibration line was determined from two different water standards, one of which has more negative isotope contents (STD10) and another with more enriched isotopic values (STD11).The averaged lines obtained during the laboratory test are as follows: δO IRMS = (0.946 ± 0.005) δO WVIA − 0.96 ± 0.89. (2) The linear relationship between IRMS and WVIA measurements has already been reported in Sturm and Knohl (2010), however the slope for both isotopes are substantially different in this study.This finding suggests that linearity calibrations will likely be instrument specific.During the period of this laboratory test, the calibration line was determined every day and then normalized to the VSMOW/SLAP scale, because each obtained calibration line significantly differed from the average values shown in Eqs. ( 1) and ( 2).The results of the water standards measurements are summarized in Table 1 (Fig. 6 for δ 18 O).The isotopic difference between known and measured values is less than 1.5 ‰ for δD and 0.2 ‰ for δ 18 O.Thus, the corrected WVIA values agree well with known values within analytical error.In summary, obtaining accurate isotope data from WVISS/WVIA systems will require, at a minimum, correcting for time-dependent isotopic drift using a frequently repeated reference standard, and normalization of the drift corrected data to the VSMOW/SLAP scale using a two standard approach.The recommended calibration interval to determine the VSMOW/SLAP scale is daily.
Concentration dependence
Previous studies have reported water vapor concentration dependence of δ values, and highlighted this factor as the primary source of analytical uncertainty (Sturm and Knohl, 2010;Rambo et al., 2011).Through the WVISS performance test a clear concentration dependence for δ 18 O arising from the calibration system was uncovered, however this effect has not been fully considered in previous studies.
The water vapor concentration effect, determined from raw WVIA isotopic values through the measurement of WVISSgenerated air, together with the isotopic changes in WVISSgenerated air collected by cold trap are shown in Fig. 7.The responses to changes in water vapor concentration are shown as differences in isotope values from those measured at 10 000 ppm. Regarding δ 18 O there is a increasing trend in δ 18 O values with decreasing water vapor concentration for the WVISS/WVIA system that is a nearly linear match to that of WVISS-generated air up to a water vapor concentration of 6500 ppm.However, in the lower range of the concentration (lower than 6500 ppm), the δ 18 O values of WVISS-generated air decreased inversely with water vapor concentration.These findings suggest that concentration dependence determined by WVIA-WVISS measurements is underestimated for low water vapor concentration.The corrected concentration effect arising from WVIA measurements was determined through subtraction of isotopic changes in WVISS-generated air.These results show that there is no effect in the concentration range of 6500 to 10 000 ppm and it gradually increases in the concentration To escape from the influence of WVIA temporal drift, this experiment was completed within a 30 min time frame after starting calibration.Moisture from WVISS exhaust air was collected using the cold trap system and analyzed off line (blue).To collect a sufficient amount of water for isotope analysis, vapor trapping continued more than two hours.A large spread in the error bar at each vapor concentration may result from isotopic changes in WVISS-generated air and analytical error.Humidity bias stemming solely from the WVIA (green line) was calculated by subtracting the mean values of water vapor generated by the WVISS from WVIAmeasured values at a given water vapor concentration.
range of 6500 to 4500 ppm.The δ 18 O values at 3000 ppm were similar to those found at 4500 ppm.This non-linearity effect for δ 18 O appears to be similar to that found by Sturm and Knohl (2010), although the amplitude of isotopic variations is smaller.
For δD, WVIA-reported raw isotopic values slightly increase with decreasing water vapor concentration from 10 000 ppm to 3000 ppm (not shown); this tendency is consistent with the result of cold trap samples (Fig. 2).However, the spread of values from cold trap samples is larger than that of the WVIA reported values in the whole concentration range (Fig. 3) and thus we cannot conclude that this water vapor concentration dependency is related to WVISS vapor production.Because the amplitude of this shift is small (1 ‰ from 10 000 ppm to 3000 ppm), we do not consider the concentration dependence in this study.The amplitude is quite small compared to that reported by Sturm and Knohl (2010).The concentration effect results likely from the spectral fitting procedures used, including the removal of interferences in the WVIA.The spectral fitting procedure of our instrument was updated in May 2011 and the upgraded instrument may be less sensitive to the water vapor concentration effect.
Calibration procedure
The calibration procedure developed from laboratory experiments was applied to the continuous measurement of water vapor isotopes.First, the WVIA-reported raw isotopic values were converted using the reference standard values to correct time-dependent isotopic drift.In the laboratory test, the reference standard was measured just once a day, however more frequent measurement of the reference is recommended for continuous measurement under routine conditions (see Fig. 4).For the measurements of outdoor air, the reference gas (which is set to a water vapor concentration of 10 000 ppm) was measured every hour for 10 min.The isotopic contents of the source water used as a reference were determined by IRMS measurement (δD = −79 ‰ and δ 18 O = −11.3‰).The hourly reference data was interpolated to 10 min steps and the data at measuring time was used for reference scale conversion.For δ 18 O, because humidity bias cannot be ignored, the observed non-linear function was applied to time-interpolated reference data and the corresponding reference value δ ref at the concentration measured in ambient air was calculated as 7 (green line).As described in the previous section, we applied this function when the water vapor concentration of ambient air dropped lower than 6500 ppm, otherwise f (H 2 O) = 1.Next, the reference scale value was converted to the VSMOW scale value and then normalized to the VS-MOW/SLAP scale value using Eqs.( 1) and (2) in Sect.3.2.2.The measurement of multiple water standards was not performed during the measurement of ambient air, thus the longterm mean slope and intercept for δD and δ 18 O value obtained from the laboratory test was used.The possible error in isotopic measurements when applying a constant slope and intercept was evaluated through a comparison with the results shown in Table 1.Without daily calibration, the standard deviation of water standards increased a little, ranging from 0.79 to 1.32 ‰ for δD and from 0.19 to 0.39 ‰ for δ 18 O.However, this error is relatively small compared to that arising from the uncertainty of the humidity bias correction.
Field data evaluation
To further test our calibration procedure, corrected WVIA data was compared with the results of cryogenic vapor collection/IRMS measurement.The continuous measurement of atmospheric vapor based on a laser instrument and ambient air sampling using the cold trap method was carried out from August to December 2011.During this period, more than 150 water vapor samples were collected using the cold trap method with water vapor concentrations ranging from 3000 to 15 000 ppm.
Even though several sources of uncertainty contribute to the degraded accuracy of water vapor isotope measurements using the WVIA, the primary source of uncertainty may be associated with non-linear dependency on the humidity bias correction.Because this function includes the uncertainties arising from both the WVIA and the WVISS (Fig. 7), the robustness of this function should be tested before applying it to field data.In Fig. 8a, the non-linear function was compared with the humidity bias obtained from ambient air measurement.For this comparison, we used WVIA data without correcting the water vapor concentration effect; these were integrated over time during vapor trapping.Although the data points are widely scattered in the low concentration range (σ > 0.35 ‰ at 4500 ppm, whereas σ < 0.30 ‰ between 6500 and 15 000 ppm), a clear negative effect of water vapor concentration on δ 18 O contents is seen in the field data.In addition, this humidity bias is in very good agreement with the calibration curve obtained from the laboratory experiment.Thus, applying the calibration function leads to a decrease in systematic offset depending on concentration (Fig. 8b).This finding emphasizes that the non-linear function reasonably represents concentration dependence arising from the WVIA.For δD, the concentration effect was not clearly seen in the field data, although the points are more scattered towards the region of decreasing water vapor concentration (Fig. 9).Over the observation period, the δ 18 O (δD) values observed from samples collected by the cold trap method varied widely from −27.1 ‰ (−199 ‰) to −13.1 ‰ (−94 ‰), and these large temporal changes were also successfully reflected by the WVIA values which in fact reveal even greater temporal dynamics than were possible to measure using the trapping approach (not shown).In addition, the time-series of the d-excess parameter (d = δD − 8 × δ 18 O), defined by Dansgaard (1964) show reasonable consistency between the cold trap and WVIA analysis over a large range (5 ∼ 25 ‰).The similarity between trap based values and WVIA based values is strong evidence that high quality high temporal resolution data can be obtained over a wide range of isotope values and humidity conditions with the WVISS/WVIA system.To further evaluate the accuracy and precision of δD and δ 18 O measured with the WVIA, we calculated the timeaveraged isotopic values during vapor trapping and then subtracted from the cold trap values for the same sampling periods (Fig. 10).The plots are scattered randomly in the range of −0.65 to 0.77 ‰ for δ 18 O and from −3.8 to 2.9 ‰ for δD, and there is no offset.The mean value of the deviations of the 154 samples is 0.06 ± 0.28 ‰ for δ 18 O and −0.3 ± 1.4 ‰ for δD, respectively.Thus, although there is substantial scatter the mean values for both isotopes are close to zero suggesting that our developed calibration procedure can provide accurate isotope data, similar to the conventional method.The relatively large random error may result from the several sources of uncertainty.As discussed earlier, there are uncertainties related to how WVIA/WVISS data were corrected and calibrated.More frequent linearity calibration may help to reducing the random error.Regarding δ 18 O the uncertainty of the isotopic value of WVISS-generated air is 0.25 ‰ and may contribute to random error.Further development of the calibration system may play a key role in reducing this random error.Finally, we evaluated the d-excess values by comparing the d-excess measured by the WVIA with that from the cold trap method (Fig. 11).Most data plot along the 1 : 1 line which indicates that our calibration procedure can provide d-excess data that are consistent with the conventional trap approach.
Summary and conclusions
Water vapor stable isotope measurements based on a commercially available laser instrument (WVIA) in conjunction with a calibration system (WVISS) was evaluated through laboratory experiments using several water standards.We found that WVIA instrument can provide accurate results if corrections are applied for: (1) temporal drift, (2) normalization to the VSMOW/SLAP scale, and (3) dependency of isotope results on water vapor concentration.Periodic VS-MOW/SLAP normalization using two standards (linearization calibration) is especially important for δ 18 O (and dexcess), because the slope of the calibration line between the WVIA-reported values and the IRMS value differs substantially from 1 in addition to drift correction for temporal variation using a frequently measured single reference standard as described by Rambo et al. (2011).Another remarkable finding of our study is that concentration dependence stems from the WVISS.The δ 18 O values of vapor generated by the WVISS vary with time and depending on the output humidity concentration when vapor concentration becomes lower than 6500 ppm.In this study, we determined the "true" concentration effect stemming from the WVIA and then produced a new calibration procedure for WVIA measurement.
To test our calibration procedure and the instrumentation, continuous measurement of water vapor isotopes in ambient outdoor air was carried out from summer to winter using the WVISS/WVIA system together with water vapor sampling using the conventional cold trap method.Despite water vapor concentrations that varied from 3000 to 15 000 ppm, the results from both methods agree well throughout the entire observation period.The precision estimated from the comparison of both measurements is 0.28 ‰ for δ 18 O and 1.4 ‰ for δD, respectively.These uncertainties allow calculation of representative d-excess values and to assess natural variability.The d-excess variations from the WVIA measurements reasonably match those obtained from cold trapped vapor.The fact that accurate d-excess values can be obtained is good confirnamtion of the overall performance of the instrument and calibration approach.
In summary, accurate water vapor isotope data, with results comparable to those achieved using conventional methods, can be obtained from commercially available systems with high time resolution.This suggests that isotopes in water vapor can be measured without special operational skills.In addition, laser based instruments do not require the manual effort and hassles.Thus, the laser based technique has substantial potential to increase the availability of water vapor isotope data which can significantly advance scientific understanding related to the water cycle.
Fig. 2 .
Fig. 2. Analytical variations in the δD and δ 18 O of cold trapped vapor sampled from a WVISS exhaust line with different H 2 O concentrations.Repeated analysis of standard water (δD = −78 ‰ and δ 18 O = −11.3‰) was run at 6 levels of vapor concentration from 3000 to 10 000 ppm.The broken line represents known isotopic values determined via IRMS measurement.The box and whisker plots of δ 18 O data represent the median (dark bar), 25-75 % quantile range (inter-quantile range [IQR] shown as the box), and maximum and minimum values (whiskers) for repeated experiments.
Fig. 3 .
Fig. 3.A comparison of cold trap vapor analysis with direct vapor measurement using the WVIA: (a) δD in a concentration range of 3000-10 000 ppm; the purple points represent values at 3000 ppm; and (b) δ 18 O at 10 000 ppm only; the red plots represent data from 27-29 November 2011.Broken lines represent the 1 : 1 relationship.These WVIA measurements are the uncorrected and uncalibrated δD and δ 18 O values.
Fig. 4 .
Fig. 4. Time series of uncalibrated δD and δ 18 O values of a single water standard (δD = −78 ‰ and δ 18 O = −11.3‰) measured using the WVIA from late August through mid-December 2011.The data represent the uncorrected and uncalibrated δD and δ 18 O values recorded by the WVIA and show the amount of instrument drift over the multi-month period.
Fig. 5 .
Fig. 5.Comparison of time-dependent δD and δ 18 O variations of six water standards with different isotopic compositions.Plotted values are expressed as a normalized anomaly, that is, a departure from the long-term mean for the analysis period, divided from the standard deviation during this period.
Fig. 6 .
Fig. 6.The difference in WVISS based δD and δ 18 O values from known IRMS based values of each water standard (blue circle).In both figures, the x-axis represents known IRMS based SMOW/SLAP scale values.For δ 18 O, collected SMOW scale values are also plotted at the top of the figure (white circle).
Fig. 7 .
Fig.7.δ 18 O anomaly with changing water vapor concentrations.Anomalies are calculated by subtracting the mean value at 10 000 ppm from measured values at a given water vapor concentration.Error bars show the standard deviation of repeated experiments.The WVISS was run with different water vapor concentrations and the resultant vapor was measured with the WVIA (red line).To escape from the influence of WVIA temporal drift, this experiment was completed within a 30 min time frame after starting calibration.Moisture from WVISS exhaust air was collected using the cold trap system and analyzed off line (blue).To collect a sufficient amount of water for isotope analysis, vapor trapping continued more than two hours.A large spread in the error bar at each vapor concentration may result from isotopic changes in WVISS-generated air and analytical error.Humidity bias stemming solely from the WVIA (green line) was calculated by subtracting the mean values of water vapor generated by the WVISS from WVIAmeasured values at a given water vapor concentration.
Fig.
Fig. The δ 18 O difference in atmospheric water vapor between WVIA data and cold trap vapor samples as a function of changing water vapor concentrations.(a) WVIA data without a water vapor concentration correction and (b) corrected data.WVIA data was integrated during vapor trapping.The broken red line with crosses shows average values at each vapor concentration from 3500 ppm to 14 500 ppm.For comparison, the water concentration bias arising from WVIA and shown as a green line in Fig. 7 was superimposed in the uncorrected version.
Fig. 10 .
Fig. 10.Comparison of δD and δ 18 O of water vapor collected via the cold trap method and directly measured using the WVIA.For improved comparison, the time-averaged isotopic values of WVIA measurements during vapor trapping periods are plotted.
Fig. 11 .
Fig. 11.Comparison of d-excess in water vapor using the cold trap and WVIA methods.The WVIA-measured d-excess values were derived from corrected and calibrated δD and δ 18 O values and then time-averaged values were calculated.
Table 1 .
δD and δ 18 O values of water standards measured using the IRMS and the WVIA.
18 O 16 O ref − 1 where D/H raw ( 18 O/ 16 O raw ) and D/H ref ( 18 O/ 16 O ref ) are the WVIA-reported isotopic ratios of ambient air and water vapor reference generated by the WVISS.The f (H 2 O) represents a calibration function for the concentration effect shown in Fig. | v3-fos-license |
2018-04-03T03:59:46.002Z | 2017-08-01T00:00:00.000 | 3315263 | {
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} | pes2o/s2orc | Organocatalyzed synthesis of fluorinated poly(aryl thioethers)
The preparation of high-performance fluorinated poly(aryl thioethers) has received little attention compared to the corresponding poly(aryl ethers), despite the excellent physical properties displayed by many polysulfides. Herein, we report a highly efficient route to fluorinated poly(aryl thioethers) via an organocatalyzed nucleophilic aromatic substitution of silyl-protected dithiols. This approach requires low catalyst loadings, proceeds rapidly at room temperature, and is effective for many different perfluorinated or highly activated aryl monomers. Computational investigations of the reaction mechanism reveal an unexpected, concerted SNAr mechanism, with the organocatalyst playing a critical, dual-activation role in facilitating the process. Not only does this remarkable reactivity enable rapid access to fluorinated poly(aryl thioethers), but also opens new avenues for the processing, fabrication, and functionalization of fluorinated materials with easy removal of the volatile catalyst and TMSF byproducts.
F luorinated materials exhibit many highly desirable properties such as increased chemical resistance, hydrophobicity, and thermal stability. One common route to introduce fluorine into polymer backbones is through the polycondensation of perfluoroarene-containing monomers ( Fig. 1) [1][2][3][4][5][6][7][8][9][10][11][12] . These perfluorinated aromatic polymers impart increased order and stability to processed materials by forming energetically favorable π-π stacking arrangements with non-perfluoroarenes 13,14 , as evidenced by their thermal properties 4 . Fluorinated aromatic polymers often possess a lower refractive index and optical loss than non-fluorinated analogs, making them excellent candidate materials for optical material applications 3,5 . Given the advantages of having perfluoroarene units in polymers, we felt that the preparation of high performance fluorinated poly(aryl thioethers) would be ideally suited for use in coating and device applications.
As with perfluoroaryl-containing polymers, poly(aryl thioethers) are attractive materials that display excellent performance characteristics such as high thermal stability and hydrophobicity 15 . Poly(aryl thioethers) are traditionally prepared via nucleophilic aromatic substitution (S N Ar) under conditions that utilize stoichiometric amounts of base, extended reaction times, or in some cases, high temperatures [16][17][18][19][20][21] . Although these conditions have been utilized for the incorporation of perfluoroaryl-containing monomers into poly(aryl thioethers) 22 , they are not ideal, given the propensity of perfluoroarenes to undergo multiple substitutions 3, 23-26 potentially leading to uncontrolled branching or cross-linking. Additionally, the stoichiometric amount of salt generated under standard S N Ar conditions could hamper further applications such as casting films to prepare hydrophobic surfaces. To circumvent the necessity of using stoichiometric base for the S N Ar reaction, we sought conditions for catalytic generation and reaction of thiolates. Here, we focused our attention on trimethylsilyl-protected thioethers, which could be cleaved by a catalyst to reveal a thiolate nucleophile. This intermediate could then undergo S N Ar with a perfluoroarene, thereby liberating trimethylsilylfluoride (TMSF) and regenerating the catalyst for subsequent reactions. Repetition of this cycle would give rise to the desired fluorinated poly(aryl thioether). Although similar protocols have been utilized for the preparation of polyethers 27, 28 , polysulfoxides 29 , polythiophenes 1, 5 , and poly(phenyleneethynylene)s 3 , there are no reports on the use of silylated dithiols as monomers. Thus, in order to take full advantage of the unique properties of fluoropolymers and poly (aryl thioethers), we developed a catalytic approach for the direct polymerization of perfluoroarenes into poly(aryl thioether) systems under mild conditions. This approach facilitates the polymerization of a variety of thiol nucleophiles and fluoroarenes and concurrent computational investigation of the reaction mechanism reveals a unique role of the catalyst in facilitating the polymerization process.
Results
Evaluation of polymerization catalysts. We began by preparing thioether 1a (Table 1) as the trimethylsilane (TMS) protected nucleophile. By reacting 1a with hexafluorobenzene using 5 mol % DBU as the organocatalyst 27 , a swift exotherm was observed, coupled with the formation of fluorotrimethylsilane (TMSF) and a rapid precipitation of polymeric material having a M n of 8456 g/mol and a dispersity of 4.88 (entry 1, Table 1). The fast rate of reaction was consistent with preliminary time course experiments at higher catalyst loadings, which revealed the complete consumption of hexafluorobenzene within several seconds (see Supplementary Fig. 1). For further comparison of different catalyst systems and catalyst loadings, we selected 15 min as a benchmark reaction time. The large dispersity observed (entry 1, Table 1) may be the result of cross-linking or branching via multiple substitutions on the arene ring and would be consistent with the high reactivity of hexafluorobenzene 3, 23-26 . However, examination of the 19 F NMR spectrum reveals a clean singlet indicative of a symmetrically substituted perfluoroarene ring and therefore the dispersity of the isolated material is more likely a result of kinetic quenching from the precipitation of the polymeric material. By lowering the catalyst loading to 1 or 0.5 mol %, the corresponding M n and dispersity of the polymers decreased while still affording short reaction times (entries 2 and 3, Table 1). Performing the reaction inside a glovebox under strictly anhydrous conditions afforded a higher M n and dispersity, indicating that the polymerization reaction is likely sensitive to water and ambient moisture (entry 4, Table 1).
Other catalyst systems in addition to DBU were also evaluated. Guanidine containing catalysts such as TBD and DMC, gave similar results as compared to DBU (entries 5 and 6, Table 1). TBD afforded a higher dispersity, presumably due to increased basicity and hence reactivity. Less basic catalysts, such as triethylamine, DIEA, and DABCO, all gave polymers with a narrower dispersity than their more strongly basic counterparts (entries 8-10, Table 1). However, the corresponding molecular weights of the resultant polymers were lower and longer reaction times were required. By heating the reaction with 10 mol % of diisopropylethylamine; however, results similar to those using DBU could be obtained (entry 11, Table 1).
Thermal properties of fluorinated poly(aryl thioethers). The thermal properties 1b were investigated by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The T 2% of 1b, which was isolated by precipitation prior to analysis, was found at 333°C (see Supplementary Fig. 2). Figure 1 shows the reverse Cp plot of the same isolated polymer (Fig. 1b), as well as DMA traces of in situ polymerized solutions of 1a and C 6 F 6 on a support braid ( Fig. 1a) in NMP. Two thermal transitions were detected on the DSC thermogram in the studied temperature range: a glass transition around −18°C and a melting endotherm around 105°C, evidence of the semi-crystalline morphology of 1b ( The sample used for DSC analysis was isolated by precipitation, while a braid for DMA was prepared with NMP-solutions of 1a and hexafluorobenzene thioethers) 10,22 . These data were in agreement with the DMA plots, where two E' drops along with two tan δ maxima were observed (Fig. 1a).
Evaluation of other monomers. Having investigated the thermal properties of 1a and identified the appropriate catalyst systems and reaction parameters, we next utilized this approach to polymerize other fluoroarene electrophiles and silyl thioethers. Decafluorobiphenyl proved to be an excellent substrate for this reaction and readily polymerized when 1a was used as the nucleophile with either DBU or TBD as the catalyst (2b, entries 1 and 2, Table 2). The TMS thioether of 4,4′-thiodibenzenethiol (2a, Table 2) was also a viable monomer for polymerization with perfluoroarenes. Interestingly, when decafluorobiphenyl was utilized as a co-monomer with 2a, no catalyst was necessary as dissolution of both monomers in DMF was sufficient to induce rapid polymerization to afford 2c (entry 3, Table 2). Presumably, this reactivity is due to the increased lability of TMS-protected aryl thioethers relative to TMS-protected alkyl thioethers. Both reactions to produce 2a and 2b (entries 1-3, Table 2) gave polymers that are comparable to those produced using unprotected thiols and stoichiometric base (see Supplementary Figs. 3 and 4), highlighting the efficacy of this approach to produce fluorinated poly(aryl thioethers) without stoichiometric salt byproducts. For synthesizing 2d, a catalyst was still needed to facilitate the polymerization of 2a and hexafluorobenzene (entry 4, Table 2). Non-perfluorinated, yet highly activated aryl electrophiles such as bis(4-fluoro-3-nitrophenyl) sulfone could also be rapidly polymerized under the reaction conditions to form 2e (Table 2). Unfortunately, the very limited solubility of polymers 2d and 2e prevented their analysis via GPC or NMR, although the T g for these materials could be obtained from DSC analysis (entries 4 and 5, Table 2).
Computational mechanistic investigation. To understand the origins of the aforementioned remarkable reactivity, we performed computational investigations with the M06-2X density functional method on the mechanisms and energetics for the TBD-catalyzed reaction of hexafluorobenzene with TMSprotected methanethiol (TMS-SMe) (Fig. 2). The nucleophilic attack of TBD on the TMS group of TMS-SMe and displacement of methanethiolate (MeS − ) from the silyl protecting group was identified as a key starting point. This process results in the formation of complex INT1 -where MeS − is hydrogen-bonded to the TBD-TMS cation-and then associates with C 6 F 6 to form the productive trimolecular complex INT2 (Fig. 2). The free energy of INT2 formation illustrates that strong enthalpic contributions of attractive supramolecular interactions largely compensate for the unfavorable entropy (ΔH = −8.4 kcal/mol, ΔG = + 4.5 kcal/mol relative to the separated components). Next, complex INT2 reacts to afford the mono-substituted product (Prod1) via TS2, in which TBD promotes the attack of MeS − on the aromatic ring as Fleaves (Fig. 2). This contrasts directly with the typical, stepwise addition-elimination mechanism of S N Ar reactions, as the TBD-catalyzed reaction proceeds in a concerted manner where formation of the C-S bond is coordinated with scission of the C-F bond 27,[30][31][32] . The TBD catalyst serves dual roles in the S N Ar process occurring in TS2: it delivers the MeS − nucleophile and assists the concomitant displacement of the fluorine atom through a hydrogen bonding interaction (Fig. 2). The free energy barrier for the TBD-assisted thioetherification and fluoride displacement is only 17.6 kcal/mol, presumably owing to synergistic interactions present in the TS, which allows for the scission of a very strong C-F bond to proceed with such a small penalty.
These synergistic interactions include the covalent attachment of TBD to the TMS group, which renders the TBD catalyst cationic, and thereby enhances the hydrogen-bond interaction Electrostatic stabilization due to such interactions has been reported to be significant, even in the absence of strong n F /σ* C-H overlap 32 . Finally, the rigid nature of the catalyst leads to a unique stereoelectronic advantage, as the N-H•••S interaction is associated with the in-plane lone pairs of sulfur, leaving the out-of-plane p-type lone pair fully available for the nucleophilic attack at the aromatic π-system (Fig. 3a). The TBD catalyst is regenerated in INT3 by attack of the hydrogen-bonded fluoride anion on the neighboring TMS group in a low-barrier process with a free energy barrier of 6.0 kcal/mol with reference to the low-lying Prod1 (Fig. 2). The catalyst reenters the cycle by activating a protected thiol to form INT4 in a mechanism analogous to INT1 formation. Complexation of INT4 with MeSPhF 5 results in the formation of INT5, a complex similar to INT2, in which the deprotected thiolate is poised for nucleophilic attack para to the thiol substituent (Fig. 2). The presence of SMe substituent in the aromatic ring lowers the free energy barrier by 2.2 kcal/mol, making the second substitution roughly~33 times faster than without the SMe (TS5, Fig. 2; Supplementary Fig. 8). The activating effect of the SMe group originates from an unusual geometry at the aryl thioether junction, with the SMe substituent rotating to the near orthogonality out-of-plane of the aromatic ring. The conformational change converts the aryl thioether from a moderate p-donor into a moderate σ-acceptor (Fig. 3b) 33,34 . This is shown in Fig. 3b, as the π CC →σ Ã SÀC interaction increases from 2.2 to 6.8 kcal/mol (4.6 kcal/mol difference), delocalizing more of the electronic density in the TS and lowering the overall free energy barrier for TS5 (Fig. 2). Following TS5, the TBD catalyst is regenerated in a similar low-barrier process as before (TS6, Fig. 2) to give the final products TMSF, TBD, and the para-disubstituted fluoroarene (INT6, Fig. 2).
Discussion
Overall, we have developed an organocatalyzed reaction for the synthesis of fluorinated and non-fluorinated poly(aryl thioethers). As opposed to standard S N Ar reactions between thiols and perfluoroarenes, our conditions avoid the generation of stoichiometric salt by-products. This advantage, in combination with short reaction times, room temperature conditions, and low to no catalyst loadings, will enable new routes for processing the prepared fluoropolymers into devices and coatings. Computational examination of the mechanistic underpinnings of this process reveals a unique, concerted mechanism for the S N Ar reaction between silyl protected thioethers and perfluoroarenes, where the organocatalyst plays a critical, dual-activation role. Future work will endeavor to leverage these mechanistic and synthetic insights to further refine control over the polymerization reaction conditions for the development of both new fluorinated materials and
Methods
General procedure for polymer synthesis. An 8 ml screw-cap vial equipped with a magnetic stir-bar was charged with the thioether monomer (1 equiv) and fluoroarene (1−1.05 equiv), if solid. Solvent was then added, followed by any monomer that is a liquid (thioether or fluoroarene). The reaction mixture was then stirred to fully mix and dissolve the monomers. The catalyst (0.5-10 mol %) was then added and the reaction mixture was stirred for the indicated time at the specified temperature. Following completion of the reaction, methanol (8 ml) was added to precipitate the polymer. The solid was collected via centrifugation and decanting of the supernatant. Additional methanol (8 ml) was added to the recovered solid and the process centrifugation and decanting was repeated a second time. Synthesis of 2c. In a nitrogen filled glovebox and in accordance with the general procedure, a mixture of 2a (197 mg, 0.50 mmol), decafluorobiphenyl (168 ml, 0.50 mmol), and DMF (0.5 mmol) were stirred at room temperature for 5 min. Following the workup in accordance with the general procedure, the polymer was isolated as a white solid (mass recovered: 263 mg, 94%). Note: Polymer retained residual DMF after workup and drying. M n = 7768 g/mol, M w = 47026 g/mol, Ð = 6.05. 1 Synthesis of 2d. In a nitrogen-filled glovebox and in accordance with the general procedure, a mixture of 2a (197 mg, 0.50 mmol), hexafluorobenzene (56 µl, 0.50 mmol), DBU (20 µl of a 0.062 M stock solution in DMF), and DMF (0.50 ml) were stirred at room temperature for 10 min. Following the workup in accordance with the general procedure, the polymer was isolated as a white solid (mass recovered: 218 mg, 75%). T g (DSC): 48°C. Note: The low solubility of the resulting polymer prevented full analysis by GPC or NMR.
Synthesis of 2e. In accordance with the general procedure, a mixture of 1a (168 µl, 0.50 mmol), bis(4-fluoro-3-nitrophenyl)sulfone (172 mg, 0.50 mmol), DBU (20 µl of a 0.125 M stock solution in DMF), and DMF (0.5 ml) were stirred at room temperature for the indicated amount of time. Following the workup in accordance with the general procedure, the polymer was isolated as a light yellow solid (mass recovered: 145 mg, 64%). T g (DSC): 28°C. Note: The low solubility of the resulting polymer prevented full analysis by GPC or NMR.
Data availability. All the data are available from authors upon reasonable request. | v3-fos-license |
2020-11-26T09:02:13.102Z | 2020-11-01T00:00:00.000 | 227169243 | {
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} | pes2o/s2orc | Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120
Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.
Introduction
Cyanobacteria use light energy to fix inorganic carbon (C i ) and nitrogen (N), harvested from their aquatic environment, into the metabolic components required for growth and propagation. Environmental sources of C i include dissolved CO 2 and bicarbonate (HCO 3 − ), while N can be supplied by nitrate (NO 3 − ), nitrite (NO 2 − ), ammonium (NH 4 + ), urea or N 2 (in diazotrophic cyanobacteria; reviewed in [1]). The tight coupling of the concentrations of C i and N taken up from the environment prevents metabolic imbalance within the cell, which allows cyanobacteria to thrive amidst varying nutritional conditions. This is achieved in large part by transcriptional modifications that are triggered by fluctuations in the cellular homeostasis of organic carbon (C) and N, which are represented by changes in the relative abundances of key metabolite signals (reviewed in [2][3][4]). One such metabolite is 2-oxoglutarate (2OG), also known as α-ketoglutarate (αKG), which is a product of the tricarboxylic acid (TCA) cycle. The metabolite 2OG provides the inorganic carbohydrate skeleton for glutamate synthesis that occurs by the incorporation of NH 4 + in the glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT) cycle. Cellular 2OG levels therefore represent the abundances of both C and N (reviewed in [5,6]), making 2OG a central signalling metabolite that triggers transcriptional adjustments to restore C/N balance [7][8][9]. 2-phosphoglycolate (2PG) is another metabolite that controls transcriptional reprogramming in response to C/N balance [10], and 2PG is formed when Rubisco catalyses the oxygenation of RuBP (photorespiration), instead of the favoured carboxylation reaction between RuBP and CO 2 (reviewed in [4,11]). An increase in 2PG concentration therefore represents CO 2 deficiency, triggering transcriptional reprogramming designed to upregulate C i import into the cell [12][13][14][15]. Nostoc (Anabaena) sp. PCC 7120 is a filamentous, diazotrophic cyanobacterium wherein C/N balance controls the formation of heterocyst cells specialised for fixing N 2 into NH 4 + , while photosynthetic CO 2 fixation is restricted to vegetative cells (reviewed in [16,17]). In Nostoc sp. PCC 7120 and other heterocystous cyanobacteria, differentiation in cell structure and function is triggered by changes in C/N homeostasis and enacted by massive transcriptional reprogramming [1]. Emphasis on the impact of N concentration on heterocyst differentiation has revealed the central roles of 2OG and several regulatory proteins in instigating transcriptional and physiological responses to N deficit [6]. However, the transcriptional response of heterocystous cyanobacteria to C i availability has drawn only little attention [18], in sharp contrast to that in non-diazotrophic species [12,13,15,[19][20][21][22]. In the current study of the global transcriptome of Nostoc sp. PCC 7120, we found that a shift from 3% CO 2 to 0.04% CO 2 for 1 h can be largely attributed to changes in the levels of the metabolites 2PG and 2OG, which has a strong effect on genes involved in the import and metabolism of C i and N. This study also identified that genes encoding factors involved in photosynthetic electron transport, glycolysis and iron homeostasis are regulated by C/N homeostasis, which is suggested to trigger a transition from efficient photoautotrophic growth and energy storage to photoinhibition and glycolysis.
Growth and CO 2 Stepdown
Nostoc sp. PCC 7120 cultures were grown in BG11 medium [23] buffered with 10 mM TES-KOH (pH 8.0) at 30 • C under constant illumination of 50 µmol photons m −2 s −1 with 120 rpm agitation, in air enriched with 3% (v/v) CO 2 . During the exponential growth phase (OD 750 = 1.0), 2 mL samples were taken from three individual replicate cultures and frozen for RNA isolation. For CO 2 stepdown, the cultures were pelleted and the pellets washed once with fresh BG11, before resuspension in fresh BG11 and growth in air containing 0.04% (v/v) CO 2 . After 1 h, 2 mL samples were collected from three replicates and frozen for RNA isolation.
RNA Isolation and Transcriptomics
Total RNA was isolated as described in [24]. Total RNA samples were submitted to the Beijing Genomics Institute (China) for library construction and RNA sequencing using Illumina HiSeq2500. RNA reads were aligned using Strand NGS 2.7 software (Avadis) using the Nostoc sp. PCC 7120 reference genome and annotations downloaded from Ensembl (EBI). Aligned reads were normalised and quantified using the DESeq package (R). Significantly differentially expressed genes were identified using a 2-way ANOVA. p-Values were adjusted for false discovery rate (FDR) using the Benjamini-Hochberg procedure.
Results
The transcriptome of Nostoc sp. PCC 7120 grown in BG11 under 3% CO 2 -enriched air was compared with that of the same strain shifted from 3% CO 2 to 0.04% CO 2 for 1 h, revealing 230 genes to be upregulated >2-fold and 211 genes to be downregulated >2-fold, in the low CO 2 condition. The RNAseq data are available at the NCBI Sequence Read Archive (submission SUB8244772). As expected, given the short period under a new metabolic condition, no differences in the growth rates, lengths of filaments or frequencies of heterocysts (approximately 4% of all cells under 3% CO 2 ) were observed between the cultures exposed to 0.04% CO 2 conditions, compared to those grown at 3% CO 2 . Therefore, statistical tests of these parameters in the different cultures were not performed.
Uptake and Metabolism of Carbon and Nitrogen Are Inversely Responsive to Low CO 2 Conditions
The operons encoding three plasma membrane-localised HCO 3 − uptake systems were among the most strongly upregulated entities in Nostoc sp. PCC 7120 following CO 2 stepdown (Table 1). In cyanobacteria, the Cmp (BCT1) system is powered by ATP hydrolysis [25], while the SbtA and BicA systems depend on Na + ions for HCO 3 − symport [21,26]. The upregulation of the operon encoding the Mrp Na + :H + antiporter upon CO 2 stepdown may also be linked to HCO 3 − uptake through the extrusion of Na + to support SbtA and BicA activity [27,28]. HCO 3 − uptake in cyanobacteria forms a major part of the carbon concentration mechanism (CCM), which also involves the concentration of cellular CO 2 into HCO 3 − by a customised NAD(P)H dehydrogenase (NDH-1) complex [29]. Genes ndhF3, ndhD3 and cupA, which encode subunits that specialise the NDH1-MS complex for inducible CO 2 uptake, were upregulated after CO 2 stepdown (Table 1), as were several other genes encoding the core NDH-1M complex (see below). Notably, a putative cupS orthologue (alr1320), which is encoded separately from the ndhF3/ndhD3/cupA cluster in the Nostoc sp. PCC 7120 genome [30], was not differentially expressed (DE) in the current work. Genes encoding Rubisco and some carboxysome subunits were mildly upregulated in low CO 2 (1.3 to 1.7 fold change (FC); not shown), while other CCM components were not DE, suggesting that these components were already in sufficient abundance before CO 2 stepdown, whereas C i uptake from the environment, especially HCO 3 − uptake, was apparently a primary concern for survival after 1 h under low CO 2 . The decrease in CO 2 was found to cause downregulation of the nir operon, which encodes subunits of an ATP-dependent nitrate (NO 3 − ) transporter, as well as NO 3 − and nitrite (NO 2 − ) reductases [31,32].
The nir operon is responsible for NO 3 − uptake and reduction to ammonia (NH 3 ), and is broadly conserved across cyanobacteria (reviewed in [5]). Unlike the nir operon, the majority of nif genes that encode subunits for the assembly and function of nitrogenase, which reduces atmospheric N 2 to NH 3 , were not DE in the current work. Exceptions were nifB and nifH2, which were downregulated (Table 1). A gene that encodes a protein similar to the C-terminus of Mo-like nitrogenase (alr1713) was strongly downregulated, along with its neighbour (asr1714); however, the function of the encoded proteins is not known. Since heterocyst development is upregulated by a high C/N ratio (reviewed in [33]), it was not surprising to see many genes involved in the structural development of heterocysts repressed by the shift to low CO 2 . In particular, the hpd, hgl and dev gene clusters that encode many components for the synthesis and export of glycolipids, which form the oxygen-impermeable heterocyst envelope (reviewed in [34,35]), and were downregulated in the current data. Notably, the hep genes encoding heterocyst outer layer polysaccharides were only moderately downregulated by the shift to low CO 2 (average FC −1.5, data not shown).
Expression of Genes Encoding Photosynthetic and Respiratory Components Responds to Low CO 2 Conditions
Expression of genes encoding photosystem II (PSII) core proteins D1 and D2 was upregulated after the shift to low CO 2 ( Table 2), indicating an increase in the damage and turnover of PSII reaction centres [36,37]. In keeping with this, the genes encoding the two FtsH proteases involved in the degradation and turnover of damaged D1 protein were also upregulated [38,39]. We also found strongly induced expression of the flv2-flv4 operon, as well as several genes encoding orange carotenoid proteins (OCPs) and early light-inducible proteins (ELIPs), all of which are associated with PSII photoprotection [40][41][42][43][44]. These expression data suggest that the shift to low CO 2 led to the over-reduction, damage and repair of PSII. Given this evidence for PSII over-reduction, it was surprising to find the gene encoding the "plastid" terminal oxidase (PTOX) as one of the most strongly downregulated in the current study (Table 2), being highly expressed under 3% CO 2 and strongly repressed in 0.04% CO 2 . PTOX is part of a water-water cycle that moves electrons from reduced plastoquinone (PQ) to O 2 , and is thought to be an electron valve for balancing the photosynthetic redox state [45], which would presumably be important under low CO 2 (discussed below).
Virtually all genes encoding subunits of photosystem I (PSI) were substantially downregulated in the current study, which is in contrast to their increased expression in Synechocystis sp. PCC 6803 and unchanged expression Synechococcus elongatus PCC 7942 in low CO 2 [13,20]. PSI downregulation in Nostoc sp. PCC 7120 points towards a decrease in PSI electron transport that may be related to the diminution of the terminal electron acceptor CO 2 . These conditions would also be expected to upregulate O 2 reduction and the subsequent formation of toxic superoxide radicals (O 2 •− ); indeed, genes encoding a superoxide dismutase (SodB; alr2938) and peroxiredoxin (all2375), involved in reactive oxygen species (ROS) scavenging, were upregulated 3.2-fold and 2.3-fold, respectively (not shown). The expression of isiB (alr2405), which encodes a flavodoxin (Fld) that accepts electrons from PSI via a flavin mononucleotide cofactor [46], was also upregulated after low CO 2 treatment ( Table 2), suggesting a shortage of oxidised ferredoxin (Fd) acceptors [47]. The upregulated expression of genes encoding F 0 -F 1 ATP synthase points to an increased demand for energy in low CO 2 , required for HCO 3 − import and CO 2 hydration (reviewed in [2]). Notably, the gene alr1004 encoding an enzyme that converts glyoxylate to glycine for the detoxification of 2PG [18] was found to be downregulated after CO 2 stepdown, while other enzymes in the glycolate metabolism pathway were not DE. Uncharacterised protein −5.8 <0.001 1 Shaded cells represent operons or clusters of neighbouring genes; 2 Fold changes of genes upregulated or downregulated in low CO 2 , compared to high CO 2 , are coloured orange or green, respectively. In cases of multiple genes, average fold changes are shown; 3 p-values determined by moderated t-test. In cases of multiple genes, largest p-value is shown. The downregulation of PSI abundance in response to low CO 2 can partially clarify the apparent PSII over-reduction discussed above. Lower PSI levels may also be linked to a decrease in the number of heterocysts, which contain a higher PSI:PSII ratio than in vegetative cells [16]. Although such a decrease in heterocysts was not observed after 1 h in low CO 2 , suppressed heterocyst development was evident in the downregulation of hgl and dev clusters (Table 1), and this can also explain the suppression of genes encoding cytochrome c6, Flv1B and Flv3B ( Table 2) that operate predominately [48] or exclusively [49] in heterocysts. The gene encoding the small subunit of the heterocyst-specific uptake hydrogenase (HupS) was also downregulated here, reflecting the downregulation of the heterocystous nitrogenase activity under low CO 2 .
We observed downregulation of the pec cluster that encodes the phycoerythrocyanin (PEC) parts of the light-harvesting phycobilisome (PBS) complex [50,51], while genes encoding the other components of the PBS were not DE, indicating that the light-harvesting cross-section of PBS in Nostoc sp. PCC 7120 is altered in response to low CO 2 . An operon encoding a subunit of the light-independent protochlorophyllide reductase (DPOR) was also downregulated after 1 h in low CO 2 , while the expression of chlG and hemH genes, involved in later stages of chlorophyll and haem synthesis, respectively, were upregulated ( Table 2).
Most genes encoding subunits of the NDH-1 complex were upregulated under low CO 2 ( Table 2), probably to fulfil their role in CO 2 uptake as part of the NDH-1MS complex described above. In contrast, ndbA encoding NDH-2 was downregulated in the current study, suggesting a decrease in NDH-2-mediated respiration after the shift to low CO 2 . In addition, the gene encoding phosphoenolpyruvate (PEP) synthase, which converts pyruvate to PEP that is consumed in the TCA cycle, was strongly downregulated ( Table 2). Many genes encoding subunits of the bidirectional hydrogenase (Hox) were among the most strongly downregulated in response to low CO 2 conditions (Table 2), being both highly expressed in 3% CO 2 and strongly repressed in low CO 2 . Hox reversibly catalyses the reduction of H + to form H 2 , powered by reduced Fd/Fld with electrons derived from either PSI or pyruvate, the latter route by way of pyruvate ferredoxin/flavodoxin oxidoreductase (PFOR), which converts pyruvate to acetyl-CoA (reviewed in [52]). The nifJ gene encoding PFOR followed a similar expression profile to Hox subunits, being another of the most strongly downregulated genes in the current study. The physiological role of the Hox enzyme is not known, but has been described as an electron valve that can maintain redox balance and store reducing power as H 2 during excess photosynthesis or fermentation [52][53][54][55]. The expression profile of Hox and PFOR genes suggests that pyruvate-powered hydrogen production is active under 3% CO 2 and inactivated by the shift to low CO 2 .
Expression of Transcription Regulators Responds to Changes in CO 2 Conditions
The current study revealed substantial changes in the expression of several genes encoding transcription regulators, providing evidence of an ongoing cascade of transcriptional reprogramming after 1 h under low CO 2 (Table 3). Upregulated expression of the LysR-type regulator (LTTR) cmpR corresponds to the strong upregulation of its target, the cmp cluster encoding the BCT1 HCO 3 − transporter (Table 1), as previously shown in Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942 [56] and Nostoc sp. PCC 7120 [57]. Two sigB-type group 2 sigma factors, which have roles in the transcriptional response to low CO 2 and C/N balance [58][59][60], were upregulated after the shift to low CO 2 (Table 3). SigB has also been implicated in response to environmental stress and resistance to photoinhibition in Synechocystis sp. PCC 6803 [61,62], which is in line with the upregulation in this study of groES and groEL genes (4.3 to 5.5 FC; not shown) and photoprotective factors such as OCPs and the flv2-flv4 operon ( Table 2). Two homologous two-component response regulator clusters, which each comprise a histidine kinase and a DNA-binding regulator, were found to be upregulated by low CO 2 . Of the two, the chromosomal hik31 (C-hik31) operon was more highly upregulated, and has been found to be involved in the responses to oxygen concentration, light and metabolism [63,64]. A TetR-family transcription regulator with unknown function was also upregulated by low CO 2 (Table 3).
Another LTTR gene that was highly expressed under high CO 2 and was strongly downregulated after low CO 2 transition (Table 3) shared substantial sequence homology with the ndhR transcription repressor (also called ccmR) of Synechocystis sp. PCC 6803 [12,27]. In other cyanobacteria, NdhR represses the expression of CCM genes, including the sbt and bicA HCO 3 − importers, the mrp cluster and the ndhF3/ndhD3/cupA cluster [12,15,20,27,65]. The rapid downregulation of a putative ndhR orthologue in Nostoc sp. PCC 7120 may reveal the mechanism behind the strong upregulation of CCM genes after CO 2 stepdown in the current study ( Table 1). Downregulation of the transcription enhancer ntcB, which increases N metabolism through upregulation of the nir operon [66], also correlates with downregulation of other N-related genes in the current work ( Table 1). Expression of ntcB is controlled by NtcA [1]. As ntcA was not DE in the current study, downregulation of ntcB and other NtcA regulons may be due to the low CO 2 -induced inactivation of NtcA (discussed below). Similarly, downregulated transcriptional regulator genes patB (also called cnfR), devH and nrrA can also be attributed to inhibited NtcA activity [67][68][69][70][71]. These genes are expressed in heterocysts, where PatB upregulates the expression of nifB [72], DevH regulates heterocyst glycolipids [48,68,73] and NrrA induces expression of both the heterocyst regulator hetR and fraF encoding a filament integrity protein [70,74]. Both nifB and the hgl cluster were downregulated in this study (Table 1), while hetR and fraF were not DE (not shown).
Low CO 2 Conditions Influence the Expression of Metal Homeostasis Genes
A number of genes and gene clusters related to cellular homeostasis of iron (Fe) and other metals were found to be DE after CO 2 stepdown (Table 4). A gene cluster encoding subunits of a periplasmic ferrous Fe (Fe(II)) transporter [75,76] was strongly downregulated in the current study (Table 4), indicating a lower uptake of Fe from the environment under low CO 2 . The suf cluster, encoding proteins involved in Fe mobilization and Fe-S cluster assembly, was also downregulated. The expression of both the Fe(II) transporter and the suf cluster is upregulated by Fe deprivation [77][78][79], suggesting a surplus of cellular Fe after low CO 2 treatment. Several neighbouring clusters of genes encoding subunits of metal cation efflux systems such as copper, nickel, zinc, cadmium and cobalt, were upregulated after low CO 2 treatment (Table 4). These genes have been implicated in heavy metal resistance [80,81], although the link to the current conditions is not clear. Taken together, low CO 2 appears to induce an active decrease in cellular metal content, which may be a strategy to avoid oxidative stress during the reducing conditions induced by an insufficient availability of photosynthetic electron acceptors.
Transcriptional Regulation in Response to CO 2 Stepdown is Triggered by Metabolites
Induction of the most strongly upregulated genes, the HCO 3 − transporters (Table 1), after CO 2 stepdown, suggests a rapid transcriptional response that is highly sensitive to cellular C i levels.
In some unicellular cyanobacteria, repression of the CCM genes by NdhR (also called CcmR) can be modulated by both 2OG and 2PG [12,15,65,82]. Increased cellular 2PG concentration caused by increased photorespiration in low CO 2 leads to increased abundance of the NdhR-2PG complex that is unable to bind DNA to repress expression [4]. Additionally, declining 2OG levels due to lower CO 2 fixation and potentially lower TCA cycle activity decrease the abundance of the NdhR-2OG repressor complex, although it is unclear whether 2OG levels would actually decrease after only 1 h in low CO 2 due to the mobilisation of stored glycogen into the TCA cycle [2,15,82,83]. NADP + , another co-repressor of NdhR [82], is also theoretically far less abundant after CO 2 stepdown, due to decreased CO 2 fixation and lower NADPH consumption despite constant light conditions. Although a putative NdhR in Nostoc sp. PCC 7120 has not been studied, it appears that the LTTR encoded by all4986 represents such an orthologue, and that the strong downregulation of all4986 after CO 2 stepdown led to de-repression of the NdhR regulon, which includes the ndhR gene itself [84]. Previous transcriptomics studies have shown ndhR expression to be upregulated in Synechocystis sp. PCC 6803 after >3 h in low C i conditions [12,13], which is in contrast to the strong downregulation of all4986 seen here after 1 h (Table 3). This suggests that NdhR de-repression in response to CO 2 stepdown may be transient, and/or that expression of the NdhR regulon is also controlled by other transcription factors [18]. In the current study, de-repression by the putative NdhR is proposed to have caused a rapid and strong increase in HCO 3 − and CO 2 uptake under C limitation, with the upregulated cmp operon (Table 1) inducing further increase in HCO 3 − uptake. The cmp inducer CmpR is activated by 2PG or RuBP [82,85], both of which are in higher concentrations after CO 2 stepdown due to decreased CO 2 fixation. Furthermore, cmpR expression is also auto-upregulated (Table 3) [57]. In Synechococcus sp. PCC 7942 CmpR additionally upregulates the expression of PSII core subunits [86,87], found upregulated also in the current study alongside factors for PSII photoprotection and turnover, and downregulation of most PSI genes ( Table 2). The overlap between cellular responses to either low CO 2 or high light stress is well documented [12,20,86,88], highlighting insufficient electron sinks similarly created by both high light and low CO 2 , and resulting in the over-reduction of photosynthetic electron carriers [2]. Notably, several PSII photoprotection factors encoded by genes that were DE in the current study, including psbAIII, flv4 and sodB, were shown to be regulated together with Rubisco and other CCM genes by another LTTR in Nostoc sp. PCC 7120 called PacR [18], suggesting the likely involvement of PacR in the transcriptional reprogramming seen here. In addition to the LTTR transcription factors, the transcriptional response to low CO 2 is also regulated by LexA and the cyAbrB paralogues [89][90][91][92]. The vast change in expression of the hox operon after CO 2 stepdown (Table 2) may be related to the activity of LexA [93] and/or cyAbrB [90,[94][95][96]. In Synechocystis sp. PCC 6803, cyAbrB2 controls the expression of many CCM components that were likewise found to be upregulated in this study (Table 1) [90], while the cyAbrB2 orthologue in Nostoc sp. PCC 7120 regulates the expression of FeSOD [96], also upregulated here. Nostoc sp. PCC 7120 cyAbrB1 has been recently implicated in transcriptional regulation of heterocyst differentiation [97], demonstrating a role close to the interface of C i and N availability that suggests cyAbrB1/2 were likely active in the transcription regulation observed in the current study.
Given the transcriptional activation of NtcA, the master regulator of N metabolism, by high levels of 2OG upon a shift to low N (high C/N ratio) [7,98,99], it is widely assumed that a decrease in CO 2 leads to a decline in NtcA activity by decreasing the abundance of the 2OG-NtcA-PipX activator complex [100]. In the current study, lower NtcA activity was indeed evident in the downregulation of nir genes encoding NO 3 − uptake and metabolism (Table 1), as well as downregulation of the nir co-activator ntcB (Table 3; reviewed in [101]). This transcriptional regulation would effectively bring N metabolism into alignment with decreased C metabolism after CO 2 stepdown, despite the presence of N sources in the BG11 media. Overall, nearly 50% of genes downregulated in the current study have NtcA-binding promoters [71], although many NtcA-regulated genes, such as those involved in the uptake of NH 3 and urea, regulation of GS-GOGAT enzymes, as well as NtcA itself [71,[102][103][104], were not DE after 1 h in low CO 2 . The current work may therefore include only the early NtcA regulon. The NtcA-activated differentiation of vegetative cells to heterocysts occurs over approximately 24 h, via a cascade of transcriptional regulation that includes early upregulation of the co-activator nrrA [33]. The rapid downregulation in the current work of some heterocyst regulators, including nrrA, may block the commencement of heterocyst differentiation in response to relative N excess over C after CO 2 stepdown, and may signal an eventual decrease in the small number of heterocysts that are known to occur under high C/N [105]. Downregulation of NtcA activity in the current study appears to indicate a decline in 2OG levels after only 1 h in low CO 2 , although an increased concentration of NH 3 derived from 2PG metabolism can also explain suppression the NtcA regulon under low CO 2 (low C/N ratio) [83]. N excess under low CO 2 is also evident in the −2.2 FC downregulated expression of cyanophycinase chb2 (all0571; not shown), which is regulated by NrrA, suggesting a decrease in the mobilisation of stored N under low CO 2 (reviewed in [106]). It can also be argued that an increase in the ADP/ATP ratio under CO 2 stepdown, caused by decreased photosynthetic electron transport and rapid changes in metabolism, increases the abundance of both the ADP-PII-PipX complex and the inactive form of NtcA [6]. The strong downregulation of operons involved in ferrous Fe import and Fe-S cluster assembly in the current work (Table 4) suggests a connection between cellular Fe homeostasis and C/N balance in Nostoc sp. PCC 7120, which has been explored [107]. The current results indicate a CO 2 stepdown-induced cellular excess of Fe and other transition metals, which may be due to downregulation of Fe-rich PSI complexes (Table 2) [108] and/or an excess of reductant caused by insufficient electron sinks. Both conditions pose the danger of ROS formation, evidenced by upregulated expression of SOD, peroxiredoxin and protein chaperones.
Altered C/N Balance Modulates the Energetic Strategy of Nostoc sp. PCC 7120
The current study shows that a stepdown from 3% CO 2 in enriched air to 0.04% CO 2 (atmospheric) for just 1 h led to substantial reprogramming of global gene expression in Nostoc sp. PCC 7120 cultures. As discussed above, most of the transcriptional changes observed here can be directly attributed to metabolite signalling instigated by the alteration of the cellular C/N balance (Figure 1), initiated by the decrease in CO 2 concentration. Furthermore, these results highlight rapid transcriptional reprogramming of photosynthesis and energy metabolism in Nostoc sp. PCC 7120 in response to CO 2 levels (Figure 2). High CO 2 in light promotes a high rate of photosynthesis and the storage of photosynthate in the form of glycogen [13,[109][110][111]. Strong expression of PEP synthase and PFOR under these conditions indicate glycolysis/gluconeogenesis through the metabolism of pyruvate, which is consumed in the TCA cycle to facilitate respiratory electron transport and to provide 2OG for amino acid synthesis (reviewed in [112,113]). Therefore, growth under 3% CO 2 somewhat resembles photomixotrophy, with photosynthetic and glycolytic metabolisms occurring concomitantly, even though glucose was not externally provided to cultures. Under these conditions, the cells experience a high C/N, which is evident in the relatively high expression of N metabolic genes, reflecting a cellular excess of 2OG [7,98,99]. In high CO 2 , strong expression of Hox, which is important under mixotrophy and N deprivation [55], and PTOX, may provide a system to maintain redox poise [45], and in the case of Hox, also store surplus energy as H 2 [53]. The transfer of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 cultures from high to low CO 2 showed that the toxic effects of 2PG transiently block Calvin-Benson-Bassham (CBB) activity [15,20,83] and this is also evident here in the upregulated expression of PSII repair, photoprotection and ROS scavenging enzymes in Nostoc sp. PCC 7120, which indicate over-reduction of the photosynthetic electron transport chain. Interestingly, detoxification of 2PG appeared to be downregulated through repression of alr1004 during CO 2 stepdown (Table 2), perhaps highlighting the importance of the metabolite for signalling during the early stages of C i deprivation. We also observed downregulation of the terminal proteins in the phycobilisomes, suggesting modified harvesting of light energy to alleviate excitation pressure on the photosynthetic system. Under these conditions, inhibition of photoassimilation is compensated by glycolysis of stored glucose and CBB intermediates, providing an important supply of substrates for anaplerosis of the CBB and TCA cycles during acclimation to the transition [83,114,115]. In the current work, enhanced glycolytic activity is indicated by the upregulation of NDH-1, suggesting an increased reliance on respiratory electron transport for ATP generation, while strong downregulation of PEP synthase and PFOR after CO 2 stepdown may prevent diversion of pyruvate away from the TCA cycle. Notably, PEP abundance increased substantially in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 after a shift from high to low CO 2 [20,83], while genes encoding both pyruvate kinase and PFOR were highly expressed in Synechococcus elongatus PCC 7942 after long-term acclimation to low CO 2 , but not directly after the transition [20]. These findings support the results of this study and suggest that the metabolism of PEP and pyruvate are tightly regulated after the transition to low CO 2 . This may be linked to the role of PEP and pyruvate as substrates to anaplerotic carbon fixation to produce TCA cycle intermediates oxaloacetate and malate (reviewed in [116]). During the adjustment to a low C/N ratio, TCA cycle activity generates 2OG for amino acid synthesis, utilising excess NH 4 + generated through 2PG detoxification [11,83].
This work has revealed rapid transcriptional reprogramming in Nostoc sp. PCC 7120 in response to a decrease in C i availability, namely strong upregulation of CCM components and photoprotection, and downregulation of N uptake and early stages of heterocyst differentiation. Despite the vast increase in HCO 3 − uptake, glycolysis of stored C apparently plays an important role in energy metabolism at low CO 2 , likely due to 2PG-induced inhibition of the CBB cycle. A majority of the transcriptional effects induced by low CO 2 in Nostoc sp. PCC 7120 can be attributed to 2PG modulation of CmpR and a putative NdhR homologue; however, the effects of changing abundance of 2OG and NtcA activity after 1 h in 0.04% CO 2 , as well as the roles of other transcriptional regulators cannot be discounted. Finally, this work highlights the sensitivity of Nostoc sp. PCC 7120 to factors that influence the cellular C/N balance and demonstrates the speed at which genetic and metabolic reprogramming can take place, allowing rapid acclimation for surviving and thriving in the fluctuating environment.
Life 2020, 10, x FOR PEER REVIEW 11 of 20 may be linked to the role of PEP and pyruvate as substrates to anaplerotic carbon fixation to produce TCA cycle intermediates oxaloacetate and malate (reviewed in [116]). During the adjustment to a low C/N ratio, TCA cycle activity generates 2OG for amino acid synthesis, utilising excess NH4 + generated through 2PG detoxification [11,83]. cycle, which produces reductant, ATP and succinate to drive respiratory electron transport through NAD(P)H-dehydrogenase (NDH) and succinate dehydrogenase (SDH), as well as other cellular processes. The TCA cycle also generates 2-oxoglutarate (2OG), which accumulates under high CO 2 due to a relative shortage of NH 4 + and glutamine. 2OG operates as a signal for upregulating genes involved in N uptake and metabolism. Strong expression of phosphoenolpyruvate (PEP) synthase converts pyruvate to PEP. Pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA, reducing oxidised ferredoxin/flavodoxin (Fd/Flv ox ) that is consumed by bidirectional hydrogenase (Hox) for storage of excess energy as H 2 . Strong expression of plastoquinone terminal oxidase (PTOX) maintains redox homeostasis of the plastoquinone (PQ) pool during high photosynthetic electron transport, while cytochrome c6 oxidase (COX) maintains the redox poise of lumenal electron carriers cytochrome c 6 (c 6 ) and plastocyanin (PC). Genes encoding factors coloured orange are highly expressed under 3% CO 2 and strongly downregulated by the shift to 0.04% CO 2 . (B) After 1 h in 0.04% CO 2 , a deficiency of CO 2 electron acceptors leads to oxygenation of Rubisco, causing photorespiration that produces 2-phosphoglycolate (2PG). The CBB cycle and other metabolic pathways are inhibited by 2PG, which also signals upregulation of the transcriptomic response to low CO 2 . Low CBB activity causes over-reduction of the photosynthetic electron transport chain, leading to the production of reactive oxygen species (ROS) at photosystem I (PSI) and downregulation of PSI subunits. Increased reducing pressure on photosystem II (PSII) also causes upregulation of the PSII repair cycle and upregulation of PSII photoprotection by flavoproteins (Flv2/4) and orange carotenoid proteins (OCPs), as well as downregulation of phycoerythrocyanin in the phycobilisome (PBS). Glycolysis triggered by decreased CO 2 assimilation provides substrates for anaplerotic supplementation of the CBB and TCA cycles, including the production of oxaloacetate from PEP and bicarbonate (HCO 3 − ). TCA cycle activity also produces 2OG for glutamate production from excess ammonium (NH 4 + ) resulting from the shift from 3% to 0.04% CO 2 . Genes encoding factors coloured orange or green are upregulated or downregulated, respectively, 1 h after the shift from 3% to 0.04% CO 2 . Black arrows indicate the movement of electrons or ATP, dashed arrows summarise multiple enzymatic reactions in carbohydrate metabolism, blue arrows indicate the movement of protons. The red arrow in (A) shows the production of 2OG by TCA cycle activity, the red arrow in (B) shows the production of 2PG by Rubisco oxygenation in the CBB cycle and the black bar in (B) indicates CBB inhibition by 2PG. | v3-fos-license |
2019-05-02T13:03:00.874Z | 2019-04-30T00:00:00.000 | 141480522 | {
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} | pes2o/s2orc | Salt tolerance in indica rice cell cultures depends on a fine tuning of ROS signalling and homeostasis
Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant’s response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant’s response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO’s possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.
Introduction
Aerobic metabolic processes such as respiration, photosynthesis and photorespiration unavoidably produce reactive oxygen species (ROS) in the mitochondria, chloroplast, and peroxisomes respectively [1][2]. These ROS are produced in a controlled amount under optimal conditions. However, under abiotic stress their level increases dramatically. Overproduction of ROS caused by abiotic stress in plants highly damages proteins, lipids, and nucleic acids leading to cell injury and death [2]. ROS are also generated across the plasma membrane and apoplastic region [1][2][3]. Under abiotic stress these apoplastic ROS might also act as signal molecules for the activation of stress responsive pathways [4].
ROS induced by salt stress have lately been gaining more attention as second messengers [5][6]. Salt-induced ROS are generally represented by H 2 O 2 [7], mainly produced at the apoplast by calcium or phosphorylation derived activation of plant NADPH oxidases (NOXs) also known as respiratory burst oxidase homologs (RBOHs) [8]. This NOX generates a ROS signal which moves to the cytoplasm via regulated aquaporin [9], and together with intracellular ROS alters the redox status of key regulatory proteins such as transcription factors (TFs) [10]. This ROS signal activates numerous signaling transduction pathways to mediate multiple biological processes, including abiotic stress response and adaptation [11][12]. The elevated levels of ROS during the early phase of stress may act as a vital signal but the regulatory components of ROS mediated stress response are unknown. Yet, a signal transduction pathway has been proposed in which a mitogen-activated protein kinase (MAPK) cascade and downstream TFs are the key regulators of ROS signaling [13,7].
Rice is a highly salt sensitive crop, and its growth is severely affected when the plant is exposed to saline stress [14]. Owing to its large genetic variability, rice species show different degrees of salt sensitivity [15]. Seedling and reproductive phases of growth are the most sensitive stages under salinity [16]. Salt exerts its toxicity by inducing osmotic, ionic and oxidative stress [17][18]. Excessive Na + entry depolarizes the plasma membrane, increases Ca 2+ influx through unknown channels causing a change in Ca 2+ signature of the cell activating a signaling cascade. In response, the cells try to minimize Na + uptake, increase Na + sequestration and extrusion and restore K + levels by the up-regulation of Na + /K + symporter and antiporter [19,20]. An enhanced detoxification of ROS is also activated at the same time to reduce the oxidative damage [21][22][23].
In rice, NOX-dependent H 2 O 2 production emerges within few minutes of salt stress [24] and generates the earliest defence response at the molecular level. Salt and H 2 O 2 responsive ethylene response factor TFs, SERF1 and Dehydration-Responsive Element Binding 2a (DREB2a) have a critical role in regulating H 2 O 2 mediated molecular signalling cascade during the early response to salt stress in rice and Arabidopsis respectively [25,26]. SERF1 is essential for the proliferation of the first ROS signal in rice roots [27] while an early and enhanced expression of DREB2a evidently controls stress responses through abscisic acid "ABA" independent signaling, improving dehydration and tolerance to salt stress in rice plant [28].
An increased understanding of ROS signals generation and propagation is required to gain further insight into the regulatory mechanism underlying responses and adaptation to salt stress in cereal crops.
The aim of the study is four-fold. Firstly, we developed cell cultures from the mature seeds of two indica rice varieties to provide a simplified model that can accurately mimic the response of rice plant under salt stress. Secondly, it attempts to quantify the amounts of redox signalling molecules H 2 O 2 & NO produced extracellularly as well as intracellularly in sensitive/tolerant cells/varieties. Thirdly, it describes the expression pattern of salt-responsive apoplastic NOX subunit OsRbohA and TFs OsSERF1 and OsDREB2A and their correlation with the ROS signature of the cell. Fourthly, we tried to explore the role of recently characterised OSCA1 family members important for initial Ca 2+ influx (OsOSCA 1.2 and OsOSCA 3.1) and genes important for Na + and K + homeostasis (i.e. OsNHX1, OsSOS1, OsTpka, OsHAK5 and OsKAT) to understand their role for survival under salt stress.
Plant material and salt treatment
Experiments were conducted with non-embryogenic cell cultures of two rice (Oryza sativa L.) varieties, KS-282 and Super Basmati. Briefly, mature seeds were dehusked and surface sterilized under the laminar hood with 70% ethanol for 1 minute followed by 15 min in 3.5% sodium hypochlorite and tween-20 on a gyratory shaker, followed by 5 min with 3.5% bleach followed by five washes with Mille-Q water, 2 min each.
After surface sterilization, seeds were sowed in solid N6 medium (3.96 g L −1 Chu(N6) Medium Salt Mixture, 30 g L −1 sucrose, 2 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D) and 8 g L −1 plant ager; pH 5.8) under dark conditions and moved to new agar plates fortnightly. After few months, the callus was friable, and ready to be transferred in the liquid medium. The resulting Cell cultures were grown at 26˚C in dark on a rotatory shaker at speed of 96rpm in liquid N6 medium (3.96 g L −1 Chu(N6) Medium Salt Mixture, 30 g L −1 sucrose and 2 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D) pH 5.8). Every ten days they were filtered to eliminate bigger clumps until quite homogeneous Cell cultures were obtained. For the next sub culturing, 2 mL of packed cell volume of cells was transferred into 50 mL fresh medium every 10 days. Cells at day 3 after subculture were treated with 75, 100 and 150 mM NaCl and used for the next experiments. To determine the growth capabilities of the two cell cultures under salt stress, cells were filtered and the fresh and the dry weight were measured.
Cell viability assay under control and stress condition
Cell death was evaluated by a spectrophotometric assay of Evans blue stain retained by cells according to Gaff and Okong'o-Ogola [29] with minor modifications. Briefly, one mL of cell cultures was sampled from the cultures at desired intervals. Evans blue dye solution was added to the cell cultures to a final concentration of 0.05% and incubated for 30 min at room temperature, followed by rigorous washing with water until the disappearance of colour. The 50% methanol containing 1% (w/v) sodium dodecyl sulfate (SDS) was added to the washed cells and then incubated at 56˚C for 30 min. The percentage of dead cells was determined spectrophotometrically by measuring/at OD 600 nm. The boiled cells (100% dead) were used for comparison.
ROS and RNS assay
Determination of extracellular hydrogen peroxide. Extracellular H 2 O 2 secreted in the medium by the cultured cells was measured as described by Bellincampi (2000) [30]. Briefly, 500ul of the culture medium from control and treatment cell cultures was added (1:1) assay reagent (500 μM (NH4)2Fe(SO4)2�6H2O, 50 mM H 2 SO 4 , 200 μM xylenol orange, and 200 mM sorbitol) and kept in dark for 45 min. The H 2 O 2 dependent oxidation of Fe 2+ to Fe 3+ was determined by measuring the absorbance Fe 3+ -xylenol orange (FOX) complex at 560nm. A calibration curve obtained by measuring the absorbance of FOX complex of H 2 O 2 standards allowed the conversion of the absorbance values into concentration estimates. All reactions were carried out at least in duplicate with four replicates to check their reproducibility.
Determination of intracellular hydrogen peroxide. Intracellular H 2 O 2 production was measured using dihydrorhodamine123 (Sigma-Aldrich, Germany) as a probe. 1ml of cell cultures was incubated with 20 μM DHR123 for 15 min in a rotating shaker, and then washed thrice with 1 mL of fresh N6 medium. The cells were analysed under a fluorescence microscope (DM5000, Leica Microsystems, Germany) with a I3 filter. Relative fluorescence was determined using the ImageJ software.
Enzyme activity assay
The activity of ROS scavenging enzyme i.e. ascorbate peroxidase (APX) (total (tAPX) and cytosolic(cAPX)), catalase (CAT), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and superoxide dismutase (SOD) was measured. 0.5 g of cells were harvested at 30 min and 24 h and ground using a sterilized pistil and mortar with liquid nitrogen. Powdered cells were homogenized at 4˚C in an extraction buffer (50 mM Tris-HCl pH 7.5, 0.05% cysteine, 0.1% bovine serum albumin). Homogenates were centrifuged at 14000 g for 15 min at 4˚C to obtain supernatant which was used for enzyme activity. Protein contents were determined according to Bradford (1976) [31] using bovine serum albumin (BSA) as a standard. The activity of SOD was measured according to Beauchamp and Fridovich (1971) [32].
Determination of intracellular Nitric Oxide
Intracellular NO was detected with the fluorescent dye 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM-DA; Alexis Biochemical). One ml of cells was incubated with 0.5 mM DAF-FM-DA for 15 min in a rotating shaker, and then cells were washed three times with 1 mL of fresh N6 medium. Fluorescence was estimated using a fluorescence microscope (DM5000, Leica Microsystems, Germany) with a green florescent protein (GFP) filter. Relative fluorescence was determined using the ImageJ software. cPTIO pre-treatment and CDP analysis 50 μM cPTIO was added directly to the cells cultures and incubated for 15 min on a rotatory shaker. Cell cultures were then exposed to 100 mM NaCl and cell death was evaluated by a spectrophotometric assay of Evans blue stain retained by cells according to Gaff and Okong'o-Ogola (1971) [29] with minor modifications.
Total RNA isolation and first strand cDNA synthesis
Total RNA was extracted from both cell cultures using the RNeasy Plant Mini Kit followed by in-column DNase treatment (Qiagen, Hilden, Germany). RNA concentrations were measured using a Nano drop ND-1000 spectrophotometer (Nano drop Technologies, Rockland, DE, USA) and 5 μg of RNA was treated with RQ1 DNase (Promega) according to the manufacturer's protocols. RNA was precipitated with 2.2 μl of CH 3 COONa 3M pH 5.2 and 60.5 μl of ethanol and incubated for 30 min at -20˚C. After centrifugation the pellet was washed with 70% ethanol, air-dried, re-suspended in 5 μl of water and used for first-strand cDNA synthesis using the Superscript II Reverse Transcriptase (Thermo Fisher) according to user instructions.
Gene expression profile analysis
RT-qPCRs were performed using GoTaq qPCR master mix (Promega, United States) on the QuantStudio 12K Flex real time PCR system with standard protocol. Ubiquitin (Os05g0160200) was used as reference gene. Primers are listed in supporting information, S1 Table.
Ion analysis
Cells from control and treatment groups were harvested at 12, 24 and 72 h. Cells were filtered and oven dried at 40˚C for three days The dry weight was recorded and then the cells were ground to a fine powder which was afterwards dissolved in 5ml of 68% HNO 3 (Sigma Aldrich, Germany). Standard calibration curve was obtained for Na + and K + using 1000ppm Stock solutions (Fisher Scientific, USA). The samples were analysed using Atomic absorption spectrophotometer (Perkin Elmer Model 303). The concentration of Na + and K + (μg/g) was determined by comparing to the known concentrations of cation standards.
Statistical analysis
The data was analysed by two-way ANOVA (p<0.05) followed by Bonferroni post hoc test. The comparative C T method was used to analyze the gene relative expression (ΔΔC T method, [33]).
Salt sensitivity/tolerance of developed rice cell cultures
Cell cultures from two rice varieties were successfully developed and stabilized to study the mechanisms of salt tolerance at the cellular level. The two cell cultures were exposed to three salt concentrations (75, 100, 150 mM), and growth parameters along with measurements of cell death were evaluated.
By comparing the growth curves of the two cell cultures, differences in salt sensitivity were observed (Fig 1). A difference in the fresh weight of the two varieties was observed under control conditions. KS-282 showed a higher growth rate ( Fig 1A) than Super Basmati ( Fig 1B) at day 10. There was significant effect of salinity levels on the average calli fresh weights of both varieties. The fresh weight of Super Basmati reduced significantly as the salinity level increased from 75 to 100 and 150 mM. No significant difference in the growth rate of KS-282 cell cultures under control and at 75mM NaCl stress was observed, however the rate of cell division was significantly affected at 100 and 150 mM NaCl starting from day 6. The sensitive Super Basmati responded to all stress levels from day 4 showing severely retarded growth under saline condition. A similar drop in the dry weight of cell cultures was observed in both cell cultures under different salt concentrations (Fig 1C and 1D).
The measurement of cell death percentage (CDP) at different time points during subculture cycles showed that in Super Basmati CDP reached 40% and 52% after day 6 of salt treatment, depending upon the salt concentration (100 and 150 mM NaCl, respectively; Fig 2B). KS-282 cells, on the other hand, showed a slow increase in the CDP (27% at 150mM NaCl) at day 6 characterizing it as the tolerant cell culture (Fig 2A). No significant differences in cell viability of KS-282 cell cultures were observed in the presence of 75, 100 mM NaCl.
Extracellular hydrogen peroxide
The H 2 O 2 produced apoplastically was measured to observe the level of H 2 O 2 production by the cell cultures under control and NaCl stress. Around 100 nmol/g dry weight of H 2 O 2 was measured as basic level produced in the culture medium of untreated cells. In case of tolerant KS 282 cells, we were able to see a burst of H 2 O 2 with a first peak at 15 min ( Fig 3A) and another at 24 h ( Fig 3B) with a decreasing amplitude after 24 h for all three concentrations (75, 100 and 150 mM NaCl; p<0.05, two-way ANOVA, between control and treated cells) of salt. However, the sensitive cell cultures produced a single peak of H 2 O 2 at 24 h ( Fig 3C). H 2 O 2 was not detected during the first 6 h of salt treatment in culture medium of sensitive Super Basmati for all salt concentrations. Notably, H 2 O 2 production occurred in a dose-dependent manner in both cell cultures.
Intracellular hydrogen peroxide
Since the level of H 2 O 2 decides the fate of cell under salt stress [4], the intracellular level of H 2 O 2 was measured. The cells of tolerant and the sensitive varieties showed two peaks of H 2 O 2 inside the cells (Fig 4). One small peak was observed at 5 min with a decrease in H 2 O 2 level within 30 min of salt treatment. In tolerant KS-282 cell cultures the level of first peak was Salt stress response in rice cell cultures lower ( Fig 4A) under all salt concentrations as compared to the sensitive variety. The second peak was observed at 3 h (Fig 4B) of salt treatment with a low amplitude in a dose dependent manner which reduced progressively and reached the basal level at 24 h. On the other hand, in the more sensitive cell culture, salt treatment induced a second delayed sustainable peak with maximum amplitude at 6 h ( Fig 4D). The level of H 2 O 2 remained high above the optimal levels at 72 h. The production of intracellular H 2 O 2 again occurred in a dose-dependent manner in both the cultivars.
Enzyme activity assays
The activity of tAPX, cAPX and CAT was relatively high in the cells of tolerant KS-282 as compared to Super Basmati (Fig 5A-5D and Fig 6A and 6B) while the activity of SOD was comparatively low in the tolerant KS-282 cells as compared to the sensitive cells (control and treated respectively) (Fig 6C and 6D). A significant increase in the activity of tAPX and cAPX was Fig and S4 Fig). The activity of SOD in control cells of tolerant and sensitive cell cultures (Fig 6C and 6D) was almost equal (8.28 ± 0.47and 8.97± 1.07-unit SOD/ml/ mg protein respectively) while under saline conditions the activity of SOD increased and was higher in sensitive cell culture at 30 min (17.11± 1.29 unit SOD/ml/mg protein) as compared Values were normalized against the levels of control cells, which are given a value of 1 and therefore have no SD. Values represent the mean ± SE (p<0.05) of at least two independent experiments. The differences reported between the control and treated groups were statistically significant according to the two-way ANOVA followed by Bonferroni post hoc test. to the tolerant KS-282 cells (12.92 ± 0.72 unit SOD/ml/mg protein). SOD activity was reduced in the tolerant KS-282 cells at 24 h to optimal level (9.21± 0.63unit SOD/ml/mg protein) while it increased to 20.04 ± 2.30-unit SOD/ml/mg protein in Super Basmati sensitive cell culture (Fig 6C and 6D) exhibiting a steady increase in H 2 O 2 level.
Intracellular nitric oxide levels
In response to salt stress, NO production was observed in both cell cultures. In case of sensitive Super Basmati cells an initial increase in NO was detected with all three salt concentrations (Fig 7B). This high level dropped after 6 h with 75mM NaCl while with 100 and 150 mM NaCl level of NO remained nearly steady up to 72 h. On the other hand, in KS-282 cells only an Salt stress response in rice cell cultures early narrow peak of NO (at 3 h) was detected in the presence of all three NaCl concentrations followed sharp reduction in NO level (Fig 7A). The differences reported were statistically significant according to the two-way ANOVA at p<0.05 (n>30).
CDP measurement after cPTIO pre-treatment
CDP was measured in cell cultures of both tolerant and sensitive varieties pre-treated with 50uM cPTIO and then exposed to 100mM and 150mM NaCl. cPTIO is a NO radical scavenger In sensitive Super Basmati cells exposed to 100 mM NaCl, on day 6 CDP reduced to 68% as compared to the non-pre-treated cells while under 150mM NaCl the percentage reduction in CDP was 71% on day 6 as compared to the non-pre-treated cells at day 6 of salt treatment (Fig 8B). In the tolerant KS-282 pre-treated cells CDP reduced to 23 and 55% at 100 and 150mM NaCl respectively on day 6 ( Fig 8B). This marked reduction in CDP demonstrate a possible role of NO radicals in inducing programmed cell death in cell cultures.
Genes involved in ROS signalling
In our study an extracellular H 2 O 2 burst was observed early in the tolerant line on salt treatment, whereas a high level of H 2 O 2 combined with sustained levels of intracellular NO was detected in the sensitive cells. These two responses to salt stress may induce signals that lead to different fates, i.e., the induction of resistant mechanisms in KS-282 cells and the process of programmed cell death (PCD) in Super Basmati cells.
To investigate this hypothesis in more detail, the expression of genes linked to ROS signalling pathway was investigated before (Supporting Information, S1C-S1E Fig) and after salt treatment. First, the expression of Salt-Responsive ERF1(SERF1), the rice TF gene known for being involved in H 2 O 2 dependent salt stress signalling [25] and its possible role in salt stress adaptation, was analysed. In Super Basmati cells, there was a small peak of expression at 30 min of exposure to salt stress, whereas OsSERF1 expression in KS-282 cells was significantly high at 30 min of stress (Fig 9A). Another TF, Dehydration Response Element Binding 2A (OsDREB2A), activated by ROS and SERF1 and known to improve dehydration and salt stress tolerance in rice [28] was analyzed. OsDREB2A was found to be not responsive in sensitive Super Basmati cell cultures while by contrast, cell cultures of tolerant KS-282 showed enhanced expression of OsDREB2A at 30 min of salt treatment (Fig 9B).
OsRbohA gene expression was analysed in both cell cultures. OsRbohA belongs to the respiratory burst oxidase homologues (RBOH; [34]) gene family playing a main role in apoplastic ROS production in plants [12]. In our system, OsRbohA was significantly upregulated in KS-282 at 30 min of salt treatment (Fig 9A), whereas in Super Basmati expression of this gene was low event at 24 h (Fig 9C).
Mechano-sensitive Ca 2+ channels mediated by oxidative burst
Ca 2+ is a signal molecule that has been proven to regulate H 2 O 2 production [35-36]. However, it has also been proposed that under stress intracellular Ca 2+ waves travel from cell to cell via unknown mechano-sensors activated by the apoplastic ROS produced by the activity of NOXs [8]. We analysed the expression pattern of Ca 2+ channels OsOSCA 1.2 and OsOSCA 3.1 up to 6 h of salt treatment. We were able to see a significantly high expression of OsOSCA 1.2 and OsOSCA3.1 at 30 min of salt treatment in tolerant KS-282 cultured cells (Fig 10A and 10B) which correlates positively to the burst of H 2 O 2 in the cell cultures. No such expression of these two channels was seen in the control cell cultures of KS-282 (Supporting Information, S1A and S1B Fig). These results suggest that the OsOSCA genes can be a target of the H 2 O 2 signalling pathway. The activation of different OSCAs genes can lead to the modulation of postperception calcium signalling leading to the acclimation of the cells to the stress. The concomitant induction of the regulatory subunit gene RbohA also suggests that NOXs and OSCAs can control and treatment groups were reported were statistically significant according to the two-way ANOVA at p<0.05 (n>30). be a part of the mechanism of transmission of the stimulus from outer to inner cell layers in whole plants, as suggested by [8]. No such response is activated in the sensitive Super Basmati cell cultures up to 6 h. This delay might be responsible for the lack of appropriate mechanisms to activate the signalling pathways necessary for adaptation to salt stress in sensitive cells cultures of Super Basmati.
Comparative expression of the candidate genes involved in sodium ion sequestration, extrusion and potassium ion homeostasis
To characterize the relationship between the oxidative burst, osmosensing Ca 2+ channels and membrane ion transport proteins with respect to salt tolerance in indica rice, relative expression of OsSOS1 (plasma membrane Na + /H + antiporter; [37]) and OsNHX1 (vacuole localized Na + /H + antiporter; [38]) was analysed (Fig 11A and 11B). The difference in expression was analysed at time points starting from 10 min up to 24 h of salt treatment using RT-qPCR.
In the tolerant KS-282 cells, an up-regulation of SOS1 was observed at 1h of salt stress generating a tolerance response at cellular level ( Fig 11A) while in the sensitive Super Basmati cells Cell death rate as measured in tolerant (a) and sensitive (b) cell cultures after 50uM cPTIO pre-treatment and 100 and 150mM NaCl stress. In the pre-treated tolerant KS282 cell cultures the CDP decreased to 23 and 55% at 100 and 150mM NaCl respectively on D6 while in the sensitive Super Basmati cells exposed to 100 mM NaCl, on D6 CDP decreased to 68% as compared to the non-pre-treated cells while under 150mM NaCl the percent decrease in CDP was 71%. The cell viability and cell death percentage was measured using Evans blue staining according to Gaff and Okong'o-Ogola (1971). Values represent the mean ± SE (p<0.05) of three independent experiments performed in triplicate. Different letters indicate statistically different expression levels according to two-way ANOVA (p<0.05). 12C). The plasma membrane located channel protein, high affinity K + transporter OsHAK5 [40] (involved in potassium uptake) and potassium channels genes important for salt tolerance, KAT1 [41] was highly up-regulated in the tolerant variety at 1h of salt treatment showing an efficient K + acquisition to maintain K + homeostasis in the cytosol under saline condition
Ion analysis
Ion analysis revealed a decrease in K + content in both cell cultures at 24 h of salt treatment however cells of tolerant KS-282 were able to increase K + concentration with a decrease in Na + content at 72 h of salt exposure. On the other hand, in case of the sensitive Super Basmati cells, a sharp increase in the Na + content with a progressive decrease in K + concentration was observed ( Fig 13A and 13B). Similarly, the tolerant cell cultures were able to maintain a higher K + /Na + ratio at 12 and 72 h after salt treatment whereas in the sensitive cells a sharp decrease was recorded.
Differential expression of genes upregulated by salt stress or Abscisic acid
In order to define whether the differences in expression profiles of ROS-and ion homeostasisrelated genes were related to difference in perception of the salt stress, we analysed the expression profile of genes known to be up-regulated by salt or ABA i.e. OsLEA19,OsRAB16, PYL4 and ABA45. LEA and dehydrins are known to accumulate upon salt and osmotic stress. OsLEA19 and OsRab16, in particular, have been demonstrated to confer salt tolerance in rice [42,43]. ABA45 harbours the GRAM motif typical of genes in the ABA signalling pathway and it is known to be upregulated by ABA administration [44]. PYL4 belongs to the family of Pyrabactins-like receptors for ABA [44].
All these genes showed differences in the expression level between the two varieties under salt conditions (100mM NaCl, Fig 14A, 14B, 14C and 14D). An early (10-30 min) and stronger response of OsLEA19, OsRAB16 and OsABA45 was measured in tolerant cells. Our data strongly suggests that very early events in the tolerant system were put in place that led to the regulation of salt responsive genes and eventually the survival of the cells.
Discussion
ROS produced by the apoplastic NADPH oxidase plays a crucial role in mediating stress tolerance during the initial phase of salt stress. These ROS might act as an acclimation signal activating H 2 O 2 dependent molecular signaling cascade. Here, we propose a salt stress induced signaling pathway in the tolerant cell cultures, positively regulated by calcium influx modulation via mechano-sensitive OSCA channels and activating ROS dependent TFs OsSERF1and OsDREB2A, which propagates the initial transcriptional response under salt stress in rice.
We generated cell cultures from the mature seeds of two indica rice varieties which have previously shown contrasting response to salt stress. We demonstrated that, on treatment with 100mM NaCl, KS-282 cells are able to set up specific tolerance mechanisms, whereas Super Basmati cells undergo cell death. Growth parameters along with measurements of cell death induced by treatment was determined by comparing the growth curves and cell death percentage (CDP) revealing differences in salt sensitivity. The cell cultures of sensitive Super Basmati showed a marked reduction in the fresh weight (FW) under salt stress when compared to the lines grown in standard conditions. By measuring the CDP at different time points during sub-culture cycles, cultured cells from Super Basmati reached a higher CDP after 6 days of salt treatment at 150 and 100 mM NaCl respectively (Fig 2B) as compared to the tolerant KS-282 Salt stress response in rice cell cultures cells validating these cell cultures to be highly tolerant to salt stress and making them suitable for studying the stress response.
Previous data indicate that under salinity stress, reactive oxygen species, notably H 2 O 2 interact with NO [45]. The possibility that NO works together with the universal second messenger Ca 2+ in plant signaling processes has also been proposed [46,47]. To understand the cross talk between these important signaling components we compared the extra and intracellular H 2 O 2 and intracellular NO levels in the two cell cultures. The two cell cultures differed with respect to extracellular and intracellular H 2 O 2 and NO accumulation upon salt treatment. In the sensitive variety, salt stress induced a delayed but high level of extracellular H 2 O 2 at 24 h (Fig 3C) after salt stress while intracellularly H 2 O 2 was generated within the first 5 min of treatment which reached at a maximum amount at 3 h (Fig 4D) together with a progressive NO increase over time (Fig 7B). The level of H 2 O 2 remained high up to 48 h intracellularly in a dose dependent manner which is consistent with the activity of SOD as measured under 100mM NaCl. On the other hand, in tolerant KS-282 cells exposed to salt stress, an initial extracellular H 2 O 2 signal was detected at 15 min (Fig 3A). While a later burst of H 2 O 2 (possibly responsible for intercellular ROS wave) was seen at 24 h. Previously, it has been demonstrated in rice that NaCl stress triggers H 2 O 2 production in roots within 5 min, and this increase depends on NADPH oxidase activity [24]. Intracellularly, at 5 min and 3 h H 2 O 2 was detected which optimised to basal level by the high antioxidant enzyme activity (tAPX/ cAPX and CAT) indicating a control over the rapid oxidative burst (Figs 5A, 5B, 6A and 6B). An increase in the NO level was observed at 1 h (Fig 7A) which declined after 3 h and reached to basal levels bringing the cell cultures to a controlled oxidative state and precluding cell death in the tolerant cells.
The initial high level of extracellular H 2 O 2 in the tolerant KS-282 were found to be consistent with the expression of H 2 O 2 -responsive and salt-specific TF SERF1 and downstream genes DREB2a and OsRbohA genes as revealed by expression analysis. This early oxidative Salt stress response in rice cell cultures burst has been previously associated with stress tolerance in plants [48] suggesting H 2 O 2 as a possible signalling molecule. This prompt increase in OsRbohA transcript is consistent with the finding that OsRbohA-overexpressing transgenic lines exhibit much greater drought tolerance [49] and A. thaliana mutants lacking RbohF (OsRbohA gene homologue) showed decreased salinity tolerance [50]. Notably, in KS-282 cells, the expression level of SERF1 and DREB2a (Figs 9A and 6B), two genes involved in salt-induced signalling processes [25,24], increased very rapidly and earlier after salt stress exposure than in Super Basmati cells. Consistent with our observation, SERF1 has been reported to act as a positive regulator of salt tolerance [25]. Along with the extracellular H 2 O 2 signal, a sharp increase in NO level was seen in tolerant KS-282 cell cultures which dropped to basal level after 3h of salt treatment while in the case of sensitive cell cultures, huge amounts of NO were generated within the first 1h which then reduced but remained above basal levels up to 72 h of salt treatment indicating its continuous production. RBOH at the plasma membrane are known to be regulated by NO [8] through S-nitrosylation suggesting interplay between NO signalling and ROS homeostasis [51] which is maintained by a higher APX/CAT activity and a low SOD activity in the tolerant cell cultures. NO plays a dual role during systemic signaling by amplifying or dampening the signal [52,53]. In case of the sensitive Super Basmati cells, no extracellular H 2 O 2 and RbohA expression was seen up to 24 h of salt stress while a high level of intracellular H 2 O 2 was observed along with a significantly high SOD activity and a low APX and CAT activity. Consistently high level of intracellular NO in the sensitive cell cultures after salt exposure suggest its role in inhibiting ROS scavenging enzymes reducing H 2 O 2 decay inside the cells causing more oxidative damage and consequent programmed cell death. NO mediated S-nitrosylation inhibiting antioxidant enzymes i.e. catalase and ascorbate peroxidase has already been proposed by several groups [54,55] and our results are consistent with previous data. NO radical when scavenged by cPTIO prevents this damage, reducing the cell death rate as evident by a significantly decreased CDP in both sensitive and tolerant cell cultures. While in the case of tolerant KS-282 cells, a single peak of NO up-regulates the NOX activity and H 2 O 2 signal within 3h of salt stress. In Arabidopsis, AtRbohD was shown to be upregulated through a NO-dependent process elicited by oligo-galacturonides in response to pathogen attack [52]. Improved salt tolerance has been reported in Arabidopsis thaliana with enhanced GST activity and a contolled ROS signal [56,57]. Under hypoxia and salt stress, NADPH oxidase (s) (RbohD) play a major role in producing and controlling ROS signal [34] via K + homeostais and reduced Na + accumulation by pumping Na + out of the cytosol [58]. Moreover, NO accumulation has been correlated with reduced growth rate under low oxygen stress predicting a cross talk between ROS and NO signal [59]. Similarly, the findings of these studies augment the observed link between NO and reduced growth rate of cell cultures in our study under salt stress.
It is well established that various biotic and abiotic stimuli trigger an increase in the intracellular Ca 2+ levels by the activation of unknown Ca 2+ channel [21,[60][61][62][63]. Calcium acts as an important second messenger in the signal transduction of many abiotic stress responses [64]. OSCAs channels, activated by hyperosmolality, have been shown to be involved in osmoticstress induced fast signaling events [65], suggesting OSCAs to be osmosensor. These mechanosensitive Ca 2+ channels, ATOSCA 1.1 and ATOSCA 1.3 have recently been characterized in Arabidopsis and reported to play an important role in osmosensing under salt stress [66]. The role of Ca 2+ influx in affecting ATRboh activity is instrumental and associated with ABA signaling, Ca 2+ modulation and eventually is involved in salt stress adaptation [67,58]. In silico characterized members of OSCA family i.e. OSCA 1.2 and OSCA3.1 genes in rice were analyzed in present study. Consistent with the expression of genes involved in oxidative burst, the expression of OSCA 1.2 and OSCA3.1 channel proteins was significantly higher in tolerant KS-282 cell cultures in the first 30 min of salt treatment while a weaker response of these channel was observed in the sensitive cell culture. Previously a mechanism involving an increase in intracellular Ca 2+ due to apoplastic oxidative burst has been proposed [8]. ROS-activated Ca 2+ channels and transporters in the plasma membrane have been identified at the electrophysiological [68][69][70] and molecular levels [42]. We propose that OSCA 1.2 and OSCA3.1 channels are modulated by H 2 O 2 and may be related to systemic signaling by increasing an influx of Ca 2+ . Our results regarding ROS wave and consequent Ca 2+ influx are in agreement to the recent study available in japonica rice plants suggesting a particular composition of OSCA and RBOH at the plasma membrane capable of responding to salinity stress and adaptation [43]. We propose that once such an initial 'priming' Ca 2+ influx has occurred, the resulting cytosolic Ca 2+ signal is amplified through vacuolar stores triggering long range signaling in which specific genes are involved (OSCA 1.2, OSCA 1.3 and RbohA).
Maintaining low cytosolic Na + levels while keeping high levels of K + inside the cells is wellreported as an effective strategy to cope with salt stress. The maintenance of a high cytosolic [K + ]/[Na + ] ratio is crucial for salt tolerance [44]. The expression analysis of genes involved in increasing cytosolic K + concentration correlated with our ion analysis data and showed that KS-282 cells upregulate HAK5 and KAT 1 earlier than Super Basmati cells. The expression of OsTPK1a in the tolerant KS-282 was seen after 24 h while its expression remained low in the sensitive cell culture. The significantly high expression of some K + transporter/channel genes has been reported to increase salt tolerance [40,[71][72][73]. Therefore, in salt-tolerant KS-282 cells, a response to salt stress seems to involve the capability to more efficiently maintain K + homeostasis. Our results are further supported by the expression analysis of genes (OsLEA19; OsRAB16; ABA45) that have been demonstrated to be induced upon salt, osmotic stresses and ABA [74][75][76]. Moreover, after a massive increase in Na + content in both varieties, only KS-282 cell cultures were able to reduce intracellular Na + levels within 72 h of salt treatment. Therefore, we can hypothesize that the higher intracellular H 2 O 2 level observed in treated Super Basmati cells undergoing programmed cell death can also be correlated with the reduced [K + ]/ [Na + ] ratio. No significant differences in the expression of NHX1 in the two cell cultures also suggest that in KS-282 cell cultures, tolerance may depend more on increased cytosolic[K + ] to sustain a high [K + ]/[Na + ] ratio than on Na + compartmentalization, while Na + exclusion does not play an important role during the early response to salt stress.
Conclusion
In essence, we demonstrate that salt tolerance in KS-282 cells depends mainly on an efficient stress perception mediating a control over ROS homeostasis via upregulated ROS scavenging enzyme activities and its ability to maintain a high [K + ]/[Na + ] ratio. The ability of KS-282 cells to generate an apoplastic H 2 O 2 burst and to maintain low intracellular levels of H 2 O 2 plays a key role in survival. The H 2 O 2 signal, generated by apoplastic NOX and synchronised with the NO signal along with a high influx of Ca 2+ (putatively through OSCA hyperosmolality-gated channels), causes an upregulation of OsSERF1 and OsDREB2A TFs and generates a vital response in tolerant cells. Conversely, the sensitive cell culture fails to produce a timely ROS and Ca 2+ signal. Intracellularly, lethal amounts of H 2 O 2 and NO trigger programmed cell death. Along with that the tolerant cell cultures have the ability to maintain a high K + concentration due to a rapid increase in the expression of genes coding for transporters/channels localized to the tonoplast and to the plasma membrane.
Supporting information S1 Table. List of primers used in this study. | v3-fos-license |
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} | pes2o/s2orc | Nitrite toxicity assessment in Danio rerio and Poecilia reticulata
Nitrite is a natural component of the nitrogen cycle in the environment. Although it usually occurs in low concentrations, elevated concentrations caused by effluents or affected nitrification process can lead to serious health deterioration of fish. Two aquarium fish zebrafish (Danio rerio) and guppy (Poecilia reticulata) are recommended to use as model organisms in toxicity tests. However, their sensitivity to nitrite can differ. The aim of this study was to define acute toxicity of nitrite by the semistatic method according to OECD No. 203 (Fish, Acute toxicity test). The series of 4 acute toxicity tests was performed, with 10 fish of both species used for each concentration and for the control. The 96hLC50 NO2 value for D. rerio and P. reticulata was 242.55 ± 15.79 mg·l-1 and 30.2 ± 8.74 mg·l-1, respectively. We have proved significant difference (p < 0.05) in sensitivity between D. rerio and P. reticulata. The results showed different sensitivities to nitrites in tested fish species, which could be related to species-specific branchial chloride uptake mechanism. This is the first study on this fish species. Acute toxicity, NO2, comparison of the sensitivity, zebrafish, guppy Nitrite occurs naturally both in freshwater and saline waters, whereas the concentrations of nitrites up to tenths of mg·l-1 have been measured in surface waters in the Czech Republic (Kroupová et al. 2008). Elevated concentrations of nitrite can be found in closed intensive systems where water-recirculating is used (Svobodová et al. 2005). Nitrite is an intermediate product in bacterial nitrification and denitrification processes in natural nitrogen cycle (Pitter 1999). Nitrification is aerobic oxidation of ammonia to nitrite (by Nitrosomonas sp.) and of nitrite to nitrate (by Nitrobacter sp.). Denitrification is reduction of nitrates to the final nitrogen by a number of facultative anaerobic bacteria (Jensen 2003). This natural process is imitated on biological filters of the water-recirculating systems. However, if the nitrification on these filters is disturbed and the nitrification is retarded, the nitrites are accumulated in the water (Svobodová et al. 2005). Nitrites are considered to be toxic to fish after acute (Pištěková et al. 2005; Weirich and Riche 2006; Park et al. 2007; Rodrigues et al. 2007; Costa et al. 2008; Ozcan et al. 2010) as well as chronic exposure (Siikavuopio and Saether 2006; Kroupová et al. 2008; Voslářová et al. 2008; Kroupová et al. 2010). The toxicity of nitrites for fish differs in dependence of many internal and external factors such as water quality, fish species, their size and individual sensitivity (Lewis and Morris 1986; Jensen 2003; Voslářová et al. 2006). The most important factor influencing nitrite toxicity is the concentration of chloride ions in water. Many authors proved in their studies the protective impact of chloride ions against intoxication of nitrites (Crawford and Allen 1977; Lewis and Morris 1986; Pištěková et al. 2005; Voslářová et al. 2006; Wang et al. 2006; Weirich and Riche 2006). Nitrite is actively taken up across the chloride cells on gills. Nitrite in the blood is bound to haemoglobin which is converted to methaemoglobin and the oxygen-carrying capacity of blood is impaired. The increased amount of methaemoglobin in blood is accompanied with brown coloured blood and gills (Cameron 1971). ACTA VET. BRNO 2011, 80: 309–312; doi:10.2754/avb201180030309 Address for correspondence: MVDr. Petra Doleželová, Ph.D. University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno Czech Republic Phone: + 420 541 562 789 Fax: +420 541 562 790 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm Toxicity tests are often used to determine a possible toxic effect of chemical substances on aquatic organisms. Particularly short-term toxicity tests on fish are suitable for evaluation of new chemical substances, effluents or wastes intended for disposal. The procedure of such tests is established by the Organization for Economical Cooperation and Development (OECD) which also specified recommended organisms used in toxicity tests. The recommended organisms include two aquarium fish species – zebrafish (Danio rerio) and guppy (Poecilia reticulata). The aim of this study is to determine the possible difference in sensitivity of these two fish species to nitrites. Materials and Methods Acute toxicity tests were carried out on juvenile aquarium fish zebrafish and guppy. Both juvenile zebrafish (aged 2 months, length 30 ± 5 mm, weight 0.3 ± 0.1 g) and juvenile guppies (aged 2 months, length 25 ± 5 mm, weight 0.3 ± 0.1 g) were obtained from a stable local commercial dealer. A total number of 210 fish of each species were used. All fish were kept in glass tanks for a minimum of 7 days for the acclimatization period (water temperature 23 ± 1 °C, with a 12 h/12 h light/dark cycle) during which they were fed commercial fish pellets. Food was withheld 24 h before the start of the test. Experimental procedures were in compliance with the national legislation (Act No. 246/1992 Coll. on the protection of animals against cruelty, as amended by Decree No. 207/2004 Coll. on the protection, breeding and use of experimental animals). The method followed OECD 203 Guideline (Fish, Acute toxicity test) in the semi-static condition with solution replacement after 24 h to ensure stable concentration of nitrites in tested solutions. Five progressive concentrations of the tested substance were prepared in 5 l tanks by dissolution of nitrite (dosed as NaNO2) in dilution water of the following quality: ANC4.5 1.0–1.2 mmol·l –1; CODMn 0.8–1.2 mg·l –1; total ammonia below the limit of determination (< 0.04 mg·l-1); NO3 – 11.2–13.5 mg·l–1; NO2 – below the limit of determination (< 0.02 mg·l-1); Cl– 18.5-19.1 mg·l–1; Σ Ca + Mg 14 mg·l–1. A series of four acute toxicity tests with five ascending concentrations of the tested substance separately for P. reticulata (10, 20, 30, 40, and 50 mg·l–1) and for D. rerio (70, 100, 200, 300 and 400 mg·l–1) were used. Concurrently, the control test with only dilution water was performed. Ten fish of each species randomly picked from the spare stock were placed into each concentration and control. The total length of acute toxicity tests was 96 h and fish were daily inspected for mortality. During the tests, the temperature, pH, the concentration of oxygen dissolved in test and control tanks and fish mortality rate were recorded. No fish died in the control tank during the experiments. Temperature of the tested solutions was 24 ± 1 °C, the concentration of dissolved oxygen did not drop below 80–94% and pH was within the range 7.89–8.62. Using the number of fish dying at individual test concentrations in the time period of 96 h, the medium lethal concentrations (96h LC50) were calculated by applying the probit analysis of the EKO-TOX 5.2 software. The significance of the difference between LC50 values for zebrafish and guppies was calculated using the nonparametric Mann-Whitney test and the Unistat 5.1 software.
Nitrite occurs naturally both in freshwater and saline waters, whereas the concentrations of nitrites up to tenths of mg•l -1 have been measured in surface waters in the Czech Republic (Kroupová et al. 2008).Elevated concentrations of nitrite can be found in closed intensive systems where water-recirculating is used (Svobodová et al. 2005).Nitrite is an intermediate product in bacterial nitrification and denitrification processes in natural nitrogen cycle (Pitter 1999).Nitrification is aerobic oxidation of ammonia to nitrite (by Nitrosomonas sp.) and of nitrite to nitrate (by Nitrobacter sp.).Denitrification is reduction of nitrates to the final nitrogen by a number of facultative anaerobic bacteria (Jensen 2003).This natural process is imitated on biological filters of the water-recirculating systems.However, if the nitrification on these filters is disturbed and the nitrification is retarded, the nitrites are accumulated in the water (Svobodová et al. 2005).
Nitrites are considered to be toxic to fish after acute (Pištěková et al. 2005;Weirich and Riche 2006;Park et al. 2007;Rodrigues et al. 2007;Costa et al. 2008;Ozcan et al. 2010) as well as chronic exposure (Siikavuopio and Saether 2006;Kroupová et al. 2008;Voslářová et al. 2008;Kroupová et al. 2010).The toxicity of nitrites for fish differs in dependence of many internal and external factors such as water quality, fish species, their size and individual sensitivity (Lewis and Morris 1986;Jensen 2003;Voslářová et al. 2006).The most important factor influencing nitrite toxicity is the concentration of chloride ions in water.Many authors proved in their studies the protective impact of chloride ions against intoxication of nitrites (Crawford and Allen 1977;Lewis and Morris 1986;Pištěková et al. 2005;Voslářová et al. 2006;Wang et al. 2006;Weirich and Riche 2006).
Nitrite is actively taken up across the chloride cells on gills.Nitrite in the blood is bound to haemoglobin which is converted to methaemoglobin and the oxygen-carrying capacity of blood is impaired.The increased amount of methaemoglobin in blood is accompanied with brown coloured blood and gills (Cameron 1971).
Toxicity tests are often used to determine a possible toxic effect of chemical substances on aquatic organisms.Particularly short-term toxicity tests on fish are suitable for evaluation of new chemical substances, effluents or wastes intended for disposal.The procedure of such tests is established by the Organization for Economical Cooperation and Development (OECD) which also specified recommended organisms used in toxicity tests.The recommended organisms include two aquarium fish species -zebrafish (Danio rerio) and guppy (Poecilia reticulata).
The aim of this study is to determine the possible difference in sensitivity of these two fish species to nitrites.
Materials and Methods
Acute toxicity tests were carried out on juvenile aquarium fish zebrafish and guppy.Both juvenile zebrafish (aged 2 months, length 30 ± 5 mm, weight 0.3 ± 0.1 g) and juvenile guppies (aged 2 months, length 25 ± 5 mm, weight 0.3 ± 0.1 g) were obtained from a stable local commercial dealer.A total number of 210 fish of each species were used.All fish were kept in glass tanks for a minimum of 7 days for the acclimatization period (water temperature 23 ± 1 °C, with a 12 h/12 h light/dark cycle) during which they were fed commercial fish pellets.Food was withheld 24 h before the start of the test.Experimental procedures were in compliance with the national legislation (Act No. 246/1992 Coll. on the protection of animals against cruelty, as amended by Decree No. 207/2004 Coll. on the protection, breeding and use of experimental animals).
A series of four acute toxicity tests with five ascending concentrations of the tested substance separately for P. reticulata (10, 20, 30, 40, and 50 mg•l -1 ) and for D. rerio (70, 100, 200, 300 and 400 mg•l -1 ) were used.Concurrently, the control test with only dilution water was performed.Ten fish of each species randomly picked from the spare stock were placed into each concentration and control.The total length of acute toxicity tests was 96 h and fish were daily inspected for mortality.During the tests, the temperature, pH, the concentration of oxygen dissolved in test and control tanks and fish mortality rate were recorded.No fish died in the control tank during the experiments.
Temperature of the tested solutions was 24 ± 1 °C, the concentration of dissolved oxygen did not drop below 80-94% and pH was within the range 7.89-8.62.
Using the number of fish dying at individual test concentrations in the time period of 96 h, the medium lethal concentrations (96h LC50) were calculated by applying the probit analysis of the EKO-TOX 5.2 software.The significance of the difference between LC50 values for zebrafish and guppies was calculated using the nonparametric Mann-Whitney test and the Unistat 5.1 software.
Results
On the basis of the results of acute toxicity tests on juvenile D. rerio, the lethal concentrations of nitrite in 96 h varied from 223.5 mg•l -1 to 256.6 mg•l -1 (mean ± SD: 242.6 ± 15.79 mg•l -1 ), whereas in juvenile P. reticulata the lethal concentrations varied from 21.9 mg•l -1 to 39.9 mg•l -1 (mean ± SD: 30.2 ± 8.74 mg•l -1 ).In both experiments, the chloride concentration was about 19 mg•l -1 .The particular lethal concentrations of nitrite with confidence intervals for each lethal concentration and with mean and standard deviation for zebrafish are shown in the Table 1 and for guppy in Table 2.
Significant difference (p < 0.05) between 96hLC50 values for D. rerio and P. reticulata was recognized.
Discussion
Acute toxicity tests on fish are important for the assessment of impact of some chemical substances on fish.Although nitrites are a natural part of the nitrogen cycle in nature, significant accumulation of nitrites (e.g.surface water pollution with waste water, increased density of fish in the stock or insufficient degradation of nitrites) usually lead in fish poisoning with signs of asphyxia.
Acute toxicity of nitrites differs according to fish species.In our study we determined the 96hLC50 of NO 2 -in aquarium fish D. rerio as 242.6 ± 15.79 mg•l -1 (mean ± SD), whereas in P. reticulata it was 30.2 ± 8.74 mg•l -1 .Significant difference (p < 0.05) in the sensitivity of these two fish species was found.Many authors investigated the effects of nitrites on different fish species.Lewis and Morris (1982) reported significant differences in interspecies sensitivity of fish to nitrites.Comparing lethal concentrations at the comparable chloride concentration of 20 mg•l -1 , the most sensitive fish was rainbow trout (Oncorhynchus mykiss) with the LC50 of 21.7 mg•l -1 , whereas in common carp (Cyprinus carpio) the LC50 value was 207 mg•l -1 , in fathead minnow (Pimephales promelas) 217 mg•l -1 and in bluegill (Lepomis macrochirus) 355 mg•l -1 .Hence it could be concluded that guppy is similarly sensitive to nitrites as rainbow trout and zebrafish is as tolerant as cyprinids, especially carps and fathead minnow.Ozcan et al. (2010) tested acute toxicity in another cyprinid fish, transcaucasian barb (Capoeta capoeta capoeta) and genotoxic and histopathologic effects were determined simultaneously.The median lethal concentration of nitrite at 96 h of exposure was 122.8 mg•l -1 , slightly lower than in previous cyprinid species, hence we can consider differences in tolerance to nitrite in one family.
The differences in tolerance to nitrite ions are connected to the mechanism of action of nitrites.Nitrites have an affinity for branchial Cl -uptake mechanism and so fish with high branchial Cl -uptake rates (e.g.trouts) are more sensitive to nitrite than species with low uptake rates (Cyprinidae).Other possible explanation includes the difference in chloride cell number or proliferation of chloride cells during nitrite exposure (Jensen 2003).
The high influence on nitrite toxicity is attributed to chloride concentration in water.Wang et al. (2006) investigated the effects of nitrite on tilapia (Oreochromis niloticus) at different chloride concentrations.According to his results, the 96hLC50 raised from 28.18 mg•l -1 at 35.0 mg•l -1 chloride level to 44.67 mg•l -1 at 70.0 mg•l -1 chloride level.The protective effect of chloride ions against nitrite toxicity was reported in D. rerio (Voslářová et al. 2006) or in chinook salmon (Oncorhynchus tshawytscha) (Crawford and Allen 1977).Svobodová et al. (2005) have suggested some measures that should be done to prevent nitrite poisoning in fish.Regarding aquaculture facilities, it is important to supply such facilities with fish gradually according to filter capacity and to monitor the indicators of the water.Both in aquaculture facilities and aquariums it is possible to raise chloride concentration when there is an increase of nitrogenous metabolites. | v3-fos-license |
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} | pes2o/s2orc | The Olfactomedin Domain from Gliomedin Is a β-Propeller with Unique Structural Properties*
Background: Gliomedin contains an olfactomedin (OLF) domain, for which structural information has been lacking. Results: The crystal structure of the OLF domain from gliomedin was solved. Conclusion: The OLF domain is a five-bladed β-propeller with broken symmetry. Significance: The results provide a structural basis for gliomedin function and shed light on mutations in the human OLF family. All members of the olfactomedin (OLF) family have a conserved extracellular OLF domain, for which a structure has not been available. We present here the crystal structure of the OLF domain from gliomedin. Gliomedin is a protein expressed by Schwann cells in peripheral nerves, important for the formation of the nodes of Ranvier. Gliomedin interacts with neuronal cell adhesion molecules, such as neurofascin, but the structural details of the interaction are not known. The structure of the OLF domain presents a five-bladed β-propeller fold with unusual geometric properties. The symmetry of the structure is not 5-fold, but rather reveals a twisted arrangement. The conserved top face of the gliomedin OLF domain is likely to be important for binding to neuronal ligands. Our results provide a structural basis for the functions of gliomedin in Schwann cells, enable the understanding of the role of the gliomedin OLF domain in autoimmune neuropathies, and unravel the locations of human disease-causing mutations in other OLF family members, including myocilin.
Rapid propagation of action potentials is important for the normal functioning of the nervous system. Myelin is a multilayered membrane that enables the fast saltatory conduction of nerve impulses along axons. Although myelin acts as an electric insulator, it also drives the localization and clustering of voltage-gated sodium (Na v ) 2 channels on the neuronal plasma membrane (1-3), a crucial feature for saltatory conduction whereby the nerve impulse "jumps" from one node of Ranvier to the next.
Gliomedin is a protein expressed by myelinating Schwann cells that is important for the formation of the nodes of Ranvier and clustering of Na v channels on the axonal plasma membrane at the node (4 -6). Gliomedin contains an extracellular olfactomedin (OLF) domain, which is able to induce channel clustering (7,8). Gliomedin molecular ligands include neurofascin and NrCAM (neuronal cell adhesion molecule) (5,9), and loss of neuronal neurofascin also results in depletion of gliomedin from the nodes of Ranvier (10). Gliomedin is a type II transmembrane protein, and in addition to its C-terminal OLF domain, it harbors an extracellular collagen-like domain, which mediates gliomedin trimerization (8). Gliomedin homologs have also been identified in invertebrates (11). In vivo, gliomedin is processed proteolytically, and the extracellular OLF domain can be released either with or without the collagen domain (7,8). The OLF domain is also glycosylated (8), although the exact sites and nature of glycosylation have not been determined.
The ϳ250-residue OLF domain was first discovered in the early 1990s (12). All members of the OLF protein family contain an extracellular OLF domain (13). OLF domains are mediators of extracellular protein interactions, and they play roles in diverse processes, such as nervous system development, intercellular adhesion, and cell cycle regulation (14). Although the structure of the OLF domain has remained unknown, it can be predicted to consist largely of -structure. The OLF domain of myocilin was suggested to form a six-bladed -propeller structure (15), but no experimental data have backed up this prediction.
Due to its direct disease linkage, the most widely studied OLF family member is myocilin, mutations in which cause primary open-angle glaucoma in humans (16 -18). Myocilin is also involved in myelination, especially in the optic nerve (19 -22). The sequence identity between the OLF domains of gliomedin and myocilin is only 22%, however, suggesting that they do not carry out overlapping functions. An interaction between gliomedin and myocilin has also been reported (20).
We determined the high-resolution crystal structure of the OLF domain from gliomedin. The structure presents an unusual -propeller fold, and it can be used both to understand the involvement of gliomedin in neurological development and disease and to obtain information on disease-related mutations in related OLF family members, such as myocilin.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification-The OLF domain of rat gliomedin (residues 260 -543; sequence numbering includes the signal sequence) was recombinantly expressed in baculovirus-infected insect cells (23). Purification consisted of nickelnitrilotriacetic acid affinity chromatography, His tag cleavage using tobacco etch virus protease, and size-exclusion chromatography (23). The OLF domain was predicted by sequence analysis to lie at the very C terminus, whereas the N-terminal boundary of the domain was difficult to pinpoint. The domain selected for expression and structural studies ranged from the end of the collagen-like domain close to the C terminus. This construct provided a good yield of crystallizable recombinant protein.
Biophysical Characterization-CD spectroscopy was used to confirm the folding state of the purified gliomedin OLF domain. Spectra were measured on an Applied Photophysics Chirascan-plus instrument at ϩ20°C in a buffer containing 1.8 mM Tris (pH 7.5) and 7 mM NaCl. The protein concentration was 0.5 mg/ml, and the cuvette path length was 0.5 mm.
Synchrotron small-angle x-ray scattering (SAXS) data were obtained on the SWING beamline at the SOLEIL Synchrotron (Paris, France). The gliomedin OLF domain (6 mg/ml) was injected onto an Agilent SEC-3 size-exclusion column using an Agilent HPLC system and eluted with 0.1 M Tris (pH 7.5) and 400 mM NaCl. The eluate was directed to the SAXS flowthrough capillary cell, and scattering data were collected online. Frames corresponding to the main peak were selected and averaged using FoxTrot. Following data processing, further analyses were performed with the ATSAS package (24). GASBOR (25) was used for ab initio model building. CORAL (24) was used in conjunction with the crystal structure to build the fragments not visible in the crystal.
Crystallization and Data Collection-Highly concentrated pure gliomedin OLF domain (23 mg/ml) in 50 mM Tris (pH 7.5) and 100 mM NaCl was used for crystallization. After 3 days of incubation at 293 K, crystals were observed in 0.1 M CHES (pH 9.5) containing 20% PEG 8000. The crystals were picked up in a LithoLoop (Molecular Dimensions), briefly immersed in mother liquor supplemented with 20% 2-methyl-2,4-pentanediol, and flash-cooled in liquid nitrogen. Diffraction data for these orthorhombic native crystals were collected on the ID23-1 beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Data collection for the monoclinic crystal form was reported previously (23).
For experimental phasing, a large number of derivatives using heavy metal compounds and halides were prepared and tested. Eventually, a strong anomalous signal was observed from a single crystal soaked in K 2 PtCl 4 . Single-wavelength anomalous dispersion data were collected on beamline 14.1 at the Helmholtz-Zentrum Berlin/Berliner Elektronen-Speicherring Gesellschaft für Synchrotronstrahlung (HZB/BESSY, Berlin, Germany). All diffraction data were processed with XDS (26).
Structure Solution and Refinement-The structure was solved using the single-wavelength anomalous dispersion protocol of Auto-Rickshaw, the European Molecular Biology Laboratory (EMBL) Hamburg automated crystal structure determination platform (27). The input diffraction data were prepared and converted for use in Auto-Rickshaw using programs of the CCP4 suite (28).
Estimated substructure structure factor amplitude values were calculated using SHELXC (29). Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was set to 2.61 Å. Heavy atoms were found with SHELXD (30), and the correct hand for the substructure was determined using ABS (31) and SHELXE (32). Initial phases were calculated after density modification using SHELXE (32). The 2-fold non-crystallographic symmetry operator was found using RESOLVE (33). Density modification, phase extension, and non-crystallographic symmetry averaging were performed with dm (34). Two chains of 242 residues each were thereafter automatically built using ARP/wARP (35,36).
The automatic pipeline further refined this partial model to 2.0 Å resolution with the platinum derivative data using CNS (37), Refmac5 (38), and PHENIX (39). The partially refined structure was then used as a template for molecular replacement against a native high-resolution data set in a different space group. The structure was further refined using PHENIX and manually built using Coot (40). The final coordinates and original structure factors were deposited in the Protein Data Bank with codes 4D77 (orthorhombic) and 4D7C (monoclinic).
Crystal Structure of the Gliomedin OLF Domain-Gliomedin
contains an N-terminal transmembrane domain, a collagenlike segment, and a C-terminal OLF domain (Fig. 1A). The structure of the rat gliomedin OLF domain was solved by single-wavelength anomalous dispersion phasing using a crystal derivatized with platinum, and the structure was refined to 1.49 Å resolution using native data ( Table 1). The OLF domain structure is composed of a five-bladed -propeller (Fig. 1B), and each blade contains four -strands (strands a-d). The overall dimensions of the OLF domain are ϳ30 ϫ 40 ϫ 40 Å. Like other -propellers, the OLF domain is disc-shaped. The propeller circle is closed by insertion of the first N-terminal -strand into the framework of the last propeller blade (Fig. 1B), which is a common mechanism in -propeller proteins, also termed Velcro closure (48). The structure also contains three short ␣-helices. The longest of these helices is located in a loop between -strands c and d of blade 1. Interestingly, normal mode analysis of the structure suggests that this helix is highly mobile, and there may be significant motions between blades 1 and 5 (data not shown). In general terms, normal mode analysis allows the identification of potential low-frequency, large-amplitude, concerted motions in a protein structure. A schematic representation of the OLF domain topology is shown in Fig. 1C.
The common fold of a -propeller structure contains 4 -10 antiparallel four-stranded -sheets (49). Upon close inspection, the OLF domain five-bladed fold lacks a regular 5-fold symmetry and appears twisted. Indeed, blades 1-4 are actually arranged according to a hexagonal setting (Fig. 1C). The remaining blade 5, which contains both the N and C termini, actually takes up the space that would be filled by two blades in a six-bladed -propeller. This arrangement is certainly unexpected and unusual, and it distinguishes the OLF domain from both five-and six-bladed -propellers, also explaining difficulties in correctly predicting the OLF domain structure.
Structural homology searches using different algorithms with the gliomedin OLF domain produced rather poor hits to -propeller structures, and both five-and six-bladed structures were detected. However, when only blades 1-4 were used in searches, better fits were obtained toward six-bladed structures. This further highlights the loss of 5-fold symmetry in the OLF domain and confirms the 6-fold symmetry obeyed by the first four blades. Superpositions on symmetric five-and sixbladed structures further proved this arrangement (Fig. 2).
Surface Properties-To obtain an insight into possible functionally important sites on the gliomedin OLF domain surface, we analyzed its surface properties. The electrostatic surface of the gliomedin OLF domain shows a high positive charge on the FIGURE 1. Overall structure of the gliomedin OLF domain. A, schematic drawing of full-length gliomedin, which has an OLF domain at its C terminus. Residue numbering for predicted domain boundaries is shown to the left. col, collagen-like domain. B, crystal structure of the C-terminal OLF domain from gliomedin, viewed from above (upper) and the side (lower). The N terminus (blue) closes the ring-like structure by providing a -strand to the C-terminal blade. C, topology diagram for the OLF domain. The structure is a -propeller with five blades, but blades 1-4 follow hexagonal symmetry.
TABLE 1 Crystallographic data collection and refinement
Pt-SAD, platinum-based single-wavelength anomalous dispersion.
Data set
Pt-SAD Native Native 2 top face, whereas the bottom is more neutral or even negatively charged (Fig. 3A). The positive charge may be important in attracting binding partners of gliomedin.
Data collection
The distribution of aromatic residues on the surface of the OLF domain (Fig. 3B) indicates a possible surface for protein interaction with several aromatic rings. For example, carbohydrate ligands would be candidates for interaction with the OLF domain.
The OLF domain structure was also refined in a monoclinic crystal form, and an apparent concerted movement of large side chains at the top face between the two crystal forms was observed (Fig. 3C). This indicates some flexibility in the structure, which could be important for interactions. A plasticity of the aromatic surface could allow binding of diverse protein and/or carbohydrate ligands.
Gliomedin is known to be glycosylated on its OLF domain (8), and a total of four possible N-glycosylation sites can be predicted to lie within the domain. The crystal structure indicates that all these possible sites of glycosylation lie on the surface. Remarkably, all the sites are on the "side" of the OLF domain, keeping the top face accessible (Fig. 3B).
Solvent Cavity and Cation-binding Site-In general, -propellers have a central cavity, where, for example, enzymatic active sites can reside. The cavity or channel size increases as the number of blades gets higher (49). Gliomedin has a much smaller central cavity due to the disrupted symmetry. Furthermore, the top of this cavity is closed by a long loop (connecting strands 2d-3a), and it contains a number of water molecules and a bound cation (Fig. 3D). (72) gives a much better fit for these blades (right), which are organized in hexagonal symmetry. Blade 5 overlaps with blades 5 and 6 of the 6-fold structure. For clarity, only -strands from the respective structures are shown. In both panels, the gliomedin OLF domain is colored, and the corresponding reference structures are shown in gray. A positively charged ion is present inside the gliomedin OLF structure, on the side of the water-filled cavity. The ion is well defined in electron density and coordinated by five oxygen atoms at distances of ϳ2.30 Å (Fig. 3E). Also taking into account the buffer composition in crystallization, this ion could be confidently built as Na ϩ . The ion was also validated by the WASP (50) and CheckMyMetal (51) servers, and clearly the best scores were obtained for the presence of Na ϩ . In particular, the Na ϩ -specific valence value of 1.1 calculated by WASP clearly indicates bound Na ϩ (50). The side chains of Asn-423 and Asn-471 are involved in the coordination, as are the backbone carbonyl groups of Ala-472 and Leu-517 and one water molecule. Notably, Asn-423 corresponds to Asp-380 in myocilin. Myocilin was shown to bind and be stabilized by calcium (52), and the D380A mutation abolished calcium binding, as well as the calcium-induced increase in protein stability. This mutation is also a disease-causing variant in myocilin (see below for more discussion on myocilin mutations).
Conservation in Gliomedin from Different Species-To get a better idea of putative functionally important parts of the OLF domain structure, sequences of gliomedin from different species were compared with regard to sequence conservation. In general, the least conserved regions include the outermost -strands in the blades. On the other hand, in addition to the protein core, residues on the top surface are highly conserved (Fig. 4A) and hence most likely involved in gliomedin function in vivo. This conservation coincides with the positive electrostatic potential and the location of the clusters of aromatic residues on the surface. The bottom face of the OLF domain is poorly conserved.
In addition to gliomedins, conservation was further checked for different representative OLF domain sequences (Fig. 4, B and C). Based on this analysis, 20 fully conserved residues were identified on OLF domains, of which three are glycine and seven are aromatic. It is obvious that these conserved residues are important for the unique structural aspects of the OLF domain fold due to their localization inside the fold. The top face of the OLF domain has none of these conserved sites, which is an indication of different binding preferences of OLF family proteins for their respective ligands.
The OLF Domain Is a Monomer Folded into -Structure in
Solution-Gliomedin contains a collagen-like domain N-terminal to its OLF domain. In line with this, EM imaging has shown that gliomedin forms trimers through its collagen-like domain, such that the three OLF domains lie rather close to each other in space (8). To validate the crystal structure, which gives no indications of specific oligomerization, we characterized the OLF domain in solution.
Correct folding of the gliomedin OLF domain in solution was proven by CD spectroscopy, which shows clear hallmarks of -structure (Fig. 5A). We further characterized the domain in solution using SAXS, and it behaves like a monomer (Fig. 5B). The SAXS data indicate a radius of gyration (R g ) of 2.4 nm and a maximum dimension (D max ) of 9 nm. These values are larger than those calculated (53) from the crystal structure (R g ϭ 1.7 nm, D max ϭ 5.3 nm), which is most likely caused by the N terminus, which is not observed in the crystal structure. The molecular mass, based on the excluded volume of the ab initio model, is 36 kDa, which is very close to that expected for a monomer (32.4 kDa). In three-dimensional modeling, good fits were obtained when the N terminus was implicitly modeled (Fig. 5, B-D). Also the OLF domain of myocilin has been characterized as a monomer in vitro (54,55).
Structural modeling based on solution scattering data indicates that the construct used in our studies includes an extension to the OLF domain; this most likely represents the disordered N terminus of the protein (Fig. 5, C and D). Although the OLF domain is sufficient to induce Na v channel clustering, this process requires a pre-clustering of OLF domains (7,8). Considering the fact that the disordered N terminus of our construct essentially lies at the end of the collagen-like trimerization domain, based on the SAXS model, we calculated the expected dimensions of the C-terminal OLF domain assembly in full-length gliomedin. Assuming the collagen-like domain indeed forms a triple helix, the interaction (top) surfaces of the OLF domains cannot be farther apart from each other than 14 nm (Fig. 5E). Of course, it is possible that they actually are closer to one another in the trimeric molecule.
Comparison with Other OLF Family Members-Within the OLF family, by far the largest body of research has been carried out on myocilin, which is understandable based on its direct involvement in human disease. Dozens of myocilin mutations in glaucoma patients have been described, and the vast majority of these map to the OLF domain (14), highlighting the crucial role of this domain in the physiological function of myocilin. Using the gliomedin OLF domain crystal structure, a homology model was made for myocilin, which allowed mapping of the OLF domain glaucoma mutations onto the three-dimensional structure (Fig. 6). A number of observations on these mutations can be made. The first striking observation is that despite the -propeller fold and reports on misfolding and/or abnormal processing of myocilin mutants (56 -60), very few of the mutations are actually located in the -strands; most of them affect loops between the strands. Analysis of mutation location in three-dimensional space further indicates hot-spot regions with highly concentrated mutations; this concerns especially the top face of blades 2-4 and, to some extent, the bottom side of blades 1 and 5 (Fig. 6). The locations of some of the most severe mutations (14) are discussed below.
DISCUSSION
OLF domain proteins are known to play essential roles in development and cell differentiation in both vertebrates and invertebrates; however, their functions are only partially known at the moment, and their high-resolution structures have remained unknown. We have presented the first crystal structure of an OLF domain, which in gliomedin is crucial for correct formation of the nodes of Ranvier in myelinated peripheral nerves. At a more general level, the structure can be used both to understand OLF domain function in different OLF family proteins and to predict and analyze the molecular effects of human disease-causing mutations affecting OLF domains.
In vertebrates, gliomedin is important for the clustering of Na v channels at the nodes of Ranvier (61), a process that is a prerequisite for saltatory propagation of the action potential along axons. On the other hand, the gliomedin homolog unc-122 in Caenorhabditis elegans is involved in neuromuscular signaling (11). Interestingly, the Na v channel-clustering function of gliomedin can also be carried out by the isolated OLF domain (7,8) as long as the OLF domain has been pre-clustered. Hence, the OLF domain crystal structure presented here directly represents the functional unit of gliomedin in the development of the nodes of Ranvier in the nervous system. Whether the clustering of three OLF domains together by the collagen triple helix is enough for this function or if larger scale aggregates are required in vivo is currently unclear.
A high-resolution structural study including the collagenlike domain N-terminal to the OLF domain would clarify how the three OLF domains are arranged in a gliomedin trimer. EM imaging provided a low-resolution view of this arrangement (8), in which the three OLF domains are close to each other in space, but not bound to each other. This fits our SAXS data and the model of a trimeric arrangement of monomeric OLF domains (Fig. 5E). In general, oligomerization via neighboring domains or disulfide bridges is a general feature of OLF domains; for example, myocilin has a coiled-coil dimerization domain instead of the collagen-like domain of gliomedin. Oligomerization of OLF domains through these adjacent regions thus appears to be crucial for their biological activity.
Gliomedin is known to bind to fibronectin type III-like domains of neurofascin and NrCAM (9). These domains contain locally concentrated, negatively charged residues, which could provide a binding site for the positively charged top face of the gliomedin OLF domain. However, the precise binding mode will remain unclear until structural information becomes available for such protein-protein complexes.
Five-bladed -propeller structures are most often carbohydrate-binding proteins and/or enzymes (49). For gliomedin, apart from its interactions with neurofascin and NrCAM (5), few functional details are known at the molecular level. No enzymatic activity has been reported for OLF domains, and gliomedin is unlikely to harbor one.
Like many other myelin proteins, gliomedin is a target for autoantibodies in peripheral neuropathies (62)(63)(64), with implications for multifocal motor neuropathy, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barré syndrome. The exact autoimmune epitopes on gliomedin are not yet known. The crystal structure of the OLF domain will now enable the identification of such epitopes to understand the effects of autoimmune attack on the nodes of Ranvier and the myelin sheath.
Myocilin is by far the best characterized OLF domain-containing protein; however, no published data exist on its threedimensional structure. The gliomedin OLF domain structure enables us to understand the locations and effects of the plethora of human myocilin mutations causing glaucoma. In threedimensional space, the human myocilin mutations clearly cluster in defined regions and are largely absent from the secondary structure elements (Fig. 6). This is surprising, as glaucoma mutations in general have been assumed to cause misfolding or abnormal processing of myocilin (56 -60). The loops are apparently important for correct OLF domain folding.
A calcium-binding site was reported in myocilin, and it was suggested to involve Asp-380 (52). The glaucoma-causing mutation D380A has been intensively studied with regard to the loss of Ca 2ϩ -binding affinity upon mutation (52). In the gliomedin structure, we detected a bound cation (most likely sodium) that is coordinated by Asn-423, corresponding in sequence to Asp-380 in myocilin (Figs. 3E and 4C). Thus, Asp-380 in myocilin is most likely located in a similar cavity and possibly chelates a calcium ion that stabilizes the structure. In all other OLF domains apart from gliomedin, this Asp is conserved (Fig. 4C), and calcium binding may be a general feature of the OLF domain, with the exception of gliomedin. Interest- ingly, calcium is also required for the massive intercellular clotting involving the sea urchin OLF protein amassin (65,66); it is not known, however, if this reflects a requirement of calcium binding for a functional amassin OLF domain. The second metal-coordinating side chain of gliomedin (Asn-471) is conserved in myocilin as Asn-428. We believe that the sodium-binding site observed in the gliomedin crystal structure spatially corresponds to the calcium-binding site of myocilin. Taking into account that the extracellular Na ϩ concentration is expected to be higher than that of our buffers, it is likely that the site in gliomedin is also occupied by Na ϩ in vivo. Furthermore, it is interesting to note that gliomedin is localized close to the nodes of Ranvier, at which Na ϩ will rush into the axon when a nerve impulse passes, and that it is involved in the clustering of Na v channels. Previously, Ca 2ϩ binding by myocilin was reported to increase the stability of the OLF domain (52), and the binding of metals could clearly be a common mechanism to increase OLF domain stability. The functional relevance of metal binding by OLF domains clearly requires further study.
The residues corresponding to the myocilin mutations P370L and C433R (responsible for severe glaucoma phenotypes) are located on the loops between -strands 2d and 3a and between -strands 4a and 4b, respectively. These residues and the conformation of the loops are probably critical to forming and maintaining a stable -propeller structure. Furthermore, Cys-433 has been suggested to form a disulfide bridge with Cys-245 in myocilin (67); in gliomedin, such a disulfide is missing. Cys-433 is conserved in all other OLF domains apart from gliomedin. Considering the myocilin homology model, Cys-433 is in close contact with Gly-246, which is the N-terminal residue of the homology model. Thus, Cys-245 would be at a perfect position to form a disulfide bond with Cys-433, and this linkage can be expected to stabilize the assembly of the myocilin OLF domain.
Another human glaucoma mutation is Y437H, which also can induce disease when injected into mice (68). The corresponding residue in gliomedin (Tyr-480) lies in the middle of -strand 4b, surrounded by many hydrophobic residues. This indicates that Tyr-480 in gliomedin, which indeed is conserved in all OLF domains (Fig. 4C), may be a key residue to stabilize the -propeller structure.
Of the 20 glycine residues in the myocilin OLF domain, a total of eight have been listed as glaucoma mutation sites (14). Essentially all these residues are located in loops between -strands in the propeller blades. It is likely that their flexibility and their ability to support tight turns are of overall importance during the correct folding of the myocilin OLF domain. It is also logical to assume that glycine residues in the loops are involved in the folding of other OLF family members.
To conclude, we have presented the crystal structure of the OLF domain from gliomedin, which forms a twisted five-bladed -propeller. The high-resolution OLF domain structure is an important step toward understanding the functions and mechanisms of OLF domains. It is likely that the tertiary structures of other OLF domains are highly similar to that of gliomedin. Hence, the OLF domain is a unique -propeller, and OLF-containing proteins can be added to the list of -propeller proteins involved in human development and disease. | v3-fos-license |
2019-11-07T14:49:13.493Z | 2019-11-01T00:00:00.000 | 226301821 | {
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} | pes2o/s2orc | EFFECT OF PHOSPHOR FERTILIZER, MAGNETIC WATER AND HUMIC ACID ON THE GROWTH, PHOTOSYNTHESIS PIGMENTS AND OIL YIELD COMPONENTS OF Nigella sativa PLANT
Pots experiment was carried out in the greenhouse of Horticulture Department College of Agriculture Engineering Sciences/ University of Duhok to investigate the influence of three levels of P2O5 fertilizer (0, 260 and 520 mg ) per pot , humic acid at (0, 0.6 and 0.8 mg.L ) and magnetic water with three group, group (1) irrigated with tap water, group (2) irrigated with magnetized water remain in the container for 12 hours and group (3) irrigated with magnetized water remain in the container for 24 hours on the growth and oil yield of Black cumin Nigella sativa L. The experimental treatments consisted of five replications in Random Complete Block Design (RCBD). The results revealed that P2O5 fertilizer at 520 mg.pot -1 significantly increased all the studied characteristics. Humic acid at 0.6 and 0.8 mg.L had no significant effect on most of the studied characteristics except total chlorophyll and volatile oil. The group of plants that irrigated with magnetic water for 24 h caused significant increasing in all studied characteristics. Double and triple interactions among studies factors showed significant influence on all the studied characteristics as compared to untreated plants including (plant height, number of branches per plant, stem diameter, number of capsule/plant, dry weight, total chlorophyll, fixed oil percentage, volatile oil percentage and total carbohydrates percentage).
INTRODUCTION
One of the most important medicinal plants is Black cumin (Nigella sativa L). It is an annual herbaceous plant belonging to the family Ranunculacea which grows in west Asia and Mediterranean region, it is one of the most studied plants extensively due to its importance in phytochemical and pharmaceutical aspects (Riaz et al., 1996).The plant acquired its Pharmacological activity and its medical value in great splendor and occupied a special place for medicinal plants in the Islamic civilization through the ideological belief in its treatment of multiple diseases the holy prophet, Mohammed (peace be upon him ) that the plant is Heals all sickness except death. (Ul-Hassan Gilani.,et al 2004). It has been used as a herbal medicine for more than 2000 years. It is also used as a food additive and flavor in many countries; it was used as natural remedies traditionally from ancient time may be from Assyrian civilization (Kamil, 2003). Traditionally the black seed and its oil show effectively range of antibacterial, antitumor, anorexia, antiinflammatory, fever, hypoglycemic, skin disease, muscle relaxant, cough and immune stimulant activities (Hosseinzadeh et al., 2007;andBuriro andTayyab, 2007 andShabnam et al., 2012). Researcher mentioned that most of these effects attributed to the essential and volatile oils of N.sativa plant seeds (Nickavar et al., 2003 andGharby et al., 2014). It was also reported that high levels of nitrogen and الــرافـديــن زراعـــة مـجـلـــة ( المجلد 47 ( العدد ) 2 ) 2019 phosphorus fertilizers (280 N and 260 P2O5 kg ha -1 ) caused significant increase in fixed oil, volatile oil, protein and phosphorus content in the seeds of Nigella sativa plant (Hammo, 2008). Al-Rubaye, (2009) concluded that providing nigella plants with foliar fertilizers during active vegetative growth increases yield significantly when compared with soil applied fertilizers.
Recently the use of physical methods for plant growth stimulation is getting more popular due to the less harmful influence on the environment. Moreover, magnetized water for irrigation is recommended to save irrigation water (Aladjadjiyan, 2007).
Magnetic water is considered one of several physical factors effects on plant growth and development. Magnetic water fields are known to induce biochemical changes and could be used as a stimulator for growth related reactions (Hameda and El-Sayed, 2014).
Magnetic Water plays important role in the growth of any plant. on the quality of water used is such as enhances the growth, good quality and quantity and good yield of plants Fard et al., 2011).The effects of magnetic treatment of irrigation water and snow pea (Pisum sativum L var. macrocarpon) and Kabuli chickpea Cicer arietinum L on the seeds emergence, early growth and nutrient contents of seedlings were investigated under glasshouse conditions the results showed that magnetic water led to a significant increase in emergence rate index 42% for snow pea and 51% for chickpea), shoot dry weight (25% for snow pea and 20% for chickpea) and contents of N, K, Ca, Mg, S, Na, Zn, Fe and Mn in both seedling varieties compared to control seedlings Maheshwari and Grewal (2010). Also studied the changes in plants with seeds subjected to electric, magnetic or electromagnetic field. Effect of high voltage field on fruits like pineapple has also been studied Dastgheib et al.,(2013).
Effect of Magnetic water on chemical composition and nutrients on the Vicia faba, L. cv. Giza 3 plant the seeds of broad bean were irrigated with water passed through magnetic device is carried out by (Hameda and El Sayed, 2014), the results showed that magnetic water treatment enhanced the growth, chemical constituents such as chlorophyll a and b, carotenoids, total available carbohydrates, protein, total amino acids, total phenol, RNA,DNA,) and inorganic minerals (K + , Na + , Ca +2 and P +3 ) contents in all parts of broad bean plant Humic acid is one of the novel materials when it applied to nutrient solution enhanced the growth of transplants also increased the minerals structure (David et al, 1994). It has efficiency in the growth of plants and the availability of the elements, the using of humic acid even though with little concentration lead to increase permeability of the cellular membrane (Solange and Rezende 2008). Humic acid promoted plant growth and induced soil microorganisms like bacteria and fungi and provide carbon as a source for the organism's humic acid as well acting as chelating good martial, (Leonard, 2008). Humic compounds are the most abundant of the complex ligands, which are found in nature. In this regard, it is well known that the humic compounds improve soil structure, increase soil microbial population, increase soil cation exchange capacity and providing some specific materials for plant root indirectly by providing macro and micro minerals, leading to the increase of soil fertility (Rizal et al.,2010). Similar to these results Gad El-Hak et al., (2012) obtained that foliar application of pea plants with humic acid is very beneficial to the crop growth and yield.
This study was done to clarify the influence of phosphor fertilizers, magnetic water and humic acid on vegetative, reproductive growth, photosynthesis pigments and seeds oil yield of Nigella sativa plant.
MATERIALS AND METHODS
Pots experiment was conducted at 2013 in greenhouse of Horticulture department /college of Agriculture Engineering Sciences /University of Duhok to investigate the effect of some agricultural factors on vegetative growth and oil seed yield of Nigella sativa L. The seeds which were bought from the herbal market of Duhok city were cultivated in 15 th Oct 2013 sowed handily in with (15 cm) diameter were filled with soil that analyzed physically and chemically in the laboratory of soil department, as showed in (Table 1). The plants were fertilized with three level of phosphor (0, 260 and 260 mg) P2O5 per pot added to the plants after 3 to 4 pairs of leaves were appeared. Humic acid at three concentrations (0 and 0.6 and 0.8 mg.L -1 ) were sprayed after one month of planting by three times within ten days intervals. The plants were watered with magnetized water was prepared by passing through a pair of strong permanent magnets disk (0.32T) with opposite polarity created in the Physics department college of science in Duhok University without side effects. Which positioned outside polymer container in opposite pole configuration. Three groups were used, group (1) irrigated with tap water, group (2) irrigated with magnetized water remain in the container for 12 hours and group (3) irrigated with magnetized water remain in the container for 24 hours, to check if the water is magnetized or not a simple test was done cardboard was placed over a pair of strong permanent magnets then few drops of magnetized water were poured on cardboard exactly above the magnets. The water if properly magnetized stayed in a circular form whereas normal water failed to stay. Weeds were removed by hand and all agriculture practices were done as needed. Harvesting was done on 15 th June 2013 manually by pulling the dry plants out of the soil. Experimental measurements concluded some vegetative growth (high of plant, stem diameter, number of branch per plant, number of capsule per plant, and dry weight vegetative growth and some photosynthesis pigments, oil yield of seeds and total carbohydrate. Fixed oil percentage measurement according to (A,O,A,C,2000), volatile oil percentage measurement according to British pharmacopeia, (Ggrainger, 1968) which was mentioned by (Ranganna,1986), total carbohydrate measurement according to Herbert et al. (1971) using the Spectra photometer . All measured Characters were subjected to variance analysis. And all data obtained were analyzed and compared statistically at a significance level of 5%, using SAS program (SAS, 2007).
RESULTS AND DISCUSION
Vegetative Growth Trails. Height of plant. (cm).
The results in Table (2) indicated that P2O5 fertilizer at 520 mg.pot -1 significantly increased the height of plant (58.82 cm) compared to (45.18 cm) at 0 mg.pot -1 P2O5, also mentioned that the effect of magnetic water represented was significantly increased the height of plants to (59.03 cm) in compression with untreated plant was (53.98 cm) .while the height of plant had no significant effect when treated with humic acid the values were (56.86, 56.61 and 56.82 cm) respectively for 0,0.6,0.8 mg.L -1 concentration . The interaction between P2O5 at 520 mg.pot -1 and magnetic water with 24 h gave the highest plants (61.08 cm) as compared to untreated (51.40 cm). On the other hand there was no significant effect on the high of plants when p2o5 with H.A used. The interaction between P2O5 fertilizer and humic acid showed significant differs when P2O5 fertilizer at 520 mg.pot -1 with all concentrations of humic acid (58.89, 58.70 and 56.87 cm) respectively as compared to (53.88 cm) when treated with P2O5 0 mg.pot -1 fertilizer combined with 0.6 mg.L -1 .The same table showed that applying humic acid at all concentration interacted with magnetic water treatments effected on the height of plant significantly (59.07,59.03 and 58.98 cm) respectively comparing to the untreated plants (54.06,53.77 and 54.11 cm) . The triple interaction among p2o5 at 520 mg.pot -1 . magnetic water with 24 h and humic acid at 0,0.6 and 0.8 mg.L -1 obtained the best values included (61.10,61.12 and 61.03 cm) respectively when compared with 0 mg.pot -1 of P2O5 , magnetic water with humic acid 1 (51.56, 51.04, and 51.60 cm) respectively. Number of Branches (branch.Plant -1 ). Table (3) showed that P2O5 at 520 mg. pot -1 concentration gave the highest number of branches (7.88 branch. Plant -1 ) as compared to (7.18 branch. Plant -1 ) with treated plants. The highest number of branches was noticed when the plants irrigated with magnetic water for 24 h it was (7.89 branch. Plant -1 ) as compared to the plants that irrigated by tap water (7.08 branch.Plant -1 ). While there are no significant effect appeared when the treated with humic acid at all concentrations. The interaction between P2O5 at mg. pot -1 and magnetic water with 24 h gave the highest value of branches number (8.24 branche. Plant -1 ) as compared to (6.72 branch.Plant -1 ) with untreated plants. While there was significant effect on the number of branches when P2O5 at 520 mg. pot -1 with humic acid at all concentrations (7.85,7.89 and 7.90 branch.Plant -1 ) respectively as compared to (7.19, 7.14 and 7.20 branch. Plant -1 ) respectively for P2O5 at 0 mg. pot -1 interacted with all humic acid concentrations. The same table showed that adding humic acid at all concentrations interacted with magnetic water 24 h significantly differs (7.85, 7.86 and 7.98 branch. Plant -1 ) as compared to (7.06, 7.12 and 7.20 branch. Plant) at magnetic water 0 h interacted with humic acid at all concentrations. Regarding to the triple interaction among P2O5 at 520 mg. pot -1 , magnetic water 24 h and humic acid at all concentrations gave the significant value (8.17,8.22and 8.32 branch. plant 1 ) in comparison with lowest value obtained from the interaction of 0 mg. pot -1 of p2o5 ,magnetic water 0 h and humic acid at all concentrations (6.73.6.73 and 6.70 branch.plant -1 ) respectively. Table (3): Effect of phosphor fertilizer, magnetic water and humic acid on the branches number of Nigella sativus plant (branch.plant -1 ). *Means followed by the same letter for each factor and interaction do not differ significantly from each other's according to Duncan's Multiple range Test at 5% level.
The data in Table (4) recorded that significant effect appeared in stem diameter of the plants when P2O5 520 mg. pot -1 concentration compared to untreated plants and the values respectively were (5.24and 4.52mm).Irrigating the plants with magnetic water 24h was significantly differed with the plans irrigated with0 and 12h tap water and they were respectively (5.33, 4.48 mm and 4.93).The results also showed that all concentrations of humic acid do not have significant effect on steam diameter. Significant effect was observed with interaction between P2O5 520 mg. pot -1 and magnetic water 24 h gave (5.65mm) as compared to 0 mg. pot -1 (4.08 mm). The interaction between P2O5 520 mg. pot -1 fertilizer with all concentrations of humic acid were significantly effected (5.20,5.28 and 5.23 mm) respectively as compared to other treatments especially at P2O5 0 mg. pot -1 for all the concentrations of humic acid the values were (4.58,4.45 and 4.52 mm) respectively. The same direction was observed with interaction between humic acid at all concentration with magnetic water 24 h obtained significant effect on the stem diameter (5.32,5.33 and 5.34mm) compared to the untreated plants by magnetic water with all concentrations of humic acid (4.49,4.50 and 4.45 mm) respectively. The triple interaction among the triple interaction among P2O5 at 520 mg. pot -1 , (2) 2019 34.60 capsules.plant -1 ) respectively that obtained from 0 mg.potall concentrations of humic acid. The interaction between humic acid at all concentration with magnetic water with 24 h treatments have significant effect on the number of capsules (38.49,38.68 and 38.94 capsule. plant -1 ) as compared to the plants treated with 0 h magnetic water interacted with all concentrations of humic acid (33.50,33.51 and 33.43 capsule.plant -1 ) respectively. On the other hand the triple interaction among P2O5 at 520 mg. pot -1 , magnetic water 24 h and humic acid at 0.6 and 0.8 mg.L -1 obtained the highest values of number of capsules (40.57 and 40.86 capsule.plant -1 ) compared to (31.31,31.35 and 31.37capsule.plant -1 ) respectively 0 of P2O5 at 520 mg.pot -1 , all concentration of humic acid and 0 h magnetic water. Dry weight of plant (g). Table (6) showed that P2O5 fertilizer at 520 mg.pot -1 significantly increased the dry weight of plants (7.205 g) compared to 0 and 260 mg.pot -1 (5.760 g and 6.309g). The plants which treated with magnetic water 24h was significantly differed than that treated with 0 and 12 h magnetic water the values were (7.117, 5.746 and 6.411g) respectively. No significant differences appeared between all the concentrations of humic acid that used. The interaction between P2O5 fertilizer at 520 mg.pot -1 and magnetic water for 24 h gave a significant effect on dry weight Although all the humic acid concentrations of do not show significant effect compared to each other when it applied with P2O5 fertilizer at 520 mg.pot -1 on the dry weight of plant (7.132, 7.293 and 7.190 g) respectively, but they significantly differed with other treatments especially of 0 mg.pot -1 P2O5and the values were respectively for all concentration of humic acid (5.797, 5.850 and 5.634 g). The data obtained that interaction between at humic acid 0 and 0.6 mg.L -1 concentrations with magnetic water with 24 h treatment have significant effect on dry weight (7.190 and 7.214g) when compared to the lowest values obtained from0 h magnetic water with all concentrations of humic acid (5.712,5.791 and 5.740 g) respectively. Significant values of dry weight were obtained when (7.857, 8.004 and 7.957g) when 520 P2O5 mg.pot -1 ,24 h magnetic water with all concentrations of humic acid as compared to other treatments especially when0 P2O5 mg.pot -1 ,0 h magnetic water with all concentrations of humic acid (5.009, 5.138 and 5.073 g) respectively. while untreated plants showed the lowest value (16.14 µg.mg -1 ). Irrigated the plants with magnetic water 24h had significant effect on the chlorophyll a content (20.24 µg.mg -1 ) as compared to other treatments (16.14 and 17.81 µg.mg -1 ) respectively for 0 and 12 h magnetic water. Despite there was no significant difference between the both concentration of humic acid (0.6 and 0.8 mg.L -1 ), but were differs with 0 mg.L -1 and the values were (17.84, 18.11 and 18.24 µg.mg -1 ) respectively. The interaction between P 2 O 5 at 520 mg.pot -1 and magnetic water with 24 h obtained significant effect on content of chlorophyll a (22. 39 µg.mg -1 ) as compared to untreated plants (14.21 µg.mg -1 ), while the interaction between P 2 O 5 fertilizer at 520 mg.pot -1 and humic acid at both 0.6 and 0.8 mg.L -1 the values were (20.24 and 20.45 µg.mg -1 ) they were significantly differs with other treatments especially P 2 O 5 fertilizer at 0 mg.pot -1 with all concentrations of humic acid (15.98,16.15 and 16.27 µg.mg -1 ). Applying humic acid at 0.8 mg.L -1 interacted with magnetic water for 24h showed significant effect (20.72 µg.mg -1 ) compared to 0 h magnetic water with all concentrations of humic acid (16.08,16.18 and 16.15 µg.mg -1 ).The triple interaction among P 2 O 5 at 520 mg.pot -1 , magnetic water for 24 h and humic acid at (0.6 and 0.8 mg.L -1 ) obtained significant values of chlorophyll a content (22.34 and 22.94 µg.mg -1 ) respectively compared to all treatments especially 0 P 2 O 5 mg.pot -1 ,0h magnetic water and all concentrations of humic acid ( 14.22,14.23 and 14.19 µg.mg -1 ) respectively. Chlorophyll b content (µg.mg -1 ).
The results in Table (8) showed that the highest value of chlorophyll b contents in the plants was conducted when p 2 o 5 fertilizer at 520 mg.pot -1 was used (6.99 µg.mg -1 ) while untreated plants gave the lowest value(5.25 µg.mg 1 ). Treating the plants with magnetic water for 24h had significant effect on the chlorophyll b contents compared to the plants irrigated with tap water (6.91 and 5.18 µg.mg -1 ) respectively. All the concentrations of humic acid had no significant effect on chlorophyll b contents. The interaction treatment between P 2 O 5 at 520 mg.pot -1 and with magnetic water for 24h gave significant effect on the content of chlorophyll b content (7.58 µg.mg -1 ) as compared to untreated plants (5.25 µg.mg -1 ). Applying P 2 O 5 at 520 mg.pot -1 with and humic acid at all concentrations had significant effect on chlorophyll b content the values were (6.98 ,6.95 and 7.06 µg.mg -1 ) respectively as compared with all the treatments especially the lowest value obtained from. P 2 O 5 at 520 mg.pot -1 interacted with all concentrations of humic acid the values were respectively (5.11, 5.25 and 5.40 µg.mg -1 ). The same direction was observed when the plants were treated with humic acid at (0.0, 0.6 and 0.8 mg.L -1 ) interacted with magnetic water for 24h had significant effects (6.80,6.99 and 6.95 µg.mg -1 ) respectively as compared to untreated plants (5.12,5.20 and 5.22 µg.mg -1 ) in chlorophyll b contents. The triple interaction of P 2 O 5 at 520 mg.pot 1 , magnetic water for 24h and humic acid at all concentrations showed significant effect
61
of chlorophyll b content obtained (7.77,7.87 and 7.92 µg.mg 1 ) as compared to P 2 O 5 at 0 mg.pot -1 , magnetic water for 0h with all concentrations of humic acid ( 4.22,4.39 and 4.53 µg.mg -1 ) respectively. Total chlorophyll content (µg.mg -1 ) The results in Table (9) pointed out that P 2 O 5 feltrizer at 520 mg.pot -1 gave the significant value of total chlorophyll (28.04 µg.mg -1 ) as compared to (21.79 and 24.61 µg.mg -1 ) were obtained with 0 and 260 mg.pot -1 P 2 O 5 . Irrigating the plants with magnetic water for 24h was significantly differed with the plans that irrigated with tap water and magnetic water for 12h they were respectively (28.04, 21.70 and 24.38 µg.mg -1 ). Applying humic acid at (0.8 mg.L -1 ) had significant effect on the content of total chlorophyll in plant (24.98 µg.mg -1 ) as compared to (24.74 and 24.73 µg.mg -1 ) respectively for (0 and 0.6 mg.L -1) . The interaction between P 2 O 5 feltrizer at 520 mg.pot -1 and magnetic water with 24 h showed significant effect on content of total chlorophyll content (31. 59 µg.mg -1 ) as compared to other treatments especially untreated plants ( volatile oil percentage (%).
The results in Table (10) indicated that p2o5 fertilizer at P 2 O 5 feltrizer at 520 mg.pot -1 significantly increased the volatile oil content (0.823%) as compared to 0 and 260 mg.pot -1 (0.795 and 0.819 %), also the same Table mentioned that (0.767, 0.766 and 0.769%) respectively for P 2 O 5 feltrizer at 0 mg.pot -1 , magnetic water with 0 h and humic acid at all concentrations. Table ( 11) conducted that treating the plants Table (12) showed that fertilizer at P 2 O 5 feltrizer at 520 mg.pot -1 significantly increased the fixed oil content (38.45%) as compared to 0 and 260 mg.pot -1 (35.74 and 36.15 %), Irrigating the plants with magnetic water for 24h to was significantly differed with the plans that irrigated with tap water and magnetic water for 12 h the values were respectively (37.06 %, 35. 04 and 37.42 %).Humic acid had no significant effect on the content of fixed oil in plant at all concentrations. The interaction between P 2 O 5 feltrizer at 520 mg.pot -1 and magnetic water for 24 h showed significant effect on content of fixed oil (39. 58%) as compared to other treatments especially untreated plants (33.99%). There was significant difference noticed when P 2 O 5 feltrizer at 520 mg.pot -1 used with humic acid at all concentrations and the values were (37.87, 38.01 and 37.85%) respectively when compared with other treatments especially when p 2 o 5 fertilizer at 0 mg.pot -1 interacted with humic acid at all concentrations (35.12,35.58 and 35.52%). respectively When the plants were treated with humic acid at all concentrations interacted with magnetic water for 24 h showed significant different with other treatments (37.29, 36.85 and 37. 05%) respectively but the lowest values were untreated plants (35.31, 34.95 and 34.84%) in fixed oil contents. The triple interaction of P 2 O 5 feltrizer at 520 mg.pot -1 , magnetic water for 24 h and humic acid at all concentrations and P 2 O 5 feltrizer at 520 mg.pot -1 , magnetic water for 12 h and humic acid at all concentrations showed the significant effect on fixed oil content the values were (39.56, 39.57 and 39.6 %) and (38.90, 39.26 Fixed oil percentage (%) This significant increase in the characteristics of research is consistent with several researchers. (Singh et al., 1999) confirmed this finding. Garg and Malhotra (2008) mentioned that he results that height of plant, number of branches, number of leaves per plant stem diameter and seed yields increased with increasing of P 2 o 5 fertilization of Nigella sativa plants. Rana (2012) also ensured these findings. This increasing may be explained due to that phosphorus known to help developing broader root system and thus helping the plants to extract water and nutrients from more depth. This, in turn, could enhance the plants to produce more assimilates which was reflected in high biomass (Gobarah et al., 2006). Researchers have been increasingly interested in using magnetic technology in agricultural fields after the positive effects of this technique on the growth and flowering of plants. This can be clear up that magnetized water has a positive effect on the characteristics of flowers, bulbs and seeds. This may be due to the physical and chemical changes of magnetically treated water, which resulted in the easy absorption of water and soluble elements by the root mass as well as improved vegetative growth characteristics resulting in an increase in the amount of photosynthesis (Al-Mu'adidi, 2006;Nasher, 2008.;Kuntyastuti and Suryantini, 2014.). The same results also obtained by Amin (2009) found that irrigating Iris bulbs with magnetically treated water resulted in an increase in stem diameter, chlorophyll and dry weight Al-Jubouri (2006) approved that by improving the flowering characters of Tagets erecta L when irrigated by magnetized water. Deshpande (2014) conferred that using the magnetic water in place of normal tap water can be seen as a promising technique for rapid and healthy growth of plants. Regarding to the humic acid it works indirectly on the speed of absorption and transfer of the rest of the elements by entering the formation of chlorophyll pigments, thus increasing the carbonation process and building the proteins of great importance in stimulating plant growth and reaching a good nutritional state, which increased the efficiency of the plant to absorb | v3-fos-license |
2021-07-26T00:05:38.303Z | 2021-06-14T00:00:00.000 | 236274739 | {
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} | pes2o/s2orc | Synthesis and antileishmanial activity of styrylquinoline-type compounds: in vitro and in vivo studies
In this research, four styrylquinolines were synthesized and evaluated in vitro and in vivo antileishmanial activity. The following compounds were obtained: 2-[(E)-2-(2chlorophenyl)ethenyl]quinoline-8-ol (3a), 4-bromo-2-[(E)-2(8-hydroxyquinoline-2-yl)ethenyl]phenyl-acetate (3b), 4-[(E)2-(8-hydroxyquinoline -2-yl)ethenyl]-2,6dimethoxyphenyl acetate (3c), 2-{(E)-2-[2-(acetyloxy)-5-nitrophenyl]ethenyl} quinoline-8-yl-acetate (3d). Of the four compounds, evaluated in vitro, 3b and 3c were active against intracellular amastigotes at median effective concentrations (EC50) of 2.8 and 3.14 μg mL -1 and exhibited toxicity over human U-937 macrophages at median lethal concentrations (LC50) of 5.5 and 3.4 μg mL -1, which showed selectivity indices (SI) of 1.96 and 1.08. The results of in vivo studies of therapeutic response in hamsters with experimental CL caused by L. (V) panamensis indicated that with topical creams at 2% of compounds 3b and 3c produced a clinical improvement of 4/5 (80%) of hamsters with 3b, and with 3c a complete cure of 1/5 (20%) and a clinical improvement of 2/5 (40%) of the hamsters, while the in vivo control drug (MA-il) produced clinical improvement in 3/5 (60%) of hamsters.
INTRODUCTION
The term leishmaniasis describes a set of diseases caused by protozoans from the Leishmania genus. These are manifested in three different clinical forms: cutaneous, mucocutaneous, and visceral in function of the carrier's immune response and of the parasite species. The leishmaniasis is a zoonosis transmitted by a female phlebotomine vector in which mammals are the most frequent host reservoirs. Visceral leishmaniasis is the most severe clinical form of the disease with mortality close to 100% if untreated; the cutaneous and mucocutaneous forms are not as severe, but can lead to disfiguring lesions (WHO,2019). Currently, treatments are carried out based on medications, like Amphotericin B (AmB), Sitamaquine, Meglumine antimoniate (MA), Miltefosine, Paromomycin, and Pentamidine; as fundamental part of the structure of these active principals, we can highlight the presence of quinolinic compounds, which in addition to demonstrating their activity against leishmaniasis (Richard and Werbovetz, 2010) have also exhibited other types of activities, such as anti-trypanosomal, anti-tumor, bactericide, anti-mycobacterial, fungicide, anti-asthmatic, anti-plasmodial and anti-VIH (Franck et al., 2004;García et al., 2010;Musiol et al., 2010;Delattin et al., 2012;Marella et al., 2013;Upadhayaya et al., 2013;Afzal et al., 2015;Kieffer et al., 2015). Today, an important number of researches on antileishmanial substances have to do with isolating -in the case of natural products -or obtaining -in the case of organic synthesis -of molecules with quinolinic or isoquinolinic nuclei that can serve as therapeutic alternatives in treating this disease. Among the most promising compounds, in in vitro and in vivo assays, the following are highlighted: 8-aminoquinoline (Sitamaquine), styrylquinolinetype compounds and derivatives reduced from them, 2-arylquinolines, quinoline dimers and quinolinetriclosan hybrids, and triazine indol-quinolines, all with antileishmanial activity (Mesa et al., 2008;Pinheiro et al., 2010;Arango et al., 2011;Sánchez et al., 2014;Sharma et al., 2014;Singh et al., 2014).
The previous aspects suggest the importance and applicability of quinolinic nuclei; therefore, this research synthesized styrylquinoline-type molecules determining their antileishmanial activity and their cytotoxicity through in vitro and in vivo trials to test their effectiveness in counteracting the pathological effects and, thus, contribute to development of new treatments and relieving the disease burden, which poses a global public health problem. In this context, the styrylquinolines were obtained using the Perkin-type condensation reaction and their structure was corroborated using the nuclear magnetic resonance spectroscopic technique in one and two dimensions. These compounds were evaluated for antileishmanial activity in vitro against intracellular amastigotes and in vivo in murines infected with the parasite and, in turn, their toxicity was monitored by measuring serum values of ALT (alanine amino-transferase), BUN (Blood urea nitrogen) and creatinine. showing that two of the synthesized compounds showed promising antileishmanial activity, sufficient to be considered LEAD compounds. Based on its potential, you should continue to optimize your formulation and therapeutic dose.
General procedure to synthesize styrylquinolines
The styrylquinolines were obtained by means of a Perkin-type condensation reaction using high purity reagents from 8-hydroxyquinoline (1) with 6.00 mL of acetic anhydride (Ac 2 O) adding the corresponding aromatic aldehyde: 2-chlorobenzaldehyde (2a), 5-bromo-2-hydroxybenzaldehyde (2b), 4-hydroxy-3,5-dimethoxybenzaldehyde (2c) or 2-hydroxy-5nitrobenzaldehyde (2d) in stoichiometric excess with respect to 1 ( Figure 2) and heated under reflux with reflux with constant stirring and the progress of the reaction was monitored by thin layer chromatography (TLC) to verify the consumption of precursor and the formation of the product and the time used for the syntheses was between 8 and 14 h. Upon completing the reaction, the mixture was left to cool at room temperature and sufficient amount of a saturated solution of NaHCO 3 was added until hydrolyzing the remnant Ac 2 O. Extraction was conducted with petroleum benzine (PB) and ethyl acetate (EtOAc) in 1 : 2 ratio; the organic phase was dried with anhydrous Na 2 SO 4 , filtered, and concentrated at reduced pressure. Finally, the raw product was purified by column chromatography using PB : EtOAc as the eluent with increasing polarity gradient and silica gel 60 F 254 (Merck) as the stationary phase. All compounds obtained were synthesized by following the methodology described by (García et al., 2010;Santafé et al., 2016) the yields were >60% (Sánchez et al., 2014).
Cells and culture conditions
The U-937 promonocytes (CRL-1593.2™) (American Type Culture Collection) were cultured in complete medium containing RPMI -1640 (Sigma) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies) and 1% antibiotics (100 U mL -1 of penicillin and 0.1 mg mL -1 of streptomycin) (Sigma). The cells were under standard culture conditions at 37 °C, 5% CO 2 with medium change every 72 h until use.
Leishmania culture conditions L. (V) panamensis (MHOM/CO/87/UA140-EGFP) was used for in vitro and in vivo assays (Pulido et al., 2012). Virulence was maintained through successive passages in golden hamsters (Mesocricetus auratus). Parasites were recovered from hamsters after aspirates of the skin lesions and culturing in biphasic Novy-MacNeal-Nicholle medium at 26 °C to obtain promastigotes. Parasites in stationary phase of growth (6-day in culture) were used to infect the U-937 cells and hamsters.
In vitro cytotoxicity
The U-937 cells in exponential growth phase were adjusted to a concentration of 1 x 10 6 cells mL -1 of complete RPMI-1640 medium. One hundred µL of cell suspension were dispensed in each well of a 96-well cell culture plate and then, 100 µL of complete RPMI-1640 medium with the corresponding concentrations of each styrylquinoline compound were added to each well. AmB, was included as cytotoxicity control. For all compounds and AmB were evaluated, six double serial dilutions from 200 to 3.125 µg mL -1 . Simultaneously, cells cultured in the absence of the compounds and kept under the same conditions were used as viability control (negative control) (Robledo et al., 1999;Weniger et al., 2001). After 72 h of incubation, cell mortality was determined by using the MTT method and reading the optical densities in a spectrophotometer at 570 nm (Sereno and Lemesre, 1997). The optical densities obtained were used to calculate the percentage of mortality. Tests were run at least twice with three replicates for each concentration evaluated.
In vitro antileishmanial activity on amastigotes of L. (V) panamensis
The U-937 cells at a concentration of 300,000 cells mL -1 kept in complete RPMI 1640 medium with 0.1 µg mL -1 of phorbol myristate acetate were seeded on 24-well cell culture plates and incubated at 37 °C and 5% CO 2 . Cells were infected with promastigotes of L. (V) panamensis-EGFP in stationary growth phase (MOI: 20 parasites per cell), and the plates were incubated for 3 h at 34 °C and 5% CO 2 . The medium was removed, the cells were washed with PBS, and fresh complete medium was added. The plates were incubated for another 24 h at 34 °C and 5% CO 2. The medium was replaced by fresh medium containing each compound at 100, 50, 25, 12.5, 6.25, 3.125 or 1.56 µg mL -1 and the plates were incubated at 34 °C and 5% CO 2 . After 72 h of incubation, the effect of the compounds was determined on the viability of the intracellular amastigotes through flow cytometry with readings at 488 nm of excitation and 525 nm emission with an Argon laser capturing the percentage of positive events for green fluorescence (Sereno and Lemesre, 1997;Varela et al., 2009). AmB was used as a positive assay control and infected but non exposed cells were used as the control of infection and a negative assay control. The tests were conducted at least twice with a minimum of three replicates per concentration evaluated. The number of positive GFP events and the median fluorescence intensity (MFI) corresponded to the amount of live parasites (parasitic load). This number was used to calculate the percentage of inhibition for each concentration evaluated with respect to the untreated control (Sereno and Lemesre, 1997;Robledo et al., 1999).
Topic formulations of 3b and 3c
Creams were prepared based on a mixture of glycerin, vaseline, emulgade® 1000, Cetyl alcohol and surfactant and then, the pure compounds 3b or 3c were subsequently incorporated into the base cream at a concentration of 2%.
In vivo therapeutic response of a cream formulation containing 2% 3b and 2% 3c
Male and female, 6-weeks old hamsters were anesthetized (ketamine 40 mg/kg and xylazine 5 mg/kg) and then inoculated in the dorsal skin with 5 × 10 8 promastigotes of L. (V) panamensis-EGFP in 100 μL PBS as described elsewhere . After a cutaneous ulcer was developed hamsters were distributed in three experimental groups (n = 5 each): two groups of hamsters were treated via topic with 40 mg of 2% 3b or 2% 3c cream, administered every day for 28 days. The third group of hamsters was treated with 200 mg of MA intralesional, administered two times a week for 4 weeks (28 days). The time points of evaluation were: before treatment (treatment day 0), end of treatment (treatment day 28), and post treatment days (PTD) 30, 60, and 90. Animal welfare was supervised daily during the study. The effectiveness of each treatment was determined by comparing the lesion sizes after treatment respect to before treatment. Clinical outcomes were recorded at the end of study as cure (healing of 100% of the area and complete disappearance of the lesion); improvement (reducing the size of the lesion in >30% of the area) or failure (increasing the size of the lesion). Toxicity of cream preparations was determined according to changes in the body weight during the study and levels of alanine amino-transferase (ALT), blood urea nitrogen (BUN), and creatinine in serum at TD0 and PTD8, measured using commercial kits (LabCAM) .
Statistical analysis
Cytotoxicity was determined according to the percentages of viability and mortality registered for each compound, including AmB and culture medium. Percentage of viability was calculated based on the ratio between O.D of exposed cells vs. control cells using the equation: % Mortality = 100-[(O.D exposed cells / O.D non-exposed cells) × 100].
The mortality % were used to calculate the LC 50 that corresponds to the concentration necessary to eliminate 50% of cells using Prism 6.0 (Graph pad Prism, San Diego, CA, USA).
On the other hand, the antileishmanial activity was determined according to the percentage of infected cells that correspond to the positive events in a dot plot analysis with the green fluorescence (parasites) in y-axis and the Forward Scatter (FSC) in x-axis. Then, the amount of parasites in infected cells was determined according to the MFI of those fluorescent parasites. Lastly, the parasite inhibition percentage was calculated by the ratio between the MFI of exposed parasites vs. non exposed infected cells using the equation: % inhibition of infection = 100-[(MFI exposed infected cells / MFI non-exposed infected cells) x 100].
Results of antileishmanial activity were expressed as EC 50 determined using Prism 6.0 (Graph pad Prism, San Diego, CA, USA). In turn, the selectivity index (SI), was calculated by the ratio obtained between the activity observed in cells (LC 50 ) and the activity obtained in parasites (EC 50 ), using the formula: SI = LC 50 /EC 50 .
Values are expressed as mean ± SD. In order to compare the different compounds in terms of their biological activity, both cytotoxicity and antileishmanial activity were classified as high, moderate or low, according to the LC 50 and EC 50 values, respectively. In this way, the cytotoxicity was considered high when the LC 50 was < 100 μg; moderate when the LC 50 was between 100 and 200 μg and low when the LC 50 was > 200 μg. On the other hand, the antileishmanial activity was considered high when the EC 50 was < 25 μg, moderate when the EC 50 was between 25 and 50 μg and low when the EC 50 was > 50 μg 8 (Murillo et al., 2019). Data from in vivo study were analyzed by a two-way ANOVA using Prism 6.0 (Graph pad Prism, San Diego, CA, USA). Differences were considered significant if p < 0.05 .
Ethical issues
The procedures involving laboratory materials were manipulated and discarded following the Institutional Biosafety Manual. The procedures involving animals were approved by the Ethical Committee for the experimentation in animals of the University of Antioquia (Act No. 91 of September 25, 2014).
In vitro cytotoxicity and Antileishmanial activity of Styrylquinolines
Cytotoxicity was evaluated against human U-937 cells with for each compound the results were expressed as the median lethal concentration (LC 50 ) ( Table 1). According the defined classification, all compounds were cytotoxic with LC 50 values lower than 11 µg mL -1 (Table 1). Compound 3a had the highest toxicity (LC 50 = 1.7 μg mL -1 ) and compound 3d had the lowest cytotoxicity (LC 50 = 10.5 μg mL -1 ).
In vitro antileishmanial activity of the compounds was evaluated against amastigotes of L. (V) panamensis obtained after infection of U-937 cells (Table 1). Only compounds 3b and 3c showed very high activity against L. (V) panamensis with EC 50 values of 2.80 and 3.14 μg mL -1 respectively. As expected, AmB was active also at an EC 50 of 0.05 μg mL -1 . All compounds exhibited a lower selectivity index than the control drug (AmB), with the best being 3b and 3c with values 1.96 and 1.08 respectively.
In vivo therapeutic response and toxicity of 3b and 3c topic formulation
Ninety days after the end of treatment (PTD90), corresponding to the end of the study, the treatment of hamsters with the 2% cream formulation of 3c during 28 days allowed healing (cure complete and reepithelialization of damaged skin) of 1/5 of the hamsters and clinical improvement of 2/5 of the hamsters; the remainder hamsters failed to this treatment (Figure 3b). On the other hand, treatment with 2% 3b cream allowed clinical improvement of 4/5 hamsters with reduction in the size of the lesions varying between 13.2% and 34.1%, while remaining two hamsters failed to show improvements with this treatment (Figure 3a). In the group of hamsters treated with intralesional meglumine antimoniate (MA-il) 3/5 (60%) hamsters showed a reduction in lesion size from 19.1% to 64.2% and two hamsters failed on this treatment (Figure 3c). Figure 3 shows the increase or decrease in the sizes of lesion during the study. y axis corresponds to area of lesion in mm 2 and x axis corresponds to days of follow up. *TD0 (Treatment-day 0); TD28 (Treatment-day 28, the last day of treatment); PTD30 (Post-treatment -day 30); PTD60 (Post-treatment -day 60); PTD90 (Post-treatment -day 90).
Treatment with 2% cream of 3b in 5 murines of different sex (Figure 3a), treatment with 2% cream of 3c in 5 murines of different sex (Figure 3b) and the treatment with MA-il in 5 murines of different sex (Figure 3c).
A decrease of the size after the treatment corresponds to a positive therapeutic response, while an increase is indicative of negative response to the treatment. A positive therapeutic response is associated to clinical improvement and cure while a negative response is associated to a failure.
The appearance of the lesions of one hamster with cure with 3c and another with clinical improvement after the treatment with 3b compound are illustrated in Figure 4a and 4b respectively.
In general, hamsters treated with 3b, 3c or MA-il gained weight during the study ranging from 0.6 % to 16.5% in hamsters treated with 3b treatment, from 5.6% to 21.7% in hamsters treated with 3c and from 4.6% to 18.8 in hamsters treated with MA-il ( Figure 5). This difference was statistically significant (p < 0.05). Figure 5 shows the evolution of the body weight of hamsters during the study measured before the treatment, at the end of the treatment and every 15 days after completing the treatment. y axis corresponds to the weight in grams and x axis corresponds to days of follow up. * p < 0.05 MA-il vs. other groups. TD0 (Treatment-day 0), TD28 (Treatmentday 28, end of treatment); PTD15 (Post-treatment -day 15); PTD30 (Post-treatment -day 30); PTD45 (Post-treatment -day 45); PTD60 (Post-treatment -day 60); PTD75 (Posttreatment -day 75) and PTD90 (Post-treatment -day 90).
Serum levels of ALT, BUN and creatinine were within the range of normal values for all animals both before (TD0) and after treatment (PTD8) (Figure 6).
The results are presented as the average ± standard error. The following biomarkers were measured in hamster serum: a alanine amino-transferase (ALT), b blood urea nitrogen (BUN), and c creatinine. p > 0.05 One-way ANOVA with Bonferroni's correction. The normal range of measurements is indicated by a gray zone. The alanine amino-transferase (ALT) levels measured at the beginning of the treatment (TD0) and after 8 days (TD8) showed slight increases with the treatment with 3c and the control drug (MA-il), however they did not exceed the critical levels (gray area) (Figure 6a). The measurement of blood urea nitrogen (BUN) at the beginning of the treatment (TD0) and after 8 days (TD8) did not show important variations with the treatment with 3b and 3c and neither with the control medicine (MA-il), thus presenting acceptable levels within the allowed (gray area) ( Figure 6b).The creatinine measured in the hamsters did not show significant variations with the treatment with 3c and with the control drug (MA-il), however it was slightly increased with the treatment with 3b, but this did not represent levels outside the normal values (gray area) (Figure 6c) .
Compound 3a due to its high in vitro cytotoxicity and a selectivity index below unity, in vivo antileishmanial activity was not evaluated. Compound 3d showed the lowest antileishmanial activity of the evaluated compounds, for this reason it was not evaluated in the in vivo phase, however, due to its low toxicity, it could be considered a good molecular basis to be synthetically modified and improve its effectiveness against the parasite and similarly, for 3a to reduce its cytotoxicity.
Although compounds 3b and 3c showed a lower value of EC 50 than AmB, both compounds are still highly active at levels below 4 µg mL -1 . On the other hand, AmB was used as an internal control of cytotoxicity and leishmanicidal activity to guarantee the correct functioning of the in vitro assay. Rather than find molecules with lower levels of activity than AmB, the purpose of projects involved in the search of novel therapeutic alternatives is find molecules that have activity in the accepted ranges (p.e < 20 µg mL -1 , according our scale). Moreover, it has been shown that all different Leishmania strains and species have different sensitivity to each one of the antileishmanial drugs (Palacio et al., 2017). Similarly, toxicity of compounds 3b and 3c was higher than that showed by | v3-fos-license |
2020-03-29T11:06:22.654Z | 2019-03-15T00:00:00.000 | 214709033 | {
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} | pes2o/s2orc | THE SPECIFICITY OF LEGAL SUBJECTS IN POLISH AND ITALIAN ENVIRONMENTAL PROTECTION LAW
It should be noted that both the Polish lawmaker and the Italian lawmaker are aware of the specificity of legal subjects in the public domain. They can see that public administration bodies and public institutions cannot be described in normative terms, definitions and expressions used in legal language. New criteria for their separate treatment are needed.In addition, it is justified to treat them separately from all public administration bodies and public subjects. This only serves as proof of the rank and significance of such authorities.
For some time changes have been observed in environmental protection law that may be referred to as changes in the context of being a legal subject. It refers to creating specific entities, characteristic of environmental law only, or established solely for the purposes of tasks or actions related to environmental protection law. However, the changes inspire deeper considerations and reflections concerning legal subjects in environmental protection law and their specificity. This is due to the fact that interesting phenomena occur in environmental protection law that must be analysed in detail. This paper is an attempt at such an analysis.
At the beginning it must be indicated that the analysis neglects issues related to legal subjects in environmental protection law such as animals being legal subjects or the environment itself being a legal subject. Firstly, these issues have already been reviewed in literature 1 . Secondly, this aspect does not apply strictly to juridical elements. Legal subjects are described in normative terms and until the lawmaker chooses not to clearly regard animals or the environment as legal subjects, the problem remains only at the axiological or ethical level. The ideas supporting the aforementioned must be treated only as de lege ferenda postulates although vivid academic discussion is underway 2 . These are also disputes on the verge of jurisprudence and philosophy. If the dispute regarding treatment of animals and perhaps the environment as legal subjects is settled, it is likely the Polish lawmaker will respond by implementing relevant regulations. De lege lata the lawmaker remains uncertain about further direction of development of non-legal sciences.
However, given the rule that being a legal subject must have clear grounds, such silence should be interpreted as a lack of consent.
The starting point for further considerations is an assumption that there is no doubt that entities whose specificity will be investigated are legal subjects. Thus, the focus will be not on analysing this normative characteristic but on identifying the specificity of such legal subjects determined by the requirements of environmental protection law. However, such legal subjects are both entities having impact on the environment as well as authorities of public administration.
The analysis of the specificity of being a legal subject in Polish environmental protection law must start with the notion of user of the environment. The legal definition of this term is included in Art. 3 par. 20 of the 1 J. Białocerkiewicz, "Legal status of animals. Animal rights or legal protection of animals",Toruń 2005; B. Rakoczy, "The State Treasury and local government units as plaintiffs in matters related to environmental protection", Gdańsk Legal Studies from 2009, 207 -215. , as well as agricultural producers in the scope of crops, livestock breeding, horticulture, vegetable growing, forestry and inland fisheries, b) an organisational unit which is not an economic operator within the meaning of the Act of 6 March 2018 -Law on Economic Operators, c) a natural person who is not the entity referred to in a) who uses the environment in the scope where its use requires a permit". This definition is not expected to introduce a new category of legal subject. None of the letters creates a new type of legal subject. It is aptly noted in literature that this term is used in the Act multiple times to denote the addressee of its provisions with regard to obligations following from the Act 4 . An identical view was presented independently by M. Bar 5 .
The term 'user of the environment' was used in the Act not in order to create a new type of legal subject but to denote the addressee of the norms of the Act, in particular with regard to obligations. These, in turn, are generally imposed on the subjects mentioned in the above definition.
A user of the environment is any economic operator regardless of any criteria related to the type of economic activity, the scale and scope of environmental impact or environmental hazard. In this case the only decisive formal criterion is the status of an economic operator. There is no doubt that the lawmaker makes a cross-reference to the concepts laid out in the definition of an economic operator presented in the Law on Economic Operators, which is proved by the fact that entities running specific agricultural activity are also included in the definition of a user of the environment.
The formal criterion also underlies the inclusion of organisational units under letter b). Also in this case the lawmaker did not adopt any other eligibility criterion. The status of an organisational unit is sufficient. Lege non distinguente it is not important whether or not the specific organisational unit is a legal person. Thus, this category of notions is inclusive both of legal persons and entities without legal personality but having legal capacity (e.g. housing cooperatives) as well as organisational units without legal personality and legal capacity. This refers to every organisational unit, irrespective of the scope of its activity and tasks, except units with the status of an economic operator.
The most varied one is the third category of entities classed by the lawmaker as users of the environment. This category includes natural persons. However, in this case the formal criterion fails because the status of a natural person alone does not provide sufficient grounds for considering such a person a user of the environment. The legislator introduces two prerequisites -one positive and one negative.
A negative prerequisite is that the natural person referred to in c) cannot be an economic operator. This is fully justified since if such a person were an economic operator, he/she would be considered a user of the environment regardless of his/her actual impact on the environment and the scope of such impact. It would not be advisable to include such a category of entities separately under letter c).
Indeed, the lawmaker performs a dichotomous division of natural persons from the point of view of their eligibility as users of the environment. One of the categories is natural persons running economic activity and the other is natural persons who are not economic operators. In the first group of natural persons their affiliation with the category of subjects classed as users of the environment is due to the status of the economic operator alone. The second group of natural persons must necessarily satisfy the positive prerequisite.
In c) the lawmaker indicates that a natural person who is not an economic operator is a user of the environment only if he/she uses the environment in the scope where its use requires a permit. A significant element of this definition is that the permit as a sine qua non condition is not linked with the natural person but with the use of the environment. Thus, this provision does not take into consideration if the specific natural person is permitted to use the environment but if the specific use of the environment requires a permit. A natural person who is not an economic operator will be a user of the environment if such use requires a permit irrespective of whether the specific natural person obtains such a permit.
The notion of the user of the environment was also used in the Act of 13 April 2007 on Preventing and Remedying Environmental Damage 6 . Apparently, the problem with identification of entities included in this category of subjects does not exist because the lawmaker itself in Article 3 (20) of the above-mentioned act makes a cross-reference to the Environmental Protection Law.
Nevertheless, the definition of a user of the environment given in the Act on Preventing and Remedying Environmental Damage differs from the definition following from the Environmental Protection Law. The definition in the Act on Preventing and Remedying Environmental Damage, apart from a cross-reference to the Environmental Protection Law, contains an additional prerequisite. According to the Act, responsibility is not incurred by every user of the environment, but only by a subject whose operations pose a risk of environmental damage or a direct threat of environmental damage. Therefore, we are dealing with an eligible user of the environment despite the fact that the name of the group of subjects used by the Act on Preventing and Remedying Environmental Damage and by the Environmental Protection Law is identical.
The construction of a state legal person, which is more and more often used by the lawmaker, must also be qualified among specific subjects in environmental protection law. According to Art. 40 of the Civil Code a state legal person is a legal subject separate from the State Treasury.
This construction is used in environmental protection law mainly for purposes connected with public finance. Using such a construction with respect to a specific organisational unit the lawmaker declares, as a rule, that this is a state legal person within the meaning of the Act of 27 August 2009 on Public Finance. Such treatment was given to the National Fund for Environmental Protection and Water Management, State Water Holding -Polish Waters and to a national park.
However, in this case the specificity of Polish legal subjects is that the state performs its constitutional and statutory tasks related to environmental protection. The Polish legislator sometimes adopts a solution different from typical solutions characteristic of administrative law. Thus, it consciously gives up regulatory forms of activity of public subjects for the sake of civil law constructions. Treating them in the first place as civil law subjects the Polish legislator appreciates the means provided by private law.
Nevertheless, it does not mean that the system of Polish law completely gives up the instruments of administrative law and regulatory activity of the state.
To this extent, the specificity of legal subjects in Polish environmental protection law is primarily manifested in the introduction of a subject category -an environmental authority.
Organisation of environmental protection not only requires making good and effective law but also, or perhaps mostly, the presence of an efficient executive apparatus. Only such an apparatus can ensure the effectiveness of legal norms in practice. Such effectiveness, as a matter of fact, determines the quality of environmental protection. The executive apparatus in the system of Polish law consists mainly of public administration authorities. As mentioned above, the Polish lawmaker chose an administrative and legal model of environmental protection law, in the first place with regard to effectiveness, possibility of undertaking measures ex officio, as well as the speed of administrative proceedings.
Adopting an administrative and legal model of environmental protection with a supplementary role of courts and, in broader terms, other judicial authorities, requires the lawmaker to create a suitable structure of such authorities and to commission tasks to them. Aware of the necessity to create such a structure and simultaneously of its complexity, the lawmaker introduced the term 'environmental authority'.
Thus, in the first place it must be explained what an environmental authority is. It is significant because firstly gmina (municipal) authorities can be undoubtedly classed as environmental authorities and secondly en-vironmental authorities are confronted with environmental institutions and these are deprived of regulatory competences.
Environmental authority is a normative term. It is defined in Art. 3 par. 15) of the Environmental Protection Law. This provision reads: "For the purposes of this Act: 5) environmental authority -means an administrative authority appointed for rendering public tasks related to environmental protection according to their level of competence described in Division I Title VII".
As shown by the above-quoted definition of the environmental authority, it is a public administration authority. In turn, this term has been defined in the doctrine. Literature also classifies authorities. Including elaborate quotes from literature regarding this term is pointless. Let me only indicate some representative ones for the purposes of this article 7 .
The definition of an environmental authority emphasises two circumstances. Firstly, the lawmaker indicates that this is a type of administrative authority. Secondly, these are authorities appointed for the purposes of public tasks in the area of environmental protection. Thirdly, such public tasks fall within the competence of such authorities as described in Title VII, Part I of the Environmental Protection Law.
Insofar as the first element is defined by the doctrine, the other two are strictly normative. The scope of public tasks in the area of environmental protection is determined by the lawmaker, who indicates competent authorities by making a cross-reference to the respective part of the Environmental Protection Law.
Such normative elements cause specific difficulty in interpretation of the term 'environmental authority'. On the one hand, the lawmaker accentuates that a characteristic of an environmental authority is the fact that it performs public tasks in the area of environmental protection. On the other hand however, the lawmaker indicates that the competence of such authorities is defined in the Environmental Protection Law.
Any public administration body performing public tasks in the area of environmental protection is an environmental authority. In this case the criterion would be performance of a public task in the area of environmental protection. Leaving out the difficulty defining the environmental protection task, it must be assumed that every authority performing at least one public task in the area of environmental protection will be an environmental authority. However, the type of authority is not significant, in particular whether it is a state or local administration body, a collegial or a monocratic body. Finally, its place in the hierarchy of public administration authorities is not important either. Its constitutive characteristic is performing public tasks (at least one) in the area of environmental protection.
Nevertheless, this clear-cut criterion is subject to certain normative distortion because the lawmaker goes beyond the statement that this is an authority performing a public task in the area of environmental protection. Further, under Title VII Part I of the Environmental Protection Law, it indicates that such as task is performed according to the competence of authorities defined in the act. Thus, the competence of such an authority must be defined in this part of the act. This criterion definitely narrows the interpretation of an environmental authority. It is not sufficient that it is a public administration body performing public tasks in the area of environmental protection, but cumulatively its competence must be described in Title VII Part I of the Environmental Protection Law. A contrario any authority not satisfying the above-mentioned requirements is not an environmental authority.
However, such a conclusion is not correct. Irrespective of the catalogue of environmental authorities listed in the Environmental Protection Law, a number of authorities perform tasks that may be deemed environmental protection tasks. For instance, these include the State Water Holding -Polish Waters, and first and foremost the director of the regional water management authority. Another example is regulatory authorities of local government units -the gmina (or municipal) council and the poviat council. Another example may be veterinary administration bodies acting mainly based on the Act of 29 January 2004 on Veterinary Inspection and in the first place the district veterinarian and the regional veterinarian.
With regard to the aforementioned, a significant problem appears, namely, what should be regarded as the criterion determining whether or not the specific authority can be deemed an environmental authority. Is it performing public tasks in the area of environmental protection or being mentioned as an environmental authority by relevant provisions of the Environmental Protection Law?
In resolving this doubt it must be pointed out that the Polish legislator adopts a cumulative criterion, which is both performing public tasks in the area of environmental protection and listing the body among environmental authorities in the Environmental Protection Law. However, this criterion is mis-leading as it does not cover a number of bodies that do perform public tasks in the area of environmental protection but are not listed in the Environmental Protection Law as environmental authorities.
This dilemma can be resolved adopting the concept of an environmental authority in the narrow and in the broad sense. In the narrow sense, the environmental authority is a body that performs public tasks in the area of environmental protection and that is simultaneously listed in the Environmental Protection Law. In the broad sense, the environmental authority is a body that performs public tasks in the area of environmental protection and that is simultaneously listed in the Environmental Protection Law.
Hence, the above-listed bodies should be deemed environmental authorities in the broad sense.
In turn, the catalogue of environmental authorities in the broad sense is provided in Art. 372 of the Environmental Protection Law. This provision reads " Having found that the causes for suspending the activity or use pursuant to decisions mentioned herein ceased, the regional inspector for environmental protection, local administrator, mayor or president of the city, at the request of the party concerned, will issue the consent to resume the suspended activity or use". This catalogue of environmental protection authorities in the narrow sense is a closed catalogue which can only be changed by amending the act. However, it has several interesting characteristics that are worth analysing in more detail.
The catalogue of environmental authorities in the narrow sense includes both state administrative bodies and local administrative bodies. The state administrative bodies are, among others, the minister of environment, the voivode, the regional director for environmental protection and the general director for environmental protection. The head of the gmina administration, the mayor of town or city and the regional council form a group of local government bodies.
With regard to the division based on whether the authority is a state or local administration body, it may be assumed that state environmental authorities and local environmental authorities exist. A state environmental authority is the minister of environment, the voivode, the general director for environmental protection and the regional director for environmental protection. On the other hand, the voivodeship marshal, the starost (poviat head), the head of the gmina (mayor of town or city) must be deemed local environmental authorities. A regional council should also be included in this group.
Within a group of state administration bodies it is also significant that this group is composed of government administrative bodies and non-combined administrative bodies. Government administration is composed of the minister of environment and the voivode. On the other hand, the general director for environmental protection and the regional director for environmental protection are non-combined administrative bodies.
In addition, a division into monocratic and collegial bodies can be observed. Except for the regional council, other authorities are monocratic bodies. The regional council is the only collegial body.
In the system of Italian law specific legal subjects can also be observed. However, here different assumptions and criteria are applied following from specific characteristics of Italian law, its traditions, culture, philosophy and axiology. Nevertheless, it does not mean that the fundamental assumptions required for comparative studies are not maintained. R. Tokarczyk is right to observe that "the object of comparative law studies is typical legal phenomena referred to herein as the units of law and forms of legal thought. The first include the following, […] : components of legal norms (hypotheses, dispositions, sanctions), legal norms, legal regulations, institutions of law, branches of law, systems of law, families of systems of law, and types of law. Moreover, one can also compare different lawmaking techniques, legal procedures, contents of legal decisions, and law enforcement rules. The forms of legal thought that are most often compared are ideas, ideologies, doctrines and law programmes" 8 .
M. Rainer views this problem differently and identifies three elements -institutional, system and global comparative law. Only within these three elements can he see more detailed levels of comparative law 9 .
Thus, despite certain discrepancies to be identified, comparative law studies can be carried out at the level of specific legal subjects in environmental protection law both in Polish and Italian law.
Insofar as in the system of Italian law no specific and special subject category modelled on the user of the environment is introduced, such specificity can be observed at the level of environmental protection organisation itself. Likewise in the system of Polish law it covers public administrative bodies.
The legislative decree fundamental for Italian environmental protection law Il Decreto Legislativo "Norme in materia ambientale" no. 152 of 3 April 2006, equivalent to the Polish Environmental Protection Law, makes use of subjects competent in environmental matters (soggetti competenti in materia ambientale), defined as public administrative bodies and public institutions that, with regard to their knowledge and responsibility in the area of environmental protection, may be interested in environmental impact following from the implementation of plans, programmes or projects 10 .
The term 'environmental subject' covers both public administration bodies and public institutions. Leaving out more precise digressions on public institutions and their significance and functioning in Italian law, which would go beyond the subject matter of this paper, it can be stipulated that public institutions are to a large extent equivalent to environmental institutions. They are non-regulatory authorities that simultaneously perform public tasks as a servicing administration. On the other hand, the doctrine of Italian law defines administrative bodies similarly to the doctrine of Polish law. Similar characteristics are emphasised among which regulatory activities are the most important 11 . 9 M. Rainer, "Course of comparative legal systems", Torino 2004, 6. However, the status of a public authority or institution alone is not sufficient to qualify specific subjects as environmental subjects. Italian literature pays attention to the criterion of competence, performance of tasks and knowledge on environmental protection. However, this is not about global treatment but only about environmental impact, whereas such impact must be a consequence of implementation of plans, project or programmes 12 .
Comparing the Italian and Polish solutions a number of significant similarities and differences can be observed, which is important from the point of view of comparative law. The key similarity between the two systems of law is the fact that both legislators can see the specificity of the subjects of environmental protection law (environmental law). This specificity of subjects is manifested in that separate categories of subjects are formed for the needs of environmental protection law. Generally, they are not new legal subjects but out of the general group of legal subjects (including public administrative bodies) the legislators select those with normatively defined characteristics and create a separate legal category. Specific distinguishing characteristics of the subjects are significant from the point of view of environmental protection. They differ in both regimes but they are characteristic of the specific regime of law.
What is significant is that in both cases the specificity of legal subjects gives separate treatment to public administrative bodies. In the system of Polish law the lawmaker chose to identify both a category of specific public administration bodies and subjects who are the addresses of environmental protection law norms and who at the same time are not public administration bodies. In the case of Polish law they are termed users of the environment.
The Italian lawmaker reduced separate treatment to public subjects only. However, it must be emphasised that the analysed scope of the term 12 used in the Italian Environmental Code is not limited to public administrative bodies only. It also includes public institutions that are not public administrative bodies.
The criteria for separate treatment of subjects are also slightly different. Polish law perceives them as public administrative bodies performing environmental protection tasks. In Italian law, the criterion for separate treatment, apart from the type of subject covered by the definition, is the link to environmental impact connected with the implementation of plans, projects, strategies etc.
The most significant comparative conclusion is that both regimes of law can see the specificity of public administrative bodies and public institutions as well as the need for emphasizing it and simultaneously giving it a separate treatment.
To sum up, it should be noted that both the Polish lawmaker and Italian lawmaker are aware of the specificity of legal subjects in the public domain. They can see that public administrative bodies and public institutions cannot be described in normative terms, definitions and expressions used in legal language. New criteria for their separate treatment are needed.
In addition, it is justified to treat them separately from all public administrative bodies and public subjects. This only serves as proof of the rank and significance of such authorities. | v3-fos-license |
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} | pes2o/s2orc | Anti-melanogenic activity of Myristica fragrans extract against Aspergillus fumigatus using phenotypic based screening
Background Aspergillus fumigatus, an opportunistic fungal pathogen is associated with a wide array of diseases. It produces 1, 8-dihydroxy naphthalene (DHN) melanin that imparts greenish grey color to conidia and is an important virulence factor. It masks various molecular patterns associated with A. fumigatus and protects the fungus from host immune system. Myristica fragrans, enriched with secondary metabolites has been traditionally used for the treatment of infectious and inflammatory diseases. The present study was aimed to explore the anti-melanogenic effect of M. fragrans extracts on A. fumigatus. Methods M. fragrans extracts (hexane, chloroform, methanol and ethanol) were prepared through polarity guided extraction. Phytochemical analysis was performed to detect the chemical constituents of the extracts. The minimum effective concentration (MEC) of the extracts against A. fumigatus melanin was determined by broth micro-dilution assay. Various virulence factors were assayed by spectrophotometric methods. Electron microscopic studies were performed to evaluate the effect of the hexane extract of M. fragrans on A. fumigatus cell surface morphology. The major active compounds of the extract were detected by gas chromatography-mass spectrometry (GC-MS). Docking was performed to study the interaction between the major identified compounds and the ketosynthase domain of polyketide synthase protein. Results The results indicated that the hexane extract of M. fragrans inhibited melanin production (76.09%), reduced ergosterol content (83.63%) and hydrophobicity of the cell (72.2%) at the MEC of 0.078 mg/mL. Altered conidial surface, disappearance of protrusions and absence of melanin layer on outer cell surface was observed in electron microscopy. Forty-two compounds were identified by GC-MS. The main constituents were identified as sabinene (12.2%), linoleic acid (11.7%), hexadecanoic acid (10.5%), safrole (8.1%) and elemicin (7.8%). Docking studies revealed that hexadecanoic acid, its derivative compound cis-9-hexadecenal and isoeugenol have lower binding energy forming proper hydrogen bond with ketosynthase domain of polyketide synthase protein. Conclusion The study concludes that the extract of M. fragrans has potential antifungal properties that can be explored in combination with available antifungals. This combination approach may be helpful for large number of patients suffering with A. fumigatus infections.
Background
Aspergillus species are the most prominent airborne fungal pathogens that account for various invasive and noninvasive infections based on the impaired immune system in humans [1,2]. They propagate by conidia which are ubiquitous and have high sporulating capacity. Once inhaled, they by-pass mucociliary clearance, germinate and produce septate vegetative mycelium that invades the lung tissue [3]. Among Aspergillus infections, more than 90% of pulmonary infections are caused by Aspergillus fumigatus [4]. Major pulmonary diseases due to A. fumigatus include allergic bronchopulmonary aspergillosis (ABPA), Aspergillus rhinosinusitis, chronic pulmonary aspergillosis, invasive aspergillosis, saprophytic aspergilloma and trachoebronchitis. These diseases manifestations can coexist in the same individual.
There are limited antifungal drugs available in the market to combat fungal infections. These drugs mainly target fungal cell wall (echinocandins) and ergosterol by either binding to ergosterol (polyenes) or inhibiting intermediary enzymes of ergosterol production (azoles and allylamines). These chemically synthesized drugs have several adverse effects such as anaphylaxis, chills, fever, headache, hepatotoxicities, nausea, neurotoxicity, and reproductive disorder [5]. These classical drugs are not cost effective [6]. Besides, the incidence of drug resistance in Aspergillus is increasing globally. Hence, there is an urgent need for affordable antifungal medications to improve the drug efficacy and reduce the side effects. The use of herbal medicines may be a suitable alternative that can open new avenues in antifungal treatment.
Various plant extracts have been reported to exhibit antibacterial, antifungal and insecticidal properties in vitro [7][8][9][10][11]. Spices contain various secondary metabolites such as flavonoids, phenolics, tannins, terpenes, quinines and are associated with antioxidant property. These bioactive compounds are involved in antimicrobial activity by the cell wall disruption, leakage of cellular components, fatty acid and phospholipid alteration [12].
Myristica fragrans is a dietary spice; belongs to the Myristicaceae family and is cultivated in the Banda Islands, Grenada, the Caribbean, South India, Sri Lanka, Malaysia, Sumatra, and Brazil [13]. The main constituents of M. fragrans have been found to be alkyl benzene derivatives (myristicin, elemicin, safrole etc.), terpenes, alpha-pinene, beta-pinene, myristic acid and trimyristin [14]. It contains about 2% of lignans (diarylpropanoids), which are non volatile dimers of phenylpropanoid [15]. M. fragrans has been traditionally used for intestinal catarrh and colic, to stimulate appetite, control flatulence and also as an abortifacient [16,17]. It has been reported as an antidepressant with antioxidant and hepatoprotective role [18].
Multiple factors contribute to virulence of A. fumigatus including its versatile metabolism, thermo-tolerance, or the production of toxins and secondary metabolites like 1, 8-dihydroxy naphthalene (DHN) melanin [19]. DHNmelanin plays a major role in the protection of Aspergilli against harsh environmental conditions such as ultraviolet irradiation, reactive oxygen species and the host immune system [20]. Melanin has been shown to provide cell wall stability and structural rigidity [21]. It binds to antifungal drugs reducing their therapeutic efficacy. Time kill assay revealed that 71 and 79% of melanised yeast cells survived on exposure to 2X the MIC of amphotericin B and caspofungin, respectively [22]. Besides, melanin prevents intracellular killing of conidia by reducing luminal acidification and resisting phagolysosomal degradation [1,3]. The role of the conidial melanin in A. fumigatus has been studied by using pksP gene mutants (ΔpksP). The pksP gene encodes the first protein polyketide synthase (PKS) that catalyse the synthesis of heptaketide napthopyrone, the first step in the melanin biosynthesis pathway. The ΔpksP produce white color conidia whereas other gene mutants produced yellowish, reddish or brown colonies. It has been observed that melanized conidia masked various A. fumigatus associated molecular patterns to protect themselves from elimination whereas, the unpigmented white conidia were unable to quench reactive oxygen species (ROS) produced by human and animal granulocytes and were effectively eradicated by the host defense system [1,23]. Therefore, restraining the DHN-melanin biosynthesis through pksP gene/gene product inhibition may be considered as a novel drug target. To the best of author's knowledge, the role of M. fragrans as melanin inhibitor in A. fumigatus has not been studied till date. Thus, the present study was envisaged to evaluate the efficacy of M. fragrans extract as a melanin inhibitor in A. fumigatus.
Fungal strains
Aspergillus fumigatus (ATCC-46645) and its ΔpksP strain were a kind gift from Prof. Axel Brakhage, Department of Molecular and Applied Microbiology, Lelbeniz Institute for Natural Product Research and Infection Biology-HKI, Germany. A. fumigatus strains were maintained by subculturing on Czapek Dox Agar (CzA) slants. The fungus was grown on CzA at 28 ± 2°C for 5 days to obtain conidial growth.
Plant collection and extraction procedure
The Myristica fragrans dried spice was procured from Spice Garden in Thekkady, Kerala, India and was authenticated by Dr. ( Cleaned and dried spice (50 g) was ground using mortar and pestle to make a fine powder. It was then extracted with 200 mL hexane under constant shaking at room temperature for 72 h. Thereafter, the extract was filtered and dried under vacuum using rotary evaporator. Further, the polarity guided extraction was done sequentially using chloroform, methanol and ethanol to yield hexane (8.54 g), chloroform (5.78 g), methanol (2.88 g) and ethanol (1.88 g) dried extracts. The dried extracts of M. fragrans were re-suspended in dimethyl sulfoxide (DMSO) to make the stock suspension of 100 mg/mL and stored at 4oC for further experiments. The prepared extracts were coded as JaH, JaC, JaM and JaE extract, respectively.
Phytochemical analysis of extracts
Preliminary qualitative phytochemical analysis of the M. fragrans extracts was carried out to detect the presence of saponins, tannins, terpenoids, carbohydrate, steroids, napthaquinones and flavonoids as per the protocol of Raaman [24].
Determination of minimum effective concentration (MEC) of extracts for demelanization
Minimum effective concentration with respect to the appearance of white colonies was determined by Clinical and Laboratory Standards Institute (CLSI) broth microdilution method [25]. The experiment was carried out in triplicates. Two-fold serial dilutions of the extracts were made in Czapek Dox Broth (CzB) over a range to give final concentrations of 5.0-0.009 mg/mL. One hundred microliters of A. fumigatus conidial suspension 0.8 × 104 conidia/mL was added to each well. Negative control comprised CzB only while the positive control was CzB and conidial suspension. The final volume in each well was 200 μl. The MECs of the extracts were detected after 5 days incubation at 28 ± 2°C. MEC was defined as the lowest concentration of the extract that produced white colored conidia based on morphological appearance [25].
Extraction, purification and UV-visible spectrophotometric analysis of melanin
The isolation of cell-wall associated melanin from wild type conidia and treated conidia was performed as described by Rajagopal et al. [26]. The cultures were grown in CzA plates for 5 days at 28 ± 2°C. The treated culture plates were supplemented with minimum effective concentration of the hexane extract (JaH). Positive control plates were also prepared. Mycelial plug (1 cm diameter) was cut from the colonies grown on CzA, boiled in 5 mL of distilled water for 5 min and then centrifuged for 5 min at 7000 rpm. The pellet was washed twice and melanin was extracted by autoclaving the pellet with 3 mL of 1 M KOH. The extracted melanin was dried overnight at 20°C in a dehumified atmosphere. Further, acid hydrolysis was done to purify the extracted melanin by adding 5 mL of 7 M HCL in a sealed glass vial for 2 h at 100°C. After cooling, the pigment was washed thrice with distilled water, dried, and stored at 4°C. Extracted and dried pigment from solid culture was dissolved in 1 M KOH and melanin was recorded as the absorption spectra at range of 200-600 nm using 1 M KOH as reference blank. The absorption spectra of the control and treated plates were determined and compared with the ΔpksP strain.
Melanin pigment isolated from the culture supernatant was assayed by harvesting the cells from culture broth by centrifugation. The pellet was dissolved in 1 M KOH and was vortexed properly. It was centrifuged at 7000 rpm for 5 min and the supernatant were purified by adding 7 M HCl and boiled for 2 h, centrifuged and pellet was washed with distilled water. Extracellular melanin was assayed by dissolving the pellet in 1 M KOH and measuring the absorbance at 585 nm [27].
Analysis of physio-chemical properties of melanin
A. fumigatus was cultured on CzA with JaH extract (at MEC) and without the extract. The color patterns of the colonies were compared with the ΔpksP strain. The pigments extracted from the isolates were confirmed as melanin on the basis of their physical and chemical properties. Characterization of melanin is based on the solubility in NaOH/ KOH, insolubility in water or organic solvents, decolorization by the oxidizing agents (H2O2/ KMnO4) and precipitation by 1% FeCl3 [28].
Effect of JaH extract on conidia production in A. fumigatus
The reduction in conidia formation was estimated spectrophotometrically at 530 nm. Agar blocks (1 cm3) was excised from the 5 day old CzA plate using a sterile surgical blade and transferred to test tube. Phosphate buffer supplemented with 0.25% Tween-20 (5 mL) was added to each tube, shaken vigorously and absorbance was measured at 530 nm. The absorbance of control and the treated conidia were compared with respect to the ΔpksP strain.
Ergosterol content estimation
Ergosterol content was measured as described by Young et al. with minor modification [29]. Briefly, 5 days old mycelia were harvested by centrifugation at 2700 rpm, washed with sterile distilled water, dried and weighed. The dried pellet was transferred into sterile glass screw cap tubes and 3 mL of 25% of alcoholic KOH solution was added. The pellets were then incubated at 85°C in water bath for 1 h. Tubes were kept at room temperature. Further, 1 mL of distilled water and 3 mL of n-octane was added, vortexed for 3 min and then allowed to stand for separating the organic layer, which was transferred to another clear screw cap tube and stored at − 20°C. For analysis, 200 μL of the extracted sterol was mixed with 800 μL absolute ethanol and was spectrophotometrically analyzed at 281.5 nm and 230 nm. The conversion from optical density to ergosterol content was calculated as follows [29]:
Cell surface hydrophobicity
Hydrophobicity of the microbial cell suspension was assayed by two phase partitioning using hexadecane as the hydrocarbon phase described by Kennedy et al., with minor modification [30]. Briefly, conidia were harvested using phosphate buffer saline (PBS) and their absorbance was set to 0.30 at 630 nm. Hexadecane (500 μL) was then added and vortexed for 2 min. The suspension was incubated for 10 min at room temperature for phase separation. The absorbance of the aqueous phase was then measured at 630 nm and was compared to the initial absorbance.
Scanning electron microscopy (SEM)
Conidia from 5 days old A. fumigatus cultures grown on CzA medium with and without JaH extract (at MEC) were harvested as described in earlier section. The conidia were fixed in 4% glutaraldehyde in PBS under vacuum for 24 h. After washing, the cells were post-fixed with 1% osmium tetroxide for 60 min and dehydrated by passage through ethanol solutions of increasing concentration. The sample were then mounted on aluminium sheet and coated with gold-palladium alloy. The observations were made using a Zeiss SEM, MA EVO − 18 Special Edition [20].
Transmission electron microscopy (TEM)
A. fumigatus culture was grown in CzA medium with and without the JaH extract for 5 days. Conidia were harvested as described in earlier section, washed in and fixed overnight at room temperature with 2.5% glutaraldehyde with 0.1 M sodium cacodylate buffer (pH 7.4). Conidia were incubated for 1.5 h at 20°C in a solution of 4% formaldehyde-1% glutaraldehyde in 0.1% PBS and then incubated in 2% osmium tetraoxide for 1.5 h. Dehydration was accomplished by serial washing in graded ethanol solutions of 50-95% for 10 min, followed by two final washes in 100% ethanol for 15 min. The cells were embedded in Spurr's resin, sectioned onto nickel grids and examined on a JEOL 2100F transmission electron microscope to obtain micrographs [31].
Chromatographic analysis
The chemical constituents of the hexane extract of M. fragrans were analyzed using gas chromatography varian-450 fitted with a fused silica capillary column TG-5 (30 m × 0.25 mm i.d., 0.25 μm film thickness) [32,33]. The oven temperature was programmed from 60 to 220°C at 3°C/ min, using nitrogen as carrier gas. The injector and the flame ionization detector (FID) temperature were set at 230 and 240°C, respectively. GC-MS analysis was employed using a Thermo Scientific Trace Ultra GC interfaced with a Thermo Scientific ITQ 1100 mass spectrometer which was fitted with a ZB-5 fused silica capillary column (30 m × 0.25 mm; 0.25 μm film thickness). The oven temperature range was programmed from 60 to 220°C at 3°C/min and helium was used as carrier gas at 1.0 mL/min for analysis. The injector temperature was set at 230°C, and the injection volume was 0.1 μL in n-hexane, with a split ratio of 1:50. MS was taken at 70 eV with a mass range of m/z 40-450 [34,35]. Identification of constituents was done on the basis of retention index (RI), determined with reference to homologous series of n-alkanes C 8 -C 25 under identical experimental conditions, by comparing with the MS literature data [36] and coinjection of commercial samples from Sigma-Aldrich, India (≥ 98% purity). The relative amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor.
Docking study through protein ligand interaction (Fig. 6b). The structure of compounds (ligand molecules) was retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/; Fig. 6a). The Lamarckian genetic algorithm was used in AutoDock 4 to perform the automated molecular dockings with default parameters. The number of run was set to 50 and the lowest binding energy conformation was selected for LigPlot analysis.
Statistical analysis
The statistical analysis was conducted using one-way ANOVA to compare the results of melanin estimation, conidiation, CSH and ergosterol assay for extract treated culture with wild type, drug treated and ΔpksP strain. All experiments were conducted in technical and biological triplicates. The statistical analysis was performed using Graphpad Prism software 8.0.2.263 version and Microsoft Excel. p < 0.05 was considered statistically significant.
Phytochemical analysis of extracts
The phytochemical tests revealed the presence of steroids, carbohydrate, tannins, alkaloids and terpenoids in the M. fragrans extracts (Table 1). However, flavonoids and saponins were not present. JaH extract showed the highest amount of steroids, tannins and terpenoids which were evaluated on the basis of the color production in the respective phytochemical test.
Determination of minimum effective concentration
White colonies of A. fumigatus were observed in the wells containing the extract whereas control wells showed greenish grey conidia. The minimum effective concentration was determined as the lowest concentration of the M. fragrans extracts showing pigmentless conidial growth as compared to greenish grey wild type conidia. The JaH extract was effective for melanin inhibition at 0.078 mg/mL concentration whereas other extracts inhibited melanin formation at higher concentration. The MEC for JaC and JaM was 1.25 mg/ mL and for JaE was 2.50 mg/mL. Therefore, JaH extract was selected for further experiments.
UV-visible spectrophotometric analysis of melanin
Melanin was extracted from wild type, JaH extract treated cultures and ΔpksP strain. The spectral study of melanin from both wild type and treated cultures showed a characteristic peak in the UV region 200-260 nm (Fig. 1). The light absorbed by melanin was maximum in the UV region and decreased gradually as the wavelength increased. The ΔpksP strain showed no characteristic peak depicting the absence of melanin. The extracellular melanin content was estimated by spectrophotometric method at 585 nm. The results indicated reduction in melanin content after treatment as compared to the solvent control plates. Melanin content was reduced by 76.09% after treatment with the extract as compared to control whereas 99.8% reduction in the melanin content was estimated in ΔpksP culture ( Fig. 2a; p < 0.05).
Physical and chemical properties of melanin
The culture plates were phenotypically visualized and white colonies were observed after the extract treatment. Figure 3b shows the effect of M. fragrans extract on melanin inhibition in A. fumigatus at MEC as compared to wild type colonies (Fig. 3a) and ΔpksP (Fig. 3c). The extracted pigment was characterized on the basis of physico-chemical tests. The extracted pigment was soluble in NaOH and KOH whereas, insoluble in water or organic solvents like chloroform, ethyl acetate, alcohol and acetone. The pigment was decolorized by the oxidizing agents H2O2 and KMnO4 and precipitated by 1% FeCl3. Effect of JaH extract on conidia production in A. fumigatus Conidia formation was spectrophotometrically measured at 530 nm. It was observed that Conidia formation reduced by 72.1% after treatment with the extract of M. fragrans whereas ΔpksP culture showed 39.78% conidial reduction ( Fig. 2b; p < 0.05).
Ergosterol content estimation
A. fumigatus hyphae were analyzed for the ergosterol content with and without extract. Amphotericin B (Amp B), a known inhibitor of ergosterol was used as positive control. A reduction of 83.63 and 85.02% was found in the ergosterol content after treatment with extract and Amp B, respectively in comparison to control group ( Fig. 2c; p < 0.05).
Cell surface hydrophobicity
CSH was assessed by two-phase partitioning with hexadecane as the hydrocarbon phase. A decrease in the CSH for both treated and ΔpksP culture was observed as compared to the control wild type greenish grey conidia. The treated conidia showed 72.20% decrease in CSH and ΔpksP culture exhibited 77.79% ( Fig. 2d; p < 0.05) CSH reduction. This decrease in hydrophobicity was also observed during the preparation of conidial suspensions.
Scanning electron microscopy
The cell surface morphology of wild type conidia, ΔpksP and the extract treated A. fumigatus conidia were analyzed and compared by SEM. The wild type conidia showed echinulate surface. The ΔpksP revealed smooth conidial surface with complete absence of any protrusions. The extract treated conidia also revealed smooth conidial surface devoid of any ornamentation (Fig. 4a-c).
Transmission electron microscopy
Lateral section of the wild type, ΔpksP and treated conidia was visualized by TEM. The lateral section of the wild type conidia showed thick electron dense inner layer indicating the presence of melanin in between the membrane (Fig. 5a). In comparison to the wild type conidia, both ΔpksP and extract treated conidial section showed visible clear inner surface. There was an absence of electron dense layer. The two clear thin layers were also devoid of cell surface ornamentations (Fig. 5b-c).
Protein ligand interaction
The LigPlot analysis depicted that despite of having low binding energy, elemicin (− 5.35 Kcal/mol) was stabilized solely by hydrophobic interaction and formed no hydrogen bond with the amino acid residues of KS domain of PKS protein. The lower binding energy was observed in hexadecanoic acid (− 4.19 Kcal/mol), its derivative compound cis-9-hexadecenal (− 5.71 Kcal/mol) and isoeugenol (− 6.14 Kcal/mol). They formed both hydrogen bonds and hydrophobic interactions with PKS. Hexadecanoic acid and cis-9-hexadecenal formed proper hydrogen bonding at Asn422 whereas in isoeugenol Ser267, His277 and Asn422 was contact residue within the docking site (Fig. 6c). Cross comparing the interaction revealed that all the test ligands interacted at Asn422 residue of KS domain of PKS.
Discussion
Herbs and spices have been used as dietary supplements and traditional medicines since ages. Previous study by Mc Fadden et al. reported that crude spice extracts exhibit good antifungal activity and the probable mechanism involves the inhibition of various cellular processes, augmenting the membrane permeability and finally, leading to leakage of ions from the cells [37]. M. fragrans is a spice widely used as flavoring agent in food industries. In the present study, polarity guided extraction of M. fragrans was done using hexane, chloroform, methanol and ethanol based on their increasing polarity. It has been reported that the type of solvent used for extract preparation impacts the antimicrobial activity because even after the solvents were removed from extracts by evaporation; chemical compounds extracted using various solvents were different. In the present study JaH extract exhibited best result which coincides with the study reported by Witkowska et al. [12]. Hexane being non polar solvent breaks the hydrophobic barriers and hexane extract contains maximum lipophilic metabolites lignin, wax, lipids, sterols and terpenoids [38][39][40]. Thus, hexane extract was used for further studies. The commonly reported phytochemical constituents of M. fragrans are volatile substances, terpenoids, phenolics, lignin compounds, protein, mucilage and starch [41]. The present study confirmed the presence of alkaloids, steroids, tannins and terpenoids in M. fragrans extract. However, Iyer et al. reported the absence of terpenoids in their extracts [42]. This variation may be attributed to the plant sources, climatic conditions and the extraction methodology [43].
While the phytochemical constituents of M. fragrans have been extensively studied for its antifungal effect [42,[44][45][46][47], the melanin inhibiting potential has not been studied till date. The present work aimed at evaluating the antifungal effect of M. fragrans extract as a melanin inhibitor against A. fumigatus. The role of DHN-melanin as an important virulent factor has been broadly described [1,3,20,28]. It has also been reported that mutation in each gene of the DHN-melanin biosynthesis cluster produced different colored colony. ΔpksP A. fumigatus produced white avirulent colonies, whereas deletion of other genes of DHN-melanin cluster produced grey, reddish pink and brown conidia [1]. Among all extracts used, JaH extract showed melanin inhibition at minimum concentration against A. fumigatus. The culture plates with JaH extract at MEC were seen to be white in color as compared to the control wild type culture which was greenish grey (Fig. 3). Hence, it was hypothesized that the target of the extract is either pksP gene or its translational product which remains to be clarified.
Melanin was extracted from A. fumigatus after JaH extract treatment as well as wild type culture. The extracted pigment showed positive result for the chemical tests used for fungal melanin diagnosis [28]. The result depicted that the absorbance declined as the wavelength increased to the visible region which is the property of aromatic organic compound. The result coincides with previous report by Raman and Ramasamy [48]. Melanin pigment plays an important role in cell surface morphology. The loss of pigment biosynthesis leads to extreme changes on the conidial wall orientation. Hence, to further study the effect of JaH extract on cell wall, ergosterol estimation was conducted. Sterols are neutral lipids of eukaryotic cells and play an essential role in the maintenance of cell membrane. Among sterols, ergosterol is the main component of fungal membrane which makes its biosynthetic pathway essential for fungal growth [49]. It is also the primary target for antifungal drug such as Amp B. A marked decrease in the ergosterol percentage was observed in treated conidia in comparison to the wild type conidia (Fig. 2c). The result indicated that JaH extract reduces the ergosterol production which may lead to changes in membrane fluidity, regulation and distribution of integral membrane proteins.
Cell surface hydrophobicity (CSH) contributes to the interaction between A. fumigatus and the host epithelial cell surface, which is an important factor for spreading infection. It has been reported that there is a direct relation between cell surface hydrophobicity and adherence to the host surface [50][51][52]. Hence, it can be concluded that decrease in cell surface hydrophobicity would result in decrease in adherence to the host cell surface. Melanin increases both negative charge and hydrophobicity of the conidia. The pigment-less conidia showed decrease in CSH as compared to the wild type conidia which suggests that blockage of the melanin biosynthetic pathway leads to the reduction of some hydrophobic components on the conidial surface contributing to the marked loss of adherence properties of the conidia. Similar results were reported on the ΔpksP strain by Pihet et al. [20].
Melanized conidia have rough outer layer with protrusions on the surface. These conidial protrusions protect the organism from phagocytosis and increase its resistance to ROS produced by phagocytic cells. In the present study, the cell surface morphology of conidia was analyzed using SEM, which clearly indicated loss in cell surface protrusions and showed smooth cell wall conidia in the treated fungus. TEM examination also confirmed protrusion less surface, thinner cell wall and reduced melanin in treated conidia in comparison to control probably due to the progressive detachment of the outermost cell wall layer. Similar results were reported by Jahn et al. and Youngchim et al. [1,53].
The GC-MS analysis of the JaH extract revealed the presence of sabinene, linoleic acid, hexadecanoic acid, safrole, elemicin, myristicin and β-pinene in varying quantities. Major constituent in essential oil of M. fragrans are myristicin, safrole, pinene, isoeugenol, 4terpenol as reported by Alcazar-Fuoli and Mellado [54]. Various studies have also reported the presence of neolignan and macelignan in M. fragrans extract [14,42]. The quantitative and qualitative divergence of plant constituents may be due to the geographical, climatic, and soil conditions, which in turn may affect the composition and/or synthesize new secondary metabolites from the same plant species [55]. The potential role of these components needs to be further analyzed.
Ligand-protein interactions can be extrapolated through docking studies. Polyketide synthase is an essential protein for synthesis of conidial pigment melanin. A. fumigatus PKS protein contains three important domains: ketosynthase (KS), acyltransferase (AT) and acyl carrier protein (ACP). KS domain is the functionally active site of the enzyme [56]. The KS accepts the acyl unit from the acyl-ACP and condenses malonyl-CoA units shuttled by the ACP onto the starter unit to add a ketide unit [57]. In the present study, structure of KS domain of PKSs protein of A. fumigatus was modelled for the docking studies with the compounds. The structural differences among the ligands may contribute to differences in the interaction [58]. Based on LigPlot analysis, the O atom located on the hydrocarbon chain was situated to form H-bonds with Asn422 in the ligands. By contrast, the structural difference in elemicin pushes the same O atom away from the H-bond forming amino acids, resulting hydrophobic interactions only (Fig. 6c).
Conclusion
The study concludes that the JaH extract of M. fragrans has potential antimelanogenic properties as depicted by inhibition of melanin synthesis, loss in cell surface protrusions, formation of smooth cell wall, reduction in ergosterol concentration and cell surface hydrophobicity. Thus, it can potentially decrease the pathogenicity of A. fumigatus and increase its susceptibility to available antifungal drugs. Hence, M. fragrans extract in combination with commonly used antifungals may increase the therapeutic efficacy. This combination approach may be helpful for large number of patients suffering with A. fumigatus infections. | v3-fos-license |
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} | pes2o/s2orc | Application of N-Dodecyl l-Peptide to Enhance Serum Stability while Maintaining Inhibitory Effects on Myometrial Contractions Ex Vivo
N-Alkylation and N-acylation of the prostaglandin-F2α allosteric modulator l-PDC31 were performed to install various alkyl, PEG and isoprenoid groups onto the l-enantiomer of the peptide. Among the different bio-conjugates studied, the N-dodecyl analog reduced prostaglandin-F2α-induced mouse myometrium contractions ex vivo. Furthermore, N-dodecyl-l-PDC31 exhibited improved stability in a mouse serum assay, likely due to protection from protease degradation by the lipid chain.
Introduction
Preterm labor is a challenging contemporary target of modern medicinal chemistry. Preterm birth (PTB), defined as less than 37 weeks of gestational age, accounts for 5-13% of all births [1]. Responsible for an increased risk of neonatal and infantile mortality [2] PTB is associated with various other complications, including developmental disorders [3], vision and hearing impairments [4,5], and chronic metabolic conditions [6]. Since 2012, PTB has been the single most expensive medical urgency per patient in the United States [7], in part due to a lack of so-called tocolytic drugs that can effectively and safely delay delivery.
In less affluent countries, PTB is more problematic: >60% of PTBs occur in Africa and South-Asia. Ineffective healthcare to treat mother and child in less affluent countries is associated with a higher percentage of neonatal and infantile mortality: 90% for very preterm babies of <28 weeks. In developed countries, <10% of newborns suffering similar PTB conditions experience the same fate [7].
The currently available medications, including the oxytocin receptor antagonist Atosiban [8], calcium channel blockers [9], β 2 agonists [10], a myosin light chain inhibitor [11], and prostaglandin-endoperoxide synthase inhibitors [12], all rarely delay labor for >48 h [13,14] and many have risks of serious side effects for both mother and infant. New approaches exploring different pathways to tocolytic agents are essential for the development of more effective treatments with minimal side effects. For ideal distribution to third-world countries, such tocolytic agents would be produced cost-effectively.
The prostaglandin F2α receptor (FP) is a promising target for delaying labor because of its relevant involvement in parturition. At the beginning of labor and preterm labor, FP expression increases [15,16]. Moreover, FP-knockout mice do not go into labor, even when induced by oxytocin [17]. Targeting FP, the tocolytic prototype PDC31 is currently undergoing clinical trials [18,19]. In a phase Ib clinical trial of women with primary dysmenorrhea, PDC31 was demonstrated to be safe in reducing intrauterine pressure and pain associated with excessive uterine contractility [20]. The development of PDC31 originated from a library of sequences based on the second extracellular loop of FP. Short lead peptides were analyzed initially using in vitro and ex vivo analyses [19]. The lead peptide PDC113 (H-ile-leu-Gly-his-arg-asp-tyr-lys-OH, small letters refer to D amino acids) was identified as an active FP inhibitor [18]. Replacement of the arginine residue with citrulline yielded PDC31 (H-ile-leu-Gly-his-cit-asp-tyr-lys-OH, cit = d-citrulline), which exhibited notable efficacy (>85%) and potency (IC 50 = 13 nM) [18]. Selectively modulating the downstream signaling of FP, the mechanism of action of PDC31 is suggested to involve allosteric inhibition at a site different from the "othosteric", native ligand-binding site [19,21].
The relatively high price of d-amino acid building blocks can influence the cost of production of PDC31, which is composed of seven of d-amino acids including d-citrulline. The l-amino acid version of PDC31 has been observed to exhibit some antagonist activity but failed to do so in a myometrial tissue-based assay, probably owing to the relatively low metabolic stability of linear l-peptides. To improve the metabolic stability of l-PDC31, the peptide N-terminal has now been modified with lipid chains. Previously, N-acetylation had abolished the activity of PDC113 analogs. Thus, instead of acylation, a strategy was pursued to maintain the basic nitrogen by alkylation with different hydrophobic moieties: linear hydrocarbons, PEG and farnesyl chains.
Polyethylene glycol grafts onto polyamide structures have demonstrated success for improving pharmacodynamic properties, particularly duration of action [22,23]. Protein PEGylation has enhanced water solubility, prolonged half-life, improved metabolic stability and diminished immunogenicity compared to the unmodified molecule. Polyethylene glycol chains are often employed as a distribution of related lengths grafted indiscriminately on various residues [24,25]. Successful examples on which polyethylene glycol grafts enhanced potency and duration of action of larger proteins include Pegasys© for hepatitis C [26] and Pegvisomant for the treatment of acromegaly (Somavert©) [27]. Moreover, modification of the glucagon-like peptide 1 (GLP-1)-based drug Liraglutide ® , which features a C16 lipid chain responsible, in part, for its longer duration of action, by adding a PEG-like spacer gave Semaglutide ® , which can be administered once weekly and is used in the treatment of type 2 diabetes and obesity [28,29]. Lipid attachment has also been used to improve metabolic stability and lipophilicity [30,31] to enhance the stability of larger peptides, as well as to improve their specificity and association with membranes [32]. Lipopeptide natural products, such as daptomycin and related antibiotics have exhibited promising activity against Gram-positive pathogens [33]. Lipid conjugation has also been applied to smaller peptides. For example, Hruby and co-workers have attached hexanoyl and decanoyl chains onto α-Melanocyte Stimulating Hormone (αMSH) [34] analogs to obtain equipotent conjugates with prolonged duration of action [31,35]. In contrast, longer chains (palmitic and myristic acids) resulted in less potent molecules, probably because of the increased lipophilicity [31,35]. Peptide-based vaccines have been created with lipid components that act as adjuvants [36]. Amphiphiles, peptides bearing both a hydrophobic lipid chain and a charged moiety have been showed to aggregate in liposomes that can act as carriers for targeted drug delivery [37,38]. The strategy called reversible aqueous lipidation (REAL) has been used to improve the bioavailability of prodrugs that release the active molecule in the organism and applied to facilitate oral delivery of small peptides [30]. Metabolic stability and membrane permeability were improved by acylation of small peptides with a palmitoyl chain that facilitated anchoring in membranes and may enhance peptide folding [39].
Prenylation is an important post-translational protein and peptide modification, which enhances typically cell membrane association [40][41][42][43]. Farnesyl transferase enzymes introduce farnesyl subunits that may serve to anchor the modified protein or peptide in the membrane. Disruption of prenylation has been suggested to play an important role in cancer because of the significance of membrane-associated proteins in the cellular life cycle [44]. Prenylation by prenyl transferases of small molecules, such as prenylflavonoids may also influence biological effects by favoring membrane association [45].
Seeking to improve the pharmacokinetic properties of l-PDC31, a variety of analogs were developed. Different N-alkyl terminal grafts were introduced by solid phase methods featuring reductive amination and sulfonamide alkylation. Among the analogs evaluated in a murine ex vivo myometrium contraction assay, the best was selected for study in a mouse serum stability test and in an in vivo PTB model.
Chemistry
The influence of the different hydrophobic chains was initially evaluated by a computational model in which logP values were predicted using the HyperChem software [46,47] and compared with PDC-31 ( Figure 1). Small PEG chains of 4 to 16 atoms had limited impact and lowered the logP of the peptide analogs. Alternatively, the addition of n-alkyl chains caused a notable increase with a nearly linear correlation in log P with increasing carbon length.
Different N-alkyl terminal grafts were introduced by solid phase methods featuring reductive amination and sulfonamide alkylation. Among the analogs evaluated in a murine ex vivo myometrium contraction assay, the best was selected for study in a mouse serum stability test and in an in vivo PTB model.
Chemistry
The influence of the different hydrophobic chains was initially evaluated by a computational model in which logP values were predicted using the HyperChem software [46,47] and compared with PDC-31 ( Figure 1). Small PEG chains of 4 to 16 atoms had limited impact and lowered the logP of the peptide analogs. Alternatively, the addition of n-alkyl chains caused a notable increase with a nearly linear correlation in log P with increasing carbon length.
The branched prenyl group increased logP with the effect appearing to be related to the longest linear chain. For example, the 15-carbon farnesyl chain produced a peptide derivative that was predicted to have a logP as that of the peptide possessing a linear 12-carbon alkyl chain. Similarly, the branched 20-carbon geranyl-geranyl group exhibited a logP lower than its 16-carbon linear alkyl chain equivalent.
Based on the above prediction The branched prenyl group increased logP with the effect appearing to be related to the longest linear chain. For example, the 15-carbon farnesyl chain produced a peptide derivative that was predicted to have a logP as that of the peptide possessing a linear 12-carbon alkyl chain. Similarly, the branched 20-carbon geranyl-geranyl group exhibited a logP lower than its 16-carbon linear alkyl chain equivalent.
Based on the above predictions, a set of analogs of the l-peptide variant of PDC31 were targeted possessing N-terminal chains consisting of 6-, 11-, 12-, 13-and 18-carbon linear alkyl, tetra-ethylene glycol [48] and farnesyl groups. In addition, N-dodeconyl-l-PDC31 (8) was synthesized as an acyl control using lauric acid.
Linear sequences of l-and d-PDC31 linked to 2-chlorotrityl resin (l-and d-1) were obtained by solid phase peptide synthesis using standard Fmoc/t-Bu protocols [49]. Peptide resin l-1 was treated with the appropriate aldehyde 2 (Scheme 1). The imine resin was washed to remove excess aldehyde, and reductive amination was performed using sodium cyanoborohydride to provide secondary amine (Table 1). Except for peptide 4e, bearing the 18-carbon alkyl chain, peptides 4 were all soluble in water. Peptide 4f required a small amount of DMSO to completely dissolve. An inseparable mixture of monoand bis-N-alkyl peptides was isolated by HPLC from reaction with aldehyde of shorter chain length.
Farnesyl peptide 7 was synthesized by a route featuring sulfonylation followed by alkylation with farnesol under Mitsunobu conditions. Resin l-1 was swollen in DMF and reacted with ortho-nitrobenzenesulfonyl (oNBS) chloride and di-iso-propylethylamine (Scheme 2). With the oNBS group installed, sulfonamide resin 5 was treated with di-iso-propyl azodicarboxylate (DIAD), triphenylphosphine and farnesol. N-Farnesyl peptide resin 6 was provided in 55% conversion as demonstrated by LC-MS analysis of material after resin cleavage [50][51][52][53]. The oNBS group was removed by treatment with thiophenol and DBU in DMF [52]. N-Farnesyl peptide resin 6 was cleaved with concomitant removal of the side chain protection using a cocktail of 95:2.5:2.5 TFA:TES:H 2 O. Purification by RP-HPLC and freeze-drying of the collected fractions gave N-farnesyl peptide 7 as white powder, which was soluble in water (Table 1).
Finally, N-dodecoyl-l-PDC31 (8) was synthesized from resin L-1 by coupling with lauric acid using HBTU and DIEA, resin cleavage and removal of side chain protection as described above. Purification by RP-HPLC gave 8 as white powder after freeze-drying ( Table 1). The poor solubility of N-dodecoyl-l-PDC31 (8) necessitated dissolving in 0.1 N HCl and freeze-drying to give the hydrochloride salt as white powder that dissolved readily in water.
Among the peptides synthesized, only peptides 4f and 8 displayed aqueous solubility issues, which were in agreement with their calculated >0 cLogP values ( Figure 1). Increased lipophilic character may promote peptide aggregation and decrease solubility in aqueous solvent. The potential to aggregate may increase due to the amphiphilic character of the N-alkyl PDC-31 analogs which possess hydrophobic and charged residues respectively at the Nand C-terminals. For biological assays, solubility issues were surmounted by forming salts and using small amounts of DMSO. The influence of their amphiphilic character during purification by RP-HPLC and double alkylation from the reductive amination sequence may account for the lower isolated yields of peptides 4, 7 and 8.
Biology
All the peptides were initially tested in an ex vivo mouse myometrial tissue contraction assay. Contractile activity was measured on tissue that was harvested post-partum and treated with prostaglandin F 2α (PGF α ) alone (control) or in the presence of peptides l-4b-4g, l-7, and l-8. Among all the peptides tested, only N-dodecyl-l-PDC31 (l-4d) exhibited a significant reduction of contractions ( Figure 2). Peptides d-4d, l-4b and l-4f, all exhibited trends suggesting protective effects against PGF 2α -induced contractions. Mean tension induced by KCl (as a positive control) was not affected by l-4d.
Biology
All the peptides were initially tested in an ex vivo mouse myometrial tissue contraction assay. Contractile activity was measured on tissue that was harvested post-partum and treated with prostaglandin F2α (PGFα) alone (control) or in the presence of peptides L-4b-4g, L-7, and L-8. Among all the peptides tested, only N-dodecyl-L-PDC31 (L-4d) exhibited a significant reduction of contractions ( Figure 2). Peptides D-4d, L-4b and L-4f, all exhibited trends suggesting protective effects against PGF2α-induced contractions. Mean tension induced by KCl (as a positive control) was not affected by L-4d. Peptide l-4d was further examined for stability in serum and compared to l-PDC31. The two peptides were incubated at 37 • C in mouse plasma. Aliquots were removed at different time intervals and analyzed by mass spectrometry (MS/MS) to detect remaining amount of peptide ( Figure 3). Although l-PDC31 was completely degraded within 5 minutes, the N-alkylated peptide l-4d exhibited a half-life of about 30 minutes and was still present after 2 hours of incubation. The N-dodecyl group substantially improved the stability of the peptide in plasma, likely by conferring resistance to proteolysis.
Peptide L-4d was further examined for stability in serum and compared to L-PDC31. The two peptides were incubated at 37 °C in mouse plasma. Aliquots were removed at different time intervals and analyzed by mass spectrometry (MS/MS) to detect remaining amount of peptide (Figure 3). Although L-PDC31 was completely degraded within 5 minutes, the N-alkylated peptide L-4d exhibited a half-life of about 30 minutes and was still present after 2 hours of incubation. The N-dodecyl group substantially improved the stability of the peptide in plasma, likely by conferring resistance to proteolysis. The ability of peptide L-4d to delay labor in vivo was next tested in mice near term and treated with lipopolysaccharide (LPS) Escherichia coli endotoxin, as a pathogen associated molecular pattern, which is known to promote a general inflammatory state resulting in prostaglandin synthesis and induced premature delivery. Following LPS injection into mice (n = 4-5) at gestational day 16, all the animals tested delivered within 12−24 h. In contrast to PDC-31 and small molecule mimics, which have previously been shown to delay labor in the PTB model [54][55][56], L-4d was unable to exhibit a statistically significant reduction in the time of delivery (Figure 4). The ability of peptide l-4d to delay labor in vivo was next tested in mice near term and treated with lipopolysaccharide (LPS) Escherichia coli endotoxin, as a pathogen associated molecular pattern, which is known to promote a general inflammatory state resulting in prostaglandin synthesis and induced premature delivery. Following LPS injection into mice (n = 4-5) at gestational day 16, all the animals tested delivered within 12−24 h. In contrast to PDC-31 and small molecule mimics, which have previously been shown to delay labor in the PTB model [54][55][56], L-4d was unable to exhibit a statistically significant reduction in the time of delivery (Figure 4).
Peptide L-4d was further examined for stability in serum and compared to L-PDC31. The two peptides were incubated at 37 °C in mouse plasma. Aliquots were removed at different time intervals and analyzed by mass spectrometry (MS/MS) to detect remaining amount of peptide (Figure 3). Although L-PDC31 was completely degraded within 5 minutes, the N-alkylated peptide L-4d exhibited a half-life of about 30 minutes and was still present after 2 hours of incubation. The N-dodecyl group substantially improved the stability of the peptide in plasma, likely by conferring resistance to proteolysis. The ability of peptide L-4d to delay labor in vivo was next tested in mice near term and treated with lipopolysaccharide (LPS) Escherichia coli endotoxin, as a pathogen associated molecular pattern, which is known to promote a general inflammatory state resulting in prostaglandin synthesis and induced premature delivery. Following LPS injection into mice (n = 4-5) at gestational day 16, all the animals tested delivered within 12−24 h. In contrast to PDC-31 and small molecule mimics, which have previously been shown to delay labor in the PTB model [54][55][56], L-4d was unable to exhibit a statistically significant reduction in the time of delivery (Figure 4).
Discussion
The d-peptide PDC-31 was previously shown to reduce both the strength and length of spontaneous and PGF2α-induced contractions in postpartum mice myometrium [18]. Examination of peptide conjugates l-4b-g, l-7 and l-8 for ability to inhibit PGF2α-induced contractions in the same assay demonstrated that N-dodecyl peptide l-4d was able to reduce uterine contractions induced by PGF2α in mice myometrial tissue [57]. Moreover, addition of the dodecyl chain enhanced plasma stability. In contrast, the parent L-peptide, l-PDC-31, did not show any inhibitory activity against PGF2α-induced contractions and was rapidly degraded in plasma. Myometrial tissue is rich in proteases [58], which may degrade and render the linear l-peptide inactive. The related N-hexanyl and N-octadecanyl peptides l-4b and l-4f exhibited trends in the myometrium contraction assay which may suggest an inhibitory potential, but the results were not statistically significant (Figure 2). Octadecanyl peptide 4f was poorly soluble in water and needed slight amounts of DMSO to be tested, which, in turn, could explain the poor activity in the ex vivo assay. Neither PEGylated peptide l-4g nor N-farnesyl peptide l-7 exhibited activity. Similarly, N-dodecanoyl peptide l-8 and its HCl salt were both inactive. The latter result was consistent with the inactivity of N-acetyl-PDC31 [19] and highlighted the importance of a basic amine terminal for peptide activity.
In addition to N-alkylation rendering the L-peptide more stable to degradation in plasma, the lipid chain may compliment the hydrophobic (Ile-Leu-Gly) N-terminus to anchor the peptide in cell membranes and enhance activity and affinity for the membrane-bound receptor. To explore the latter hypothesis, the dodecyl chain was added onto PDC-31 to give D-4d. Although it exhibited a tendency similar to the activity of l-4d, d-4d affected myometrial contraction with a lower potency than the unmodified d-peptide, PDC-31 [18]. Thus, the alkyl chain appears to interfere with receptor binding [18] and likely exhibits its effect by enhancing resistance to proteolytic degradation.
Although activity was maintained and plasma stability was improved by the addition of a dodecyl chain to l-PDC-31, l-4d failed to prolong labor in vivo in the LPS-induced mouse model. The high lipophilicity of the dodecyl chain may inhibit delivery to the target receptor; instead, l-4d may be trapped in adipose tissue or cellular membranes, an effect that has been reported in other lipid-peptide conjugates [59].
Automated Peptide Synthesis
Peptide synthesis was performed using a CEM Liberty microwave peptide synthesizer (CEM, Matthews, NC, USA), employing the following protected amino acids: Fmoc-Lys(Boc)-OH, Fmoc-Tyr( t Bu)-OH, Fmoc-Asp( t Bu)-OH, Fmoc-Cit-OH, Fmoc-His(Tr)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, and Fmoc-Ile-OH. The Fmoc group was removed by using two treatments with 20% piperidine in DMF with microwave irradiation at 75 • C (35W) at first for 30 sec, and then for 180 sec in the second treatment. After the resin was washed (4 × 25 mL DMF), couplings were performed in DMF with N α -Fmoc amino acid (300 mol%, 0.2 M), HBTU (300 mol%, 0.5 M) and DIEA (600 mol%, 2 M). Typically, the N α -Fmoc amino acid solution (30 mL, 6 mmol) was added to the resin, followed by the HBTU (12 mL, 6 mmol) and DIEA (6 mL, 12 mmol) solutions. For all the amino acids except histidine, the coupling cycle was performed with irradiation at 75 • C (25W) for 300 sec. In the case of histidine, the coupling cycle was performed without irradiation initially at 50 • C for 120 sec, followed by irradiation at 50 • C (25W) for 240 sec. The resin was washed (3 × 25 mL DMF) prior to each coupling step. All the residues were coupled using a single reaction cycle. Upon completion of the sequence, the resin was washed with 10 mL of DCM and transferred to the receiving flask using 3 × 10 mL volumes of DCM. The final Fmoc group removal was done manually by swelling the resin in a solution of 20% piperidine in DMF (25 mL) and agitating for 30 min. The resin was filtered and washed with DMF (3 × 25 mL), isopropanol (3 × 25 mL), dichloromethane (3 × 25 mL) and ethyl ether (1 × 30 mL), dried under vacuum and stored at −20 • C.
Serum Stability Study
Serum was warmed and kept on ice throughout the procedure. Two blanks were prepared by adding 5 µL of DMSO to 95 µL of serum. These were incubated for 2 h and submitted to the same treatment as the other samples. Two series of vials containing 95 µL of serum were prepared and spiked with either 5 µL of l-PDC31 [0.4 mM] or l-4d [0.4 mM], and incubated for 120, 60, 30, 15, 10 and 5 min, respectively, after which 100 µL of 4% phosphoric acid was added. Two vials were also spiked with 5 µL l-PDC31 and L-4d, respectively, and immediately treated with phosphoric acid (time = 0 min). The vials were incubated for 5 min at 95 • C, cooled on ice for 2 min, treated with 600 µL of cold MeCN containing l-4f (0.02 mM) as internal standard, and incubated at -20 • C overnight. After the incubation period, the vials were vortexed for 2 min and centrifuged at 4 • C for 15 min. The supernatant was transferred into LC-MS vials and analyzed.
Results were expressed as: AN and IS refer, respectively, to the analyte and internal standard MS signals. Both values are compared with initial injection at which time AN 0 and IS 0 were not incubated.
Ex Vivo Study
Pregnant CD-1 mice (16-17 days gestation, term 19 days) were obtained from Charles River Inc. and used according to a protocol of the Animal Care Committee of Hôpital Sainte-Justine according to the principles of the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The animals were maintained on standard laboratory chow under a 12:12 light:dark cycle and allowed free access to chow and water.
The uterus from the CD-1 mouse was obtained from the animal immediately after term delivery under anesthesia (2.5% isoflurane). Briefly, a midline abdominal incision was made and the uterine horns were rapidly excised, cleansed carefully of the surrounding connective tissues, and removed. Longitudinal myometrial strips (2 to 3 mm wide and 1 cm long) were dissected free from the uterus and mounted isometrically in organ tissue baths. The initial tension was set at 2 g. The tissue baths contained 20 mL of Krebs buffer of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl 2 , 0.9 MgSO 4 , 1 KH 2 PO 4 , 11.1 glucose, and 23 NaHCO 3 (pH 7.4). The buffer was equilibrated with 95% oxygen/5% carbon dioxide at 37 • C. Isometric tension was measured by a force transducer and recorded by a BIOPAC data acquisition system (BIOPAC MP150, Montreal, PQ, Canada). Experiments were begun after 1 h equilibration. Mean tension of spontaneous contractions was measured using a BIOPAC digital polygraph system (AcqKnowledge); the same parameters were also determined after addition of PGF 2α in the presence or the absence of a 20 min-pretreatment with different FP inhibitors. At the start of each experiment, mean tension of spontaneous myometrial contractions was considered as a reference response. Changes in mean tension (g) were expressed as percentages of the initial reference response (% of baseline).
At the start of each experiments, mean tension of spontaneous myometrial contractions was considered as a reference response. Increase in mean tension (%) was expressed as percentages of (X/Y)-100, where X is the change in mean tension (g) induced by PGF 2α and Y is the initial reference response (g). Data are representative of 4-6 experiments per treated group. All results are expressed as means ± SEM and were compared by Independent t-tests. Statistical tests were performed with GraphPad Prism 4.3 software and p < 0.05 was considered statistically significant.
In Vivo Study
Timed-pregnant CD-1 mice at 16 days gestation (normal term is 19.2 days) were anesthetized with isoflurane (2%). Primed osmotic pumps (Alzet pump, Alzet, Cupertino, CA) containing either saline (n = 4) or compound l-4d (20 mg/day/animal, n = 5) were respectively subcutaneously implanted on the backs of the animals; infusion of peptide was immediately preceded by bolus injection of peptide (0.1 mg/animal intraperitoneally). Within 15 min after placement of the pumps, animals were injected with lipopolysaccharide (LPS) Escherichia coli endotoxin (10 µg/animal intraperitoneally) to mimic the inflammatory/infectious component of human preterm labor. Animals were inspected every hour for the first 18 h and every 2 h thereafter to document the timing of birth. Results are expressed as percentages of animals delivered following the injection of LPS [57]. All the experiments were approved by the Animal Care Committee of Centre Hospitalier Universitaire Sainte-Justine (Montreal, QC, Canada).
Conclusions
Herein, we report effective solid-phase methods for installing alkyl, PEG, farnesyl and alkanoyl N-terminal grafts onto small peptides. Evaluation of the peptide derivatives ex vivo has identified that N-dodecyl peptide l-4d exhibited a significant reduction of PGF2α-induced contractility in post-partum ex vivo assay versus vehicle. Moreover, in mouse plasma, the dodecyl chain prolonged stability of l-4d due likely to a protective effect against proteases. Although the strategy proved successful ex vivo and in vitro, N-dodecyl peptide l-4d was inactive in vivo when given by subcutaneous injection to induced mice. Considering the proof that l-PDC31 can exhibit activity when conjugated to a dodecyl chain, further research is merited to study alternative means of administration and conjugation towards the development of a cost-effective tocolytic agent for treating preterm labor. | v3-fos-license |
2018-04-22T17:05:56.164Z | 2013-05-31T00:00:00.000 | 5028516 | {
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} | pes2o/s2orc | Research of Quality Standards for Stachydrine Hydrochloride in Chinese Medicine TJF Granule
A high-performance liquid chromatographic method was applied to the determination of stachydrine hydrochloride concentration in TJF granule (Chinese name: Tiao-Jing-Fang), using a mobile phase of methanol-acetonitrile (50:50, v/v) by the Agilent Kro-masi NH2 column (250 mm × 4.6 mm, 5 μm, S/N: 22N25110). Detection wavelength was 201 nm. The result revealed good linearity of stachydrine hydrochloride and was obtained within the range of 0.20 1.98 μg/mL (R = 0.9995). The average recovery was 97.01%; the relative standard deviation (RSD) was 0.19%. To the best of our knowledge, this is the first report dedicated to the determination of stachydrine hydrochloride by the evaporative light scattering detector-high-performance liquid chromatographic (ELSD-HPLC) method.
Since any natural product considered for its therapeutic value must be evaluated for consistency, a proper identification of the herbal ingredients in each preparation or medical product is an obvious requirement.Concentrated pharmaceutical herbal products and the decoction herbal products have been widely adopted for clinical use in most Asian countries like: China, Malaysia, Mongolia, Japan, Korea, and even in certain countries of the whole world.
Herbal products offer an alternative to Western medicines and are often considered to be non-toxic by the general public.A major criticism of herbal products is that they fail to meet modern quality assurance standards established for conventional medicines.Thus, quantitative determination of the active or marker components in medicinal herbs is a compulsory element for quality con-trol.Several high-performance liquid chromatographic (HPLC) methods have been developed for the determination of stachydrine hydrochloride in herbs using silica columns [10,12] or reversed-phase C18 columns [11,12].The aim of this current study is to develop a simple, reliable routine analytical method for the quality control of herbal medicine preparations by determining the stachydrine hydrochloride concentration in Herba Leonuri, and a traditional herbal preparation named TJF (Chinese name: Tiao-Jing-Fang) granule.
The mobile phase consisted of methanol-acetonitrile (50:50, v/v), its flow-rate was 1 ml /min.The mobile phase was filtered through a Millipore 0.45 μm filter and degassed prior to use.The detection wavelength was set at 201 nm.
Herba Leonuri Identification by TLC
Preparation of reference solution: To get stachydrine hydrochloride standard substance, add ethanol and make a solution into the concentration of 2 mg/1 mL.
Preparation of TJF Granule sample solution: To get 6 g TJF Granule, add ethanol 30 mL, extracted 60 minutes twice by hot reflux extraction method, filters and combine the filtrate, concentrates evaporation to dryness, dissolves the residue in 0.5 mL ethanol for analysis by TLC as TJF Granule sample solution.
Preparation of negative control product solution: To get 6 g Negative control product granule (TJF Granule without Herba Leonuri), add ethanol 30 mL, extracted 60 minutes twice by hot reflux extraction method, filters and combine the filtrate, concentrates evaporation to dryness, dissolves the residue in 0.5 mL ethanol for analysis by TLC as negative control product solution.
Preparation of chromogenic agent (diluted iodized bismuth potassium solution): To get 0.85 g Bismuth subnitrate, adds 10 ml glacial acetic acid and dissolves into 40 ml Distilled water-that is the former solution.Take 5 ml former solution before use, add 5 ml potassium iodide solution (4 → 10) and 20 ml glacial acetic acid, dilute into 100 ml solution by Distilled water.(China Pharmacopoeia Committee, 2005).
According to the thin-layer chromatography (TLC) test in appendix VIB of the first section of Chinese Pharmacopoeia 2005 Edition, accurate absorb 20 μL negative reference substance solution and TJF Granule sample solution respectively, respectively point in the same silica gel plate, developing with the developing solvent N-butanol-Hydrochloric acid-Distilled water (4:1:0.5),developing distance is about 8 cm, remove and dry, spraying with the test solution of diluted iodized bismuth potassium solution, heating in 105˚C until characteristic spots appear clearly.
Result: In the identification test there's the same colour speck in the preparation Chromatography while there's no the same colour speck in the control sample.The result is shown in Figure 2.
Number of theoretical plates calculated by chromatographic peak of stachydrine hydrochloride would be no Preparation of sample solution: to get TJF Granule 3 g, weighting accurately, adding ethanol 30 mL, heating to reflux for 2 times, I hour for each time, filtering after being cooled, merging of filtrates, concentrating the mixed filtrate to a moderate amount, adding ethanol to dissolve, moving into a 5 ml volumetric flask, dilute with ethanol to volume, and mix, filter, take the subsequent filtrate as the sample solution.
Preparation of negative control product solution: To weight all the ingredients of TJF Granule except the Herba Leonuri, preparing the negative reference substance solution by the same way of TJF Granule sample solution preparation.
3) Determination method Respectively accurate absorb reference substance solution 10 μL, 20 μL; TJF Granule sample solution 10 μL; and negative reference substance solution 10 μL; inject into the high performance liquid chromatograph according to the chromatographic condition which above-mentioned and test.
4) Linear relationship inspection
To get moderate stachydrine hydrochloride standard substance (The National Institute for Food and Drug Control, batch number: 110712-200709), weighting accurately, adding ethanol to make a solution with concentration of 0.2 mg/mL.Respectively accurate absorb above-mentioned solution 1000 μL, 500 μL, 125 μL, 63 μL and 32 μL, move them into 5 mL volumetric flasks respectively, dilute with ethanol to volume and mix, prepare these solutions as the series of standard substance solutions.
Series of standard substance solutions are injected respectively, and analysis according to the chromatographic condition based on the "Determination of Stachydrine Hydrochloride by HPLC" item, record peak area, calculate regressively through sample concentration (μg/mL) as the abscissa, and logarithm of peak area as the ordinate.Result displays a good linear relation is shown within the detection range of stachydrine hydrochloride.Linear equation is: y = 0.041x + 2.882, R = 0.9995; linear range is: 0.20 -1.98 μg/mL.Since the standard curve is not pass the origin, external standard dual point method is adopted to determinate the content of Stachydrine hydrochloride standard substance in products.The result is shown in Figure 3 and Table 1.Reference substance solution has been absorbed accurately, solutions are injected 5 times continuously.The intra-day coefficient of variation (CV) is calculated by testing the relative standard deviation (RSD) for each chromatographic peak's logarithm of peak area (lgA).The result is shown in Table 2.
6) Stability Test
To get TJF Granule sample solution from same batch number, solutions are injected in 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours respectively.Test result shows that TJF Granule sample solution is stable within 24 hours.The result is shown in Table 3.
7) Repeatability Test TJF Granule has been weighted accurately, preparing the sample solution by the same way of TJF Granule sample solution preparation to test the repeatability of determination method.RSD is 0.66%, which shows a good repeatability.The result is shown in Table 4.
8) Recovery Test
To get 0.5 g TJF Granule which content has been calculated as a sample, weighting 9 samples; accurately absorb moderate reference substance solution; preparing the sample solution by the same way of TJF Granule sample solution preparation; calculate the recovery of Stachydrine hydrochloride.The result is shown in Table 5.
9) TJF Granule Determination
To get TJF Granule 5 g, weighting accurately, adding ethanol 40 mL, heating to reflux for 2 times, 1 hour for each time, filtering after being cooled, merging of According to the determination results of Stachydrine hydrochloride for different batch number of TJF Granule, select the average number with a 20% decrease of the Stachydrine hydrochloride content in 3 batch numbers of TJF Granule, temporary define no less than 3.80 mg Stachydrine hydrochloride should be tested for each bag of TJF Granule.
Experimental Result of Stachydrine
Hydrochloride in TJF granule RESULT: Good linearities of stachydrine hydrochloride and was obtained within the range of 0.20 -1.98 μg/mL (R = 0.9995; the average recoveries were 97.01%; RSD were 0.19%.
Discussion and Conclusion
DISCUSSION: Determination method of stachydrine hydrochloride: scanning result of stachydrine hydrochloride standard substance tested by the ultraviolet spectrophotometer displays the wavelength of maximum absorption is 201 nm, which is less than the cut-off wavelength of most mobile phases like methanol.Hence the ultraviolet detector is not suitable for the test, while the evaporative light scattering detector (ELSD) should be adopted for determination.
In the first section of Chinese Pharmacopoeia 2005 Edition, determination method of Extractum Leonuri Liquidum is thin-layer chromatography scarmlng method (TLCS method).The ELSD-HPLC method is more convenient on operation and takes less time.Through the experiment, when the C18 reversed phase column is used on determination, chromatographic peak of Stachydrine hydrochloride has a severe zone tailing phenomenon, therefore, the NH 2 column is adopted instead of the C18 reversed phase column.
The developing distance of Herba Leonuri Identification by TLC according to the first section of Chinese Pharmacopoeia 2005 Edition is not satisfied, the detection condition is not complete yet, and needs screening conditions continuously.
CONCLUSION: This method can be used for quality control of TJF granule.
Table 5 . The result of recovery test.
twice, calculate the content according to the external standard dual point method and select the average.The result is shown in Table 6 and Figures 4-6. | v3-fos-license |
2018-04-03T05:48:46.908Z | 2017-01-24T00:00:00.000 | 23803117 | {
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} | pes2o/s2orc | Enhanced Oral Bioavailability of Domperidone with Piperine in Male Wistar Rats: Involvement of CYP3A1 and P-gp Inhibition.
- Purpose: Domperidone is a commonly used antiemetic drug. The oral bioavailability of domperidone is very low due to its rapid first pass metabolism in the intestine and liver. Piperine, the main alkaloid present in black pepper has been reported to show inhibitory effects on Cytochrome P-450 (CYP-450) enzymes and P-glycoprotein (P-gp). In the present study we investigated the effect of piperine pretreatment on the intestinal transport and oral bioavailability of domperidone in male Wistar rats. Methods : The intestinal transport of domperidone was evaluated by an in-vitro non-everted sac method and in-situ single pass intestinal perfusion (SPIP) study. The oral pharmacokinetics of domperidone was evaluated by conducting oral bioavailability study in rats. Results : A statistically significant improvement in apparent permeability (Papp) was observed in rats pretreated with piperine compared to the respective control group. The effective permeability (Peff) of domperidone was increased in the ileum of the piperine treated group. Following pretreatment with piperine, the peak plasma concentration (C max ) and area under the concentration- time curve (AUC) were significantly increased. A significant decrease in time to reach maximum plasma concentration (T max ), clearance and elimination rate constant (K el ) was observed in rats pretreated with piperine. Conclusions: Piperine enhanced the oral bioavailability of domperidone by inhibiting CYP3A1 and P-gp in rats. This observation suggests the possibility that the combination of piperine with other CYP3A4 and P-gp dual substrates may also improve bioavailability. Further clinical studies are recommended to verify this drug interaction in human volunteers and patients.
INTRODUCTION
Oral administration has been the most common and convenient route for administration of drugs because of its high patient compliance in combination with a relatively simple and cost efficient manufacturing process. The design of a new dosage form generally depends on the pharmacokinetic and pharmacodynamic parameters of the drug (1). Some drugs remain sub-therapeutic when administered orally if they are poorly bioavailable. This poor oral bioavailability of a drug may be due to metabolism by their corresponding enzymes like CYP450 and efflux from their site of action by P-glycoprotein (P-gp). The major portion of a given dose may never reach the systemic circulation to exert its pharmacological effect until and unless very large doses are given. Lowering the dose and dosing frequency of such drugs can be achieved by significantly improving the oral bioavailability of drugs. Plant-based molecules are most widely used as bioavailability enhancers in combination therapy (2). Following the use of natural bioenhancers, the dose of the drug may be reduced, the risk of drug resistance may be minimized and also the dose-dependent toxicity of the drug, especially with anticancer drugs may be reduced.
The increase in consumption of herbal products as complementary and/ or alternative medicine has been observed during the last decade. Herbal products are generally considered as natural and safe with minimal adverse effects (3,4). The concomitant use of herbal drugs along with prescription drugs may lead to possible herb -drug interactions. _________________________________________ The ability of intestinal and hepatic drug transporters to efflux drugs and drug metabolizing enzymes to metabolize drugs is often responsible for poor oral bioavailability. This may lead to herbdrug and food-drug interactions for numerous drugs. The risk of herbal drug interactions possesses two major extreme challenges -adverse toxic reactions and therapeutic failure. Drug toxicity can result from inhibition of drug efflux transporters (Pgp) and metabolic enzymes (CYP-450) responsible for the increase in systemic exposure of drugs. Therapeutic failure can result from induction of efflux transporters and metabolic enzymes leading to faster efflux and metabolism of drugs (5). The close chromosomal location of P -glycoprotein and CYP3A4 (analogous enzyme in rat is CYP3A1) genes, their expression and similar substrate specificities in matured enterocytes suggest that these two proteins may be complementary in nature to form a coordinated intestinal barrier (6).
Black pepper (Piper nigrum) is one of the most widely used spices in human diets. Piperine, the main active constituent present in black pepper, is an alkaloid contributing pungency to the pepper. Black pepper is considered as king of spices because of its high volume of international trade (7). Bhardwaj et al., studied the influence of piperine on human CYP450 by human liver microsomes and reported that piperine inhibited the major drug metabolizing enzyme CYP3A4 (8).
Domperidone oral bioavailability is reported to be around 20-25 %. The innate low oral bioavailability of domperidone is due to drug metabolizing enzymes (CYP3A4) and efflux transporters (P-gp) which are located in epithelial cells (enterocytes) lining the small intestine and in the parenchymal cell of the liver (hepatocytes) (9). Consequently, orally administered domperidone can be metabolized twice before reaching the systemic circulation. Thus, oral bioavailability can be markedly attenuated. Significant difference in absorption of domperidone on pretreatment with silymarin was observed due to the inhibition of P-g and CYP3A (10). Domperidone stimulates gastrointestinal motility and is used as an antiemetic for the short-term treatment of nausea and vomiting of various etiologies, including that associated with cancer therapy and with levodopa or bromocriptine therapy for Parkinson's disease (11,12).
The aim of the present study was to observe the influence of piperine pretreatment for 8 days on the intestinal permeation of domperidone in male Wistar rats in vitro, ex vivo and to observe the effect of piperine pretreatment for 8 days on the oral bioavailability of domperidone in male Wistar rats.
MATERIALS AND METHODS
Domperidone was kindly donated by Sun Pharmaceuticals, Baroda, India. Propranolol and verapamil were procured from Lupin Labs Pharmaceuticals Inc, Pune, India). Phenol red was purchased from Himedia (Ghatkopar West, Mumbai, India), and methanol HPLC and acetonitrile HPLC (E. Merck Ltd Vikrolo, East Mumbai, India) were used. All other chemicals used were of AR grade.
Male Wistar rats weighing about 220-240 g were purchased from Mahaveera Enterprises, Ghatkesar road, Hyderabad, India. Rats housed in cages were kept in a room under controlled temperature (20-22º C) and 12 h day-night cycle. Animals were used for studies after 1-week acclimatization with free access to water and feed. The animal study protocol was reviewed and approved by Institutional Animal Ethics Committee of Kakatiya University (Vidhyaranyapuri, Warangal, IAEC, TS, and India). All the animals were grouped and treated with the following regimens.
The rats were divided into three groups Group I(control): All the rats in this group were pre-treated orally with 0.25% w/v sodium carboxy methyl cellulose (Sod.CMC) for 7 days and also on the 8 th day, 1hr prior to the study (non-everted and perfusion studies). Group II (piperine treated): The rats received piperine (20mg/kg/oral) suspension (0.25%w/v Sod.CMC) for 7 days and also on the 8 th day 1hr prior to the study (non-everted and perfusion studies). Group III (verapamil treated): Verapamil (100µM) was co-administered along with buffer containing domperidone (100µg/ml).
Non-everted sac (normal sac) study
Male Wistar rats were fasted overnight with free access to water before the experiment. Their intestines were flushed with 50 mL of ice-cold saline with the animal under anesthesia using thiopental sodium (50mg/kg/ip). The rat was exsanguinated, the ileum was isolated and a segment was cut into a length of 10cm for preparation of the sac. One hundred microgram per milliliter (100µg/mL) of domperidone (probe drug) was prepared by dissolving domperidone in pH 7.4 isotonic Dulbecco's Phosphate Buffer Saline (D-PBS) containing 25 mM glucose and 0.5 % of DMSO. The probe drug solution (1mL) was introduced into the non-everted sac (mucosal side), and both ends of the sac were ligated tightly. The sac containing the probe drug solution was immersed into 40 mL of D-PBS containing 25 mM glucose and the same concentration of DMSO was used. The medium was pre-warmed at 37 O C and pre-oxygenated with 5% CO 2 / 95% O 2 for 15 minutes. Under bubbling with a CO 2 /O 2 mixture gas, the transport of the domperidone from mucosal to serosal surfaces across the intestine was measured by sampling the serosal medium periodically for 120 minutes. Samples of 1 mL were collected at pre-determined time intervals from the serosal medium and replenished with fresh buffer (13). The drug transported was measured using high performance liquid chromatography (HPLC).
Apparent permeability coefficient (P app ):
The apparent permeability coefficient for domperidone was calculated from the equation given below.
where, dQ/dt is the rate of drug transport (domperidone) from mucosal to serosal medium, A is the surface area of the intestinal sac used for the study and C0 is the initial concentration of drug present in the intestinal sac (14).
In situ, single-pass intestinal perfusion (SPIP) study
The in situ single-pass intestinal perfusion (SPIP) study was performed according to the previously reported methods (15,16,17). Briefly, the rats were divided into three groups: control, standard inhibitor and pretreatment consisting of six animals each. Piperine suspension was administered orally to the pretreated group at a dose of 20 mg/ kg for 8 days and verapamil(100µM) solution was concomitantly administered along with domperidone (100mcg/mL), and the other group was kept as control. The perfusion study was conducted on the 8th day after piperine pretreatment. Rats were anesthetized with thiopental sodium (50 mg/kg, ip) and they were placed on a warm pad to maintain normal body temperature. A midline incision of 3-4 cm was made on the abdomen of rats and an ileum segment of approximately 8-12 cm was isolated using the ileo-caecal junction as a distal marker. Semicircular incisions were made at each end of the ileum and the lumen was rinsed with normal saline (37 ºC) and both ends were cannulated with polyethylene tubing and ligated using a silk suture. Subsequently, blank perfusion buffer (phosphate buffer saline, pH 7.2) was first infused for 5 min at a flow rate of 1 mL/min using a syringe pump (NE-1600, New Era Syringe Pumps, Inc. NY, USA), followed by perfusion of phosphate buffer saline (pH 7.2) containing domperidone (100µg/ml), propranolol (100 µM) and phenol red (50 mg/mL) at a constant flow rate of 0.2 mL/min for a period of 90 min and the perfusate was collected at 10 min intervals. After completion of cannulation, the ileum segment was covered with isotonic saline-wet gauze (37ºC). At the end of the perfusion, the length of the ileum segment was measured following the last sample collection. Perfusion samples were collected from the control and pretreatment groups at predetermined time points and stored at -80ºC until analysis. Domperidone concentrations in perfusion samples were analyzed by HPLC (10).
Phenol red water flux correction
The corrected outlet concentration (C out (corr)) for domperidone was calculated from the following equation (15).
where, C out is outlet concentration of the drug, whereas CPR in and CPR out are the concentrations of phenol red entering and exiting the rat intestinal segment, respectively.
Effective permeability coefficient (P eff )
The effective permeability coefficient of domperidone was calculated from the following equation.
Where, Q is the perfusion flow rate, , C in is the inlet drug concentration, r is the radius of the rat small intestine and L is the length of the perfused intestinal segment (13).
Peff was estimated from the steady state concentration of compounds which is considered to be attained when the concentration of phenol red in the perfusate samples is stable. Generally, steady state was reached at 30-40 min after the beginning of the experiment.
In vivo bioavailability study in male Wistar rats
The animal study protocol was reviewed and approved by the institutional animal ethical committee, University College of Pharmaceutical Sciences, Kakatiya University, India. Male Wistar rats weighing 200 to 225g were selected for the study. The bioavailability of domperidone after pretreatment with piperine (dose of 20 mg Kg -1 for eight days) was compared with an oral dispersion (domperidone 10 mg Kg -1 in 0.25%w/v Sod.CMC suspension). The rats were allowed free access to food and water, until the night prior to dosing and were fasted for 10 h. In the first group, oral domperidone solution (2.5 mg mL -1 ) was administered through a feeding needle, the second group was pretreated with piperine for 8 days and for the third group, verapamil was used. Blood samples (0.5 mL) from a retro-orbital vein were collected at preset intervals of 0, 0.5, 1, 2, 3, 6, 12 and 24 h respectively, after administration of oral solution and after pretreatment with piperine. All blood samples were allowed to clot and centrifuged for 10 min at 4000 rpm. The serum was separated and transferred into clean micro-centrifuge tubes and stored at -20° C until HPLC analysis. The concentration of domperidone in the samples was determined by HPLC.
Serum and perfusion samples analysis
Domperidone in serum and perfusion samples were estimated by a reverse phase HPLC method (10).
Extraction procedure for serum samples
To 250 µl serum, 20 µl of propranolol (50µg/ml) was added as an internal standard and vortexed for 2 minutes. An equal volume of methanol was added to serum samples for protein precipitation and vortexed on a cyclomixer (Remi Elektrotechnik Limited, Valiv, Thane, India) and centrifuged at 35000 rpm for 8 minutes using a Biofuge Fresco (Buckinghamshire MK169QS, England) centrifuge. Twenty microliters of serum supernatant were taken up into a Hamilton syringe (100µL, Spinco Biotech, Habsiguda, Hyderabad, India) and injected directly into the HPLC.
Analysis of phenol red by spectroscopy
The concentration of phenol red in the perfusate was measured using a UV-Visible spectrophotometer (UV-Visible spectrophotometer, Elico, Sanathnagar, Hyderabad, India) at a maximum absorption wavelength of 560nm (15).
Analysis of pharmacokinetic parameters
All the pharmacokinetic parameters were analyzed using Phoenix WinNonlin version 6.2 software (Certara, Pharsight Corporation, L.P, USA). The statistical analysis was performed using one-way analysis of variance (ANOVA) and GraphPad Prism version (6.07) (GraphPad Software Inc., San Diego, CA, USA) at a significance level of p < 0.05.
RESULTS
The piperine pretreatment for 8 days resulted in a significant increase in the mean cumulative concentration of domperidone from 0.91±0.02 to 1.88±0.07µg/mL, whereas for the verapamil (standard inhibitor) treated group, the mean cumulative concentration of domperidone was observed to be 2.45±0.14 µg/mL. The transport of domperidone was increased 2.1 and 2.7 times after pretreatment with piperine and verapamil respectively compared to their respective controls in the ileum region (Fig.1). A statistically significant (P<0.05) difference was observed in both cases. The piperine pretreatment for 8 days significantly increased the apparent permeability of domperidone. The apparent permeability of domperidone was increased 2.2 and 3.3 times after pretreatment with piperine and verapamil respectively compared to their respective controls (Table 1). A statistically significant difference was observed in both cases (P<0.05, Fig.2).
Effect of Piperine and verapamil on intestinal permeability of domperidone
Domperidone and propranolol intestinal effective permeability (Peff) were determined in the rat ileum segment using a single pass intestinal perfusion technique. Effective permeability values were calculated from the steady-state concentrations of compounds in the perfusate collected from the outlet. As shown in Fig. 3, pretreatment with piperine for 8 days (piperine treated group) and concomitant administration with standard inhibitor verapamil (verapamil treated group) resulted in a significant increase in effective permeability (p < 0.05) of domperidone while there was no significant change observed in the effective permeability of propranolol in the control, piperine pretreated and verapamil treated groups, respectively (Table. 1). The increase in effective permeability of domperidone in the ileum was found to be 4.1and 5.9-fold in pretreated and standard inhibitor groups, respectively as compared with that of the control groups (Fig.3). Propranolol was shown to have no interaction with P-gp as it is a highly permeable marker.
In vivo Study
All the rats tolerated the treatments well and there were no cases of severe adverse effects during the study period. The serum concentration-time profile of domperidone after oral administration of domperidone (10 mg/kg) in the control, piperine (20mg/kg) pretreated and verapamil treated groups were characterized and are depicted in Fig. 4. The mean pharmacokinetic parameters of domperidone are summarized in Table 2. Domperidone oral pharmacokinetics were found to be significantly (p < 0.05) altered with piperine pretreatment for 8 days compared to the control group. The increases in C max , AUC 0-24 and AUC 0-∞ of domperidone were found to be 1.6, 1.3 and 1.5 -fold respectively in the piperine pretreated group compared to that of the control group. T max decreased from 2 to 1 hrs (Fig.4). There was a statistically significant difference observed in the pharmacokinetic parameters, C max , T max, AUC 0-∞ , AUC 0-24, k el, and clearance.
DISCUSSION
Dietary supplements and foods, including fruits, vegetables, herbs, spices and teas that contain complex mixtures of phytochemicals have the greatest potential to induce or inhibit the expression and activity of drug-metabolizing enzymes. CYP450 enzymes may be particularly vulnerable to modulation by the multiple active constituents of foods, including dietary supplements (18). CYP3A4 is known to be involved in the most common fooddrug interactions, as demonstrated by reports of clinically important interactions involving orally administered drugs that are substrates of this enzyme (19).
The increased bioavailability is due to reduced metabolism which in turn may be due to a combined effect of inhibition of metabolizing enzymes belonging to CYP group and inhibition of the efflux transporter P-gp. Such enhanced absorption has been reported earlier by many researchers for different drugs with pretreatment with plant products containing anthocyanins, polyphenols and flavonoids (20).
From the last decade, the use of phytochemicals along with drugs as complementary and alternative medicine is increasing enormously considering that they are safe with minimal adverse effects. These phytochemicals can modulate various drug transporter systems and drug metabolizing enzymes which lead to possible herbal-drug interactions (3,21). Piperine, the main alkaloid present in black pepper and long pepper shows inhibitory effects on drug efflux transporter (P-gp) and one of the major drug metabolizing enzymes CYP3A4. Piperine has been reported to show an inhibitory effect on P-gp mediated efflux of digoxin and cyclosporine in Caco-2 cells and CYP3A4 mediated metabolism of verapamil in human liver microsomes (8). In the present study, the influence of piperine pretreatment on the intestinal transport and oral bioavailability of domperidone was observed.
The interaction between domperidone and piperine has been studied using in-vitro, in-situ and in-vivo models. The in-vitro non-everted rat intestinal sac model is the most direct method of identifying the transport of drug from apical to basolateral direction. A significantly lower permeability in the apical to basolateral direction provides evidence that some form of efflux transporter such as P-gp may be inhibiting the transport of test component (21). The results from a non-inverted sac study revealed that the transport of domperidone across the rat intestine is very much affected by piperine. In the present study, the mean± SD cumulative concentration of domperidone and apparent permeability were shown to increase after pretreatment with piperine for 8 days. Piperine showed a significant increase in oral bioavailability of fexofenadine by inhibiting P-gp mediated drug efflux in the rat intestine (22). This observation indicated the role of P-gp, an efflux pump, on domperidone absorption.
In situ intestinal perfusion studies were carried out on live animals to study the transport of compounds in a physiologically relevant environment in which the integrity of the organ is preserved (21). From this technique, we can study the actual extent of P-gp efflux that can be expected in-vivo. In the present study, the effective permeability of domperidone was significantly increased after pretreatment with piperine. Phenol red, a non-absorbable marker and propranolol, a highly passive permeable marker were used to provide information on the integrity of the intestinal membrane (13,15,17). Statistically significant differences were not observed in the permeability of propranolol for the control, verapamil and piperine treated groups which indicates that there was no change in membrane integrity during the perfusion study.
The results from the in-vivo oral pharmacokinetic study showed a significant increase in AUC, C max and volume of distribution. The higher plasma levels in the absorption phase and earlier T max of domperidone may be due to inhibition of P-gp and Cyp3A1 across the rat intestine and liver. A significant decrease in clearance and elimination rate constant indicates that piperine could inhibit the hepatic elimination of domperidone. Piperine has been reported to decrease the clearance of midazolam due to an inhibitory effect of piperine on CYP3A4 (23). Pretreatment with piperine significantly enhanced the AUC and C max of domperidone which may be due to increased intestinal absorption of domperidone via the inhibition of P-gp mediated drug efflux, and CYP3A4 mediated drug metabolism. Piperine has been reported to decrease the metabolism of pioglitazone as a result of CYP3A4 and CYP 2C8 inhibition (24).
The issue of cardiotoxicity of oral domperidone has arisen recently. Domperidone has been reported to prolong the QTc interval and result in a predisposition to ventricular arrhythmias similar to that of class III anti-arrhythmic agents (25). Domperidone was found to be the drug of choice for treating gastrointestinal symptoms in patients with Parkinson's disease because there is a minimal risk of developing extrapyramidal side effects compared to other drugs. They reported that patients receiving a daily dose of above 30mg should be treated with special caution, considering its potential cardiac effects (26).
CONCLUSION
The improvement in absorption of domperidone may be due to the inhibition of P-gp and Cyp3A1 in the intestine and liver by piperine. Piperine enhanced the oral pharmacokinetics of domperidone, suggesting that combined use of piperine and domperidone may be useful in reducing the dose of domperidone and require close monitoring of potential drug interactions in cardiac patients. The combination of piperine with any other CYP3A4 and P-gp dual substrate may improve absorption of drugs which have poor oral bioavailability. Further studies are recommended to verify their influence in humans. | v3-fos-license |
2018-11-13T05:24:54.342Z | 2013-01-01T00:00:00.000 | 53629406 | {
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} | pes2o/s2orc | Nanoemulsions as a Vehicle for Drugs and Cosmetics
Nowadays, pharmaceutical and cosmetic industries have had great interest in nanoemulsions due to their characteristics, such as high kinetic stability; skin delivery efficiency of active substance or drugs; excellent sensorial and esthetic aspects, besides the need for less surfactant (5-10%) when compared to microemulsions, which reduces the possibility of skin irritation and production costs [1,2].
Introduction
Nowadays, pharmaceutical and cosmetic industries have had great interest in nanoemulsions due to their characteristics, such as high kinetic stability; skin delivery efficiency of active substance or drugs; excellent sensorial and esthetic aspects, besides the need for less surfactant (5-10%) when compared to microemulsions, which reduces the possibility of skin irritation and production costs [1,2].
Nanoemulsions are special systems with uniform and extremely small droplet size, in the range of 20-500nm.Due to their characteristic size, nanoemulsions can be optically transparent or translucent and have low viscosity that results in excellent spreadability and humectation.These benefits make them very interesting to be used in many applications, for example, in cosmetic and pharmaceutical field as personal care or skin care formulations and as drug delivery systems, respectively.
Different oils can be used in order to obtain nanoemulsions, like andiroba and copaiba oils.Both oils are widely used in cosmetics due to their emollient properties and compatibility with the lipids of the stratum corneum.Moreover, these oils have attractive properties to be used in skin care products.The andiroba oil (Carapa guianensis) and copaiba (Copaifera sp.) have been documented containing repellent action and may represent a safe alternative to synthetic repellents considering the risk of toxicity in certain cases.Andiroba oil is a natural product used by Amazonian Indians to ward off insects [3].Several studies have identified the presence of limonoids in the seeds of Carapa, which present insecticidal and repellent activity [4].Thus, it is interesting because thousands of people get sick and even are death victims each year, especially from mosquitoes that transmit diseases such as dengue and yellow fever.The mosquito-borne diseases affect more than 700 million people each year [5].According to the World Health Organization, two fifths of the population is at risk of being infected with dengue virus, and more than 100 countries have been affected by epidemics of dengue and dengue hemorrhagic fever [6].The use of repellents against Aedes aegypti is one of the strategies used to prevent and reduce the incidence of diseases transmitted by these arthropods [7].
Our nanoemulsion research is based on andiroba, copaiba and other vegetable oils, such as bran rice, green coffee, raspberry, sesame indicum, passion fruit, peach and urucum oils.Some additives like polyhydric umectants (glycerol, propylenglycol, sorbitol), lanolin derivatives (ethoxylated or acethylated), some specific cosmetic components (areca, licorice and portulaca extracts) and essential oil (lavender oil) have been used to obtain nanoemulsions.
The emulsification process can affect the droplet size, the distribution and the stability of nanoemulsions [8,9].Nanoemulsions can be obtained by high energy emulsification methods, where a shear rate is promoted in order to deform the particle [10,11,12] or by low energy emulsification methods, which explore the systems' physicochemical properties through the change in the spontaneous curvature of the surfactant with temperature variation (PIT-Phase Inversion Temperature) or the composition variation (PIC-Phase Inversion Composition) [8,13,14].Another method employs dilution of formulation that presents cubic phase structures.These nanoemulsions show the same size (nm) in all dilution [15].
In order to study the process of nanoemulsion obtainment, the following parameters [16] may be evaluated: 1) effect of different surfactant quantity; 2) influence of the order of component addition: the surfactant, oil and aqueous phase, 3) influence of temperature emulsification; 4) effect of agitation speed; 5) influence of the surfactant amount; 6) determination of the phase inversion temperature-determination of Emulsion Phase Inversion (EPI).
However, some studies show that surfactant blends are more effective than isolated ones, since the blends disperse and solubilize more the internal phase within the continuous one, being therefore possible to employ a smaller ratio of surfactant and produce more long-term stable emulsions [14,17].Some of the surfactant systems employed in our nanoemulsion research were based on castor oil derivatives with different molecules of ethylene oxide (15 to 50).Different nanosystems obtained in our research were characterized according some parameters cited before and also evaluated for anti-inflammatory, antioxydant and insect repellent activity and/or cosmetic applications.
Nanoemulsions containing sesame indicum and raspberry oils
Very stable nanoemulsions were obtained with sesame indicum and raspberry oil and non-ionic surfactants at HLB value 8.0 (Figure 1).These nanoemulsions showed stability for 90 days (Table 1).
In this study, with other data not shown, we could conclude that the surfactant type, composition concentration, addition order, temperature, speed and time of emulsification were critical factors in obtaining low polydispersity index of nanoemulsions.
Antioxidant activity:
The nanoemulsions of sesame oil or sesame and raspberry oil presented antioxidant activity of 68.71% and 67.75%, respectively [18].(Data not published).
In vitro evaluation: The Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method was performed in order to evaluate irritancy of nanoemulsions from blend of sesame and raspberry oil.The results revealed that nanoemulsions showed slightly irritating power and can be used as cosmetics [19,20].
In vivo evaluation:
The topical use of nanoemulsions containing andiroba and copaiba oils [19] displayed repellent activity compared to DEET (commercial product) (Figure 2).
The test repellent revealed that the nanoemulsions composed of andiroba oil and blend of oils (andiroba oil + copaiba oil) are able to repel mosquitoes Aedes aegypti for a period of 30 minutes.These results are statistically significant in the control group.
Nanoemulsions containing rice bran seed oil
In vitro evaluation: One of the evaluations performed in nanoemulsions containing rice bran seed oil was HET-CAM method.According this method, the nanoemulsions are also suitable to be used as cosmetics, since they presented slightly irritating power [19,20].
In vivo evaluation:
The nanoemulsions containing rice bran seed oil were evaluated by topical use.When they applied to the skin of volunteers, the nanoemulsion increased the relative hydration and the oiliness of skin and maintained normal pH values of skin.These nanoemulsions could serve as an alternative treatment for skin diseases, such as atopic dermatitis and psoriasis [20].
Addition of areca, licorice and portulaca natural extracts in babassu nanoemulsion:
The addition of these natural extracts (3.0%) in babassu nanoemulsion influenced the antioxidant activity.The nanoemsulsion added of these natural extracts presented antioxidant activity of 34.19 ± 1.03 compared to the same emulsion added of BHT (41.87 ± 0.77) (data not yet published).However, the addition did not influence the initial droplet size.These nanoemulsions showed high kinetic stability in 3 months (Figure 3).Moreover, the babassu nanoemulsions showed slightly irritating power according to HET-CAM method, being able to be used as cosmetic products [19,20].
Addition of lavender essential oil in the nanosystem containing passion fruit oil:
The addition of lavender essential oil (2.0%) in this nanosystem caused a reduction in the droplet size comparing to the nanoemulsions composed with only passion fruit oil (Figure 4).
Addition of lanolin derivatives in nanoemulsions based
on raspberry seed oil+ peach kernel oil+ passion fruit oil: the addition of lanolin derivatives, ethoxylated (EL) or acethylated (AL), in these nanoemulsions influenced droplet size and electrical conductivity of the systems, however, such changes did not compromise their stability, since the droplet size remained within the nanometer range (20-200nm) (Figure 5).Nanoemulsions added with EL were more stable than those with AL (Figure 6).Moreover, they are recommended to be employed as vehicle for pharmaceuticals and cosmetics.
Conclusion
According to this short communication -It is possible to observe the importance of studying the parameters that can influence the nanoemulsion obtainment;
-
The composition of nanoemulsion, and mainly, the oily phase can provide attractive properties for the products; -Nanoemulsions are an interesting and promising vehicle for drugs and cosmetics, as well as they hold wide development possibilities and numerous benefits in the pharmaceutical and cosmetic field.
Figure 1 :
Figure 1: Nanoemulsion containing Sesame indicum and raspberry oils (a) compared with purified water (b).Particle size: The sesame indicum and raspberry nanoemulsions had their droplet diameter size determined.The diameter sizes were determined with Delsa light scattering 440DX.The nanoemulsions presented diameter size between 50 and 150 nm.A mean diameter size of the nanoemulsions was 50nm (Table1).
Figure 4 :
Figure 4: Nanoemulsion particle size obtained with passion fruit oil (5.0%) and lavender essential oil (2.0%) exposed at different conditions as time function.
Figure 5 :Figure 6 :
Figure 5: Droplet mean size of nanoemulsions as a function of lanolin derivatives concentration.(A) EL and (B) AL.
Table 1 :
Droplet diameter size (nm) and polydispersity index of sesame nanoemulsions prepared at different temperature as function of time: 7, 15 and 90 days.ω= polidispersity index. | v3-fos-license |
2020-04-30T09:08:07.359Z | 2020-04-28T00:00:00.000 | 218474091 | {
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} | pes2o/s2orc | In Vitro Characterization of a Novel Human Acellular Dermal Matrix (BellaCell HD) for Breast Reconstruction
In the past, acellular dermal matrices (ADMs) have been used in implant-based breast reconstruction. Various factors affect the clinical performance of ADMs since there is a lack of systematic characterization of ADM tissues. This study used BellaCell HD and compared it to two commercially available ADMs—AlloDerm Ready to Use (RTU) and DermACELL—under in vitro settings. Every ADM was characterized to examine compatibility through cell cytotoxicity, proliferation, and physical features like tensile strength, stiffness, and the suture tensile strength. The BellaCell HD displayed complete decellularization in comparison with the other two ADMs. Several fibroblasts grew in the BellaCell HD with no cytotoxicity. The proliferation level of fibroblasts in the BellaCell HD was higher, compared to the AlloDerm RTU and DermACELL, after 7 and 14 days. The BellaCell HD had a load value of 444.94 N, 22.44 tensile strength, and 118.41% elongation ratio, and they were higher than in the other two ADMs. There was no significant discrepancy in the findings of stiffness evaluation and suture retention strength test. The study had some limitations because there were many other more factors useful in ADM’s testing. In the study, BellaCell HD showed complete decellularization, high biocompatibility, low cytotoxicity, high tensile strength, high elongation, and high suture retention strengths. These characteristics make BellaCell HD a suitable tissue for adequate and safe use in implant-based breast reconstruction in humans.
ADM S
The research used BellaCell HD by Hans Biomed Corporation (Daejeon, Korea) and two human ADMS for comparison, i.e., AlloDerm Ready to Use (RTU) by LifeCell Corp. (Branchburg, NJ, USA) and DermACELL by LifeNet Health (Virginia Beach, VA, USA). Below are the processes that were conducted on the three human ADMS for evaluation of the BellaCell HD. The BellaCell HD is a novel human ADM.
Decellularization Assessment
For the study, paraffin-embedded ADMs were sectioned at 5 µm thickness, and after removing the paraffin, the sections were rehydrated with a reducing series of alcohol concentration to water. The standard protocol for H & E was followed. The samples were evaluated by light microscopy at different range magnifications to inspect the presence of the cells and collagen fibers. The light microscope was 40, 100, and 200 magnifications.
Cell Culture
For experimental purposes, the ADM specimens were divided into 1 by 1 cm pieces, put into 24-well cell culture dishes, washed with PBS twice, and later incubated in media at 37 • C and 5% carbon dioxide for ten minutes, before the cell seeding process. NIH3T3 and L-929 mouse fibroblasts were grown in Dulbecco's modified Eagle's medium added with 100 µg/mL streptomycin, 100 U/mL penicillin, and 10% heat-inactivated fetal bovine serum. The cells were cultured at 37 • C in 5% CO 2 /95% air.
Proliferation Assay
For cell proliferation assay, NIH3T3 fibroblasts were seeded on the ADMs at 5 × 10 4 cells/mL and incubated for 1, 7, and 14 days. Cell proliferation was assessed with the use of an MTT assay. MTT solution at the 100 µL/well was added and incubated for 4 hours at 37 • C. The formazan crystals were liquified in dimethyl sulfoxide (DMSO), and the optical densities were calculated at 570 nm by an ELISA reader.
Cytotoxicity Assay
Cytotoxicity assays with the cultured cells were used for the testing chemicals and drug screening. For this study, sterile physiological saline were added and eluted at 37 ± 2 • C for 72 ± 2 h. Minimum essential medium (1×) with Earle's salts (MEM-E, Flow Labs., Rockville, MD, USA) was used as a negative control after cell exposure to a similar environment with the test sample and DMSO as a positive control, and sterile physiological saline as the solvent control. The eluate was centrifuged at 3000 rpm for 5 min, and the supernatant was used as the test group. L-929 fibroblasts were seeded at a concentration of 5 × 10 4 cells/well and incubated for 24 hours in a 37 • C saturated incubator with 5% CO 2 . The medium was detached after one day, and the eluate from the negative control group, the positive, the solvent regulator cluster, and the test assembly was replaced with a medium mixed in the same amount, with a 2-fold content of the MEM medium. After incubation for two days at 37 • C in a humidified incubator with 5% CO 2 , cell morphology was examined under a light microscope at 200× magnification, and cell viability was evaluated by MTT assay.
Uniaxial Tensile Testing
Biological soft tissue is a non-linear material, and the mechanical features affect the quality of life [7]. For this research, seven specimens (n = 7) measuring 10 mm × 7 mm were prepared from each of the ADMs (based on the American Society for Testing and Materials (ASTM) specification #D638-03). The average thickness of the specimens for each ADM was 1.97 mm for BellaCell HD, 1.3 mm for AlloDerm RTU, and 1.9 mm for DermACELL. The sample used in this study had different thickness. Each sample was oriented vertically in the Instron material testing system with a 3.0 cm gauge length, and approximately 2.0 cm of the specimen was fixed firmly in each pneumatic grip. Samples were pulled to a uniaxial pressure at a rate of 30 mm/min until failure. When the ADM was broken, the elongation ratio (%), the maximum length divided by original length, and the maximum load (N) was determined. Tensile strength was evaluated by the division of maximum load with the cross-sectional area (mm 2 ) of the sample to yield the value in megapascal (MPa) units, and 1 N/mm 2 equaled 1 MPa.
Stiffness Testing
Most of the biomaterials were viscoelastic, showing time dependence in the cell's response to the loads. In this study, stiffness testing was conducted using a custom test fixture. The custom test fixture was fabricated based on American Society for Testing and Materials (ASTM) specification #F1306. Three specimens (BellaCell HD n = 3, AlloDerm RTU n = 3, DermACELL n = 3) measuring 5 cm × 5 cm were prepared. The average thickness of each ADM specimen was 1.88 mm for BellaCell HD, 1.16 mm for AlloDerm RTU, and 1.3 mm for DermACELL. The sample used in this study had different thickness. The specimen was fixed between the upper and lower jigs, and the probe moved downward to compress the sample at a degree of 25 mm/min. The stiffness was evaluated through division of the load sustained with sample (N) in the stiffness examination by the movement (mm) of the probe.
Suture Retention Strength Testing
The cell width and the distance of the joint bite from the sample free edge are the most significant geometrical parameters [8]. For this work, four samples (2 cm × 4 cm) were prepared from each ADM. From the bottom of the sample, a suture was passed through the ADMs (1.0 cm). A pulling rate of 20 mm/min was applied as the suture tore out of the ADMs. The maximum load (N) was recorded as mean ± standard error of the mean (SEM).
Statistical Analysis
All figures are reported as mean ± SEM. Statistical analyses were conducted using SPSS statistical software, and it was beneficial in providing the frequencies and bivariate statistics. For all data, significant differences were established using an unpaired t-test. For all analyses, p < 0.05 was explained as statistically substantial.
Decellularization Assessment
BellaCell HD and DermACELL displayed complete decellularization under a light microscope after staining. Complete decellularization showed the compatibility of the BellaCell HD for use in breast reconstruction. Some cellular debris between collagen fibers were visible on the AlloDerm RTU, as shown ( Figure 1).
Bioengineering 2020, 7, x FOR PEER REVIEW 4 of 10 All figures are reported as mean ± SEM. Statistical analyses were conducted using SPSS statistical software, and it was beneficial in providing the frequencies and bivariate statistics. For all data, significant differences were established using an unpaired t-test. For all analyses, p < 0.05 was explained as statistically substantial.
Decellularization Assessment
BellaCell HD and DermACELL displayed complete decellularization under a light microscope after staining. Complete decellularization showed the compatibility of the BellaCell HD for use in breast reconstruction. Some cellular debris between collagen fibers were visible on the AlloDerm RTU, as shown ( Figure 1).
Biocompatibility Assessment
Cell proliferation in BellaCell HD was highest in the one day, though not statistically significant. On the 7 and 14 days, BellaCell HD was higher than in the AlloDerm RTU ( Figure 2A). There was no significant difference in cell proliferation between BellaCell HD and DermACELL. After the 1, 7, and 14 days of incubation with NH3T3 fibroblasts, invasive cells on the ADM's surface were photographed with the use of a light microscope at 100× magnification. All samples displayed the same degree of cell adhesion on the first day, as demonstrated ( Figure 2B). Many cells overgrew in the BellaCell HD in comparison to the other matrices. Cell proliferation was not constant in the AlloDerm RTU, but growth was mainly seen on the dent surface ( Figure 2B).
Biocompatibility Assessment
Cell proliferation in BellaCell HD was highest in the one day, though not statistically significant. On the 7 and 14 days, BellaCell HD was higher than in the AlloDerm RTU ( Figure 2A). There was no significant difference in cell proliferation between BellaCell HD and DermACELL. After the 1, 7, and 14 days of incubation with NH3T3 fibroblasts, invasive cells on the ADM's surface were photographed with the use of a light microscope at 100× magnification. All samples displayed the same degree of cell adhesion on the first day, as demonstrated ( Figure 2B). Many cells overgrew in the BellaCell HD in comparison to the other matrices. Cell proliferation was not constant in the AlloDerm RTU, but growth was mainly seen on the dent surface ( Figure 2B).
Cytotoxicity Assay
MTT assay showed that all three products had a cell viability of over 90%, indicating no cytotoxicity as illustrated in Figure 3A. In the cytotoxicity assay, a significant decline in cell count was observed in the positive control using DMSO. There was a rise in cell count in the negative control, as seen under a light microscope at 200× magnification ( Figure 3B). In all test groups using BellaCell HD, AlloDerm RTU, and DermACELL, there was a growth with a spindle-like structure ( Figure 3B).
Uniaxial Tensile Test
Uniaxial tensile testing showed that the maximum load at the ADM break was 444.94 N for BellaCell HD, which was higher than that for AlloDerm RTU (181.92 N), and marginally lower for DermACELL (492.11 N), which was not statistically significant ( Figure 4A). The tensile strength of BellaCell HD was 22.44 MPa. It was significantly higher than the 14.34 MPa observed for AlloDerm RTU and not substantially different from the 26.12 MPa observed for DermACELL ( Figure 4B). The elongation ratio at the ADM break was 118.41% for BellaCell HD, 126.38% for AlloDerm RTU, and 104.13% for DermACELL ( Figure 4C).
Cytotoxicity Assay
MTT assay showed that all three products had a cell viability of over 90%, indicating no cytotoxicity as illustrated in Figure 3A. In the cytotoxicity assay, a significant decline in cell count was observed in the positive control using DMSO. There was a rise in cell count in the negative control, as seen under a light microscope at 200× magnification ( Figure 3B). In all test groups using BellaCell HD, AlloDerm RTU, and DermACELL, there was a growth with a spindle-like structure ( Figure 3B). Figure 3A shows 90% cell viability and no cytotoxicity. In figure 3B, cells grew in a spindle-like material. Each datum represents the ±SEM of three independent experiments.
Uniaxial Tensile Test
Uniaxial tensile testing showed that the maximum load at the ADM break was 444.94 N for BellaCell HD, which was higher than that for AlloDerm RTU (181.92 N), and marginally lower for DermACELL (492.11 N), which was not statistically significant ( Figure 4A). The tensile strength of BellaCell HD was 22.44 MPa. It was significantly higher than the 14.34 MPa observed for AlloDerm RTU and not substantially different from the 26.12 MPa observed for DermACELL ( Figure 4B). The Figure 2A is a representation of cell adhesion, while Figure 2B shows the cell proliferation rate and incubation days. The cells overgrew with time in the BellaCell HD in comparison to the other two matrices, and the AlloDerm RTU cell growth was viewed at the dent. Each datum represents the ±SEM of three independent experiments. Figure 2A is a representation of cell adhesion, while figure 2B shows the cell proliferation rate and incubation days. The cells overgrew with time in the BellaCell HD in comparison to the other two matrices, and the AlloDerm RTU cell growth was viewed at the dent. Each datum represents the ±SEM of three independent experiments.
Cytotoxicity Assay
MTT assay showed that all three products had a cell viability of over 90%, indicating no cytotoxicity as illustrated in Figure 3A. In the cytotoxicity assay, a significant decline in cell count was observed in the positive control using DMSO. There was a rise in cell count in the negative control, as seen under a light microscope at 200× magnification ( Figure 3B). In all test groups using BellaCell HD, AlloDerm RTU, and DermACELL, there was a growth with a spindle-like structure ( Figure 3B). Figure 3A shows 90% cell viability and no cytotoxicity. In figure 3B, cells grew in a spindle-like material. Each datum represents the ±SEM of three independent experiments.
Uniaxial Tensile Test
Uniaxial tensile testing showed that the maximum load at the ADM break was 444.94 N for BellaCell HD, which was higher than that for AlloDerm RTU (181.92 N), and marginally lower for DermACELL (492.11 N), which was not statistically significant ( Figure 4A). The tensile strength of BellaCell HD was 22.44 MPa. It was significantly higher than the 14.34 MPa observed for AlloDerm RTU and not substantially different from the 26.12 MPa observed for DermACELL ( Figure 4B). The Figure 3A shows 90% cell viability and no cytotoxicity. In Figure 3B, cells grew in a spindle-like material. Each datum represents the ±SEM of three independent experiments.
Stiffness Testing
A biomaterial requires mechanical strength to uphold integrity until full regeneration of the tissue and has to sustain space for the cell ingrowths and nutrient absorption in vitro, thereby supporting the loadings in vivo [9]. The scaffold should be able to match its characteristics to those of initial tissues to avoid pressure shielding and offer the cell appropriate mechanical cues [10]. The Figure 4B). Figure 4C represents the elongation ratio of 118.41%, 126.38%, and 104.13% for BellaCell HD, Alloderm RTU, and DermACELL products, respectively. Each datum represents the ±SEM of three independent experiments.
Stiffness Testing
A biomaterial requires mechanical strength to uphold integrity until full regeneration of the tissue and has to sustain space for the cell ingrowths and nutrient absorption in vitro, thereby supporting the loadings in vivo [9]. The scaffold should be able to match its characteristics to those of initial tissues to avoid pressure shielding and offer the cell appropriate mechanical cues [10]. The stiffness testing showed that DermACELL had the highest stiffness of 0.90 N/mm, while those of AlloDerm RTU and BellaCell HD were measured 0.28 N/mm and 0.44 N/mm, respectively, as illustrated in Figure 5.
Stiffness Testing
A biomaterial requires mechanical strength to uphold integrity until full regeneration of the tissue and has to sustain space for the cell ingrowths and nutrient absorption in vitro, thereby supporting the loadings in vivo [9]. The scaffold should be able to match its characteristics to those of initial tissues to avoid pressure shielding and offer the cell appropriate mechanical cues [10]. The stiffness testing showed that DermACELL had the highest stiffness of 0.90 N/mm, while those of AlloDerm RTU and BellaCell HD were measured 0.28 N/mm and 0.44 N/mm, respectively, as illustrated in Figure 5.
Suture Retenstion Strength Testing
The ADM must withstand tearing at the point of the suture when tension pulls at the suture. Break starting strength is firm against the assessment parameter discrepancies, and it is dependent on the geometry of the sample [11]. The contrast of suture preservation and mode one crack opening evaluations shows the linear relationship between break starting and tearing energy [12]. The results of suture retention strength testing showed that the maximum load for BellaCell HD was 97.06 N, which was higher than that for AlloDerm RTU and for DermACELL, as shown in Figure 6. However, these differences were not statistically significant (p > 0.05).
Suture Retenstion Strength Testing
The ADM must withstand tearing at the point of the suture when tension pulls at the suture. Break starting strength is firm against the assessment parameter discrepancies, and it is dependent on the geometry of the sample [11]. The contrast of suture preservation and mode one crack opening evaluations shows the linear relationship between break starting and tearing energy [12]. The results of suture retention strength testing showed that the maximum load for BellaCell HD was 97.06 N, which was higher than that for AlloDerm RTU and for DermACELL, as shown in Figure 6. However, these differences were not statistically significant (p > 0.05).
Discussion
The present study used the novel ADM, BellaCell HD, in an in vitro environment, contrasting it with two commercially available human ADMs. The aim was for the ADM to be successfully utilized in expander-based breast reconstruction. The BellaCell HD showed complete decellularization, viewed using H & E staining under a light microscope [13]. The BellaCell also showed high cell
Discussion
The present study used the novel ADM, BellaCell HD, in an in vitro environment, contrasting it with two commercially available human ADMs. The aim was for the ADM to be successfully utilized in expander-based breast reconstruction. The BellaCell HD showed complete decellularization, viewed using H & E staining under a light microscope [13]. The BellaCell also showed high cell adhesion and cell proliferation without cytotoxicity in the biocompatibility assessment. The BellaCell HD displayed a high tensile strength, elongation, low stiffness, and high suture retention power in the mechanical property assessment.
The manufacturing process of ADMs consists of decellularization, preservation, and sterilization stages. The most important phase is decellularization using physical, chemical, or biological techniques [6]. Manufacturing of each product is done differently. The purpose of the decellularization process is the removal of antigenic material while preserving extracellular matrix biochemistry and structure [14]. The presence of residual DNA in the biological scaffold materials results in an inflammatory response. Previous research found that the presence of cells within a biomaterial is linked with increased macrophage M1 polarization, increased proinflammatory cytokines, and weak remodeling results in a primate model [15]. Reasons for complication development in the ADMs in breast reconstruction are multifactorial [16]. Hence, this research holds that BellaCell HD is immunologically safe for implantation in the human body.
Immune cells like lymphocytes, granulocytes, macrophages and mast cells, fibroblast, and myofibroblasts recolonize the original ADM during implantation in the breast reconstruction process [14]. Capsule and capillaries are formed by the fibrosis and neovascularization [17]. It should be compatible and capable of inducing the biological responses like host cell adhesion and cell proliferation with no cytotoxicity to the host tissue. Lack of biocompatibility leads to an imbalance resulting in implant mobility that causes infection, reconstructive failure, and seroma. High compatibility of BellaCell HD might result in a favorable outcome when employed in breast reconstruction.
The benefit of using ADM in expander-based breast reconstruction is because it offers physical support to the implant hence preventing shifting or bottoming out [18]. High tensile strength ADM is a requirement for breast implantation [18]. Using a low tensile ADM the tissue would be vulnerable to matrix rupture, which could result in implant malposition [19]. After implantation, the expander was inflated for some months to get an adequate amount of skin, similar to that in the contralateral breasts. Therefore, ADM should have sufficient tensile strength to withstand the inflating pressure of the expander [18]. BellaCell HD showed a high tensile strength; thus; it was capable of providing sufficient physical support when used in breast reconstruction.
The paramount result of this research was that BellaCell HD showed both a high tensile strength and elongation ratio, which meant elasticity and flexibility, respectively. The AlloDerm RTU had low stiffness rate, and lower tensile strength [1]. The tensile power strength of DermACELL was high, like that of BellaCell HD, though with high stiffness. The elements helped the surgeons in handling ADM efficiently and overcoming the size discrepancy between the standardized ADM material and the spaces that required coverage for every patient [20]. Most surgeons create a vertical or horizontal stab incision to the ADM, whereas others mesh the ADM using a skin graft Mesher [21]. However, all of these techniques have the shortcoming of increasing the contact area between the implant and the mastectomy flap [22]. Therefore, the high elasticity and pliability of the BellaCell HD would help in bridging the gap, particularly by stretching it. Currently, prepectoral implant placement with complete coverage by ADM has become popular because of low postoperative pain and low animation deformity [23]. Since more complex techniques are needed for complete wrapping with ADM, the high flexibility of the ADM will help safe handling.
In the suture retention strength test, BellaCell HD had the uppermost suture retention strength of the three human ADMs examined. When the ADM was implanted as a hammock shape in implant/expander-based breast reconstruction, ADM was sutured with the elevated pectoralis major superior and the chest wall at the inframammary and lateral mammary fold inferolateral [24].
Although few human research studies have been conducted, it is speculated that the suture site wound is more vulnerable to dehiscence than the ADM itself, until it is fully integrated with the host tissue [25]. Therefore, the high suture retention strength of BellaCell HD will aid in preventing implant herniation through the suture site. For BellaCell HD to expand its indications like other common ADMs, it must have appropriate suture retention strength that can withstand high tensions such as abdominal walls, which is supported by these findings.
Indeed, AlloDerm RTU and DermACELL have been used in clinical applications such as breast reconstruction. DermACELL is an appropriate adjunct to the post-mastectomy outcome of reconstruction with expanders. Histological observation showed early graft integration at 6 weeks post-implantation [26]. Clinical data on AlloDerm RTU used in breast reconstruction showed full incorporation and integration into the host tissue [27]. Based on the present study, BellaCell might also reduce complications that lead to reconstruction failure.
The limitation of this study is that biocompatibility and mechanical properties of ADM were tested only in an in vitro setting. We used only NIH3T3 and L-929 mouse fibroblasts to assess cell proliferation and cell viability. However, not only fibroblasts but also myofibroblast, lymphocytes, macrophages, granulocytes, and mast cells were involved in the ADM integration [28]. Moreover, antibodies, complement, and cytokines also perform a significant part in the host response to ADM [29]. Therefore, to identify the exact mechanism through which ADM integrates into the human body and to use it to create an ideal ADM, further detailed studies are recommended. The ADM facilitates the biochemical change through a process called "stretching" after implantation, which varies from product to artefact [30]. However, it is challenging to manufacture individual multi-vector forces in an in vitro setting. In vivo studies would be required to address this, to help surgeons predict the need for an ADM sling overcorrection in implant/expander-based breast reconstruction.
Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions might also be highlighted.
Conclusions
The BellaCell HD showed high compatibility, low cytotoxicity, high tensile strength, and complete decellularization, thus, displaying its suitability for use in breast reconstruction. | v3-fos-license |
2021-07-26T00:06:36.869Z | 2021-06-03T00:00:00.000 | 236269044 | {
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} | pes2o/s2orc | Aqueous Ammonia With Sodium Sulte Pretreatment For Enhancing Enzymatic Saccharication and Bioethanol Production of Sugarcane Bagasse
Background: Bioethanol is considered as a promising alternative fuel. Lignocellulosic biomass can be used for the production of bioethanol, but its recalcitrant structure makes it difficult to be utilized. Thus, proper pretreatment is a crucial step to break this structure and enhance enzymatic saccharification. Aqueous ammonia with sodium sulfite pretreatment (AAWSSP) was first applied to enhance the enzymatic saccharification and bioethanol production of sugarcane bagasse (SCB) in this research. Results: Response surface methodology was applied to optimize the conditions of pretreatment. Under optimal parameters, 16.92 g/L of total sugar concentration (P1 SCB: 202.08 ℃ , 11.06% aqueous ammonia, 13.37% sodium sulfite, 1.22 h) and 0.51 g/g of total sugar yield (P2 SCB: 199.47 ℃ , 10.17% aqueous ammonia, 13.11% sodium sulfite, 1.17 h) were achieved, respectively. The results of ethanol fermentation showed that separate hydrolysis and fermentation performed better than that of simultaneous saccharification and fermentation, and the maximum ethanol yields of 143.30 g/kg for P1 SCB and 145.33 g/kg for P2 SCB, were obtained, respectively. Conclusions: This research indicated that aqueous ammonia and sodium sulfite in pretreatment solution might have a synergistic effect on delignification and enzymatic saccharification. AAWSSP might be a prospective method for enhancing enzymatic saccharification and bioethanol production of SCB, which provided new guidance for the bio-refinery of lignocellulose.
Compared with acid pretreatment, alkali pretreatment is conducted under relatively mild conditions, leading to less carbohydrates losses while providing higher digestibility of lignocellulose because of the lignin removal or decomposition of lignin-carbohydrates complex [11]. It enhances the accessibility of enzyme to lignocellulose mainly by modifying and removing lignin, solubilizing hemicellulose, and swelling cellulose [11]. The mechanism of delignification involves the destruction of aryl-ether bonds and the solubilization of lignin after ionization and solvation of aromatic hydroxyl groups [12]. Pretreatment aims to change or remove structural and compositional barriers of enzymatic saccharification and to enhance the enzymatic digestibility [13]. Alkali pretreatment might produce intermediate products such as black liquor containing lignocellulosic material, acids greases, polyphenolic compounds, aliphatic acids, and resinous compounds, which cannot be reused, but direct discharge will cause environmental pollution [14].
Ammonia pretreatment has been applied to the delignification of lignocellulose successfully. It can enhance the surface area of cellulose, destroy the crystalline structures, retain most of carbohydrates, and has shown better delignification along with production of minimum toxic compounds, so as to improve the enzymatic efficiency [15]. Ammonolysis of lignocellulose occurs during ammonia pretreatment, which acts specifically on the lignin-carbohydrates complex by cleaving ether and ester bonds for purpose of removing lignin and increasing the accessibility of cellulase to holocellulose [16,17]. It has higher selectivity with lignin than that of carbohydrates. Delignification of lignocellulose results in the increasing content of cellulose which can be transformed into fermentable sugar and then bioethanol [17]. Ammonia is an effective expansion reagent for biomass and one of the most widely used chemicals with lower cost [18].
Furthermore, ammonia is a pollution-free and non-corrosive reagent, and its high volatility makes it easier to be recycled [18]. In conclusion, ammonia is a suitable reagent for pretreatment because of a large amount of excellent features. However, the delignification and enzymatic saccharification of biomass pretreated by ammonia alone are still not as good as that of strong alkali.
It was found that pretreatment system containing a higher proportion of sodium sulfite could soften the lignocellulose better via delignification, hemicellulose degradation, and lignin sulfonation [19]. Sulfonation can increase the hydrophilicity of lignin and the swelling of substrate, reduce the tendency of non-productive combination of lignin and cellulase, which will improve the enzymatic saccharification [20]. Sulfite pretreatment in alkaline environment can also increase the sulfonation and dissolubility of lignin [21].
In this study, sugarcane bagasse was subjected to aqueous ammonia with sodium sulfite pretreatment (AAWSSP), in order to obtain higher total sugar concentration (TSC) and total sugar yield (TSY) while selectively removing lignin and remaining most of carbohydrates. Besides, aqueous ammonia can be recycled. Later the pretreatment conditions, including pretreatment temperature, aqueous ammonia concentration, sodium sulfite concentration and pretreatment time, were optimized using response surface methodology (RSM) based on single factor experiments.
Ultimately, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were carried out for the production of bioethanol. To our best knowledge, it is the first time to pretreat sugarcane bagasse by aqueous ammonia with sodium sulfite.
Results and discussion
Effect of pretreatment temperature on components and enzymatic saccharification of pretreated SCB Temperature acts an critical part in the pretreatment of biomass for it has a direct impact on the energy input, the capacity of pretreatment and pretreatment cycle [22]. Hence, the impact of temperatures of 120℃, 140℃, 160℃, 180℃, 200℃, 220℃ on SCB with AAWSSP was first investigated. The biomass recovery, components, TSC and TSY were compared (Additional file 1: Table S1). Most of the pretreatment groups had a significant influence on the biomass recovery and the components. Besides, AAWSSP improved the enzymatic saccharification of pretreated SCBs compared to native SCB.
As demonstrated, adding sodium sulfite (Na2SO3) to the aqueous ammonia pretreatment could increase the lignin removal and enzymatic saccharification to some extent at 120℃ when compared with aqueous ammonia pretreatment alone. When the temperature increased from 120℃ to 220℃, the biomass recovery declined from 75.39% to 60.93%, principally because of the lignin removal. A large proportion of carbohydrates were remained, the cellulose recoveries were higher than 89% and hemicellulose recoveries were within the range of 67.31-87.71%, which implied that AAWSSP could selectively remove lignin while remaining most carbohydrates [23].
Hemicellulose was more likely to be degraded at high temperature compared to cellulose, indicating that the structural intensity of hemicellulose is more weaker [24].
In addition, the contents of cellulose and hemicellulose after AAWSSP were significantly higher than native SCB (p < 0.05). The delignification rates of pretreated SCBs were 51.63-91.13%, and the values increased with the elevation of temperature from 120℃ to 220℃, indicating that high temperature resulted in the higher removal of lignin [25]. As is well-known, higher temperature could cleave α-ether and ester bonds between lignin and hemicellulose, contributing to the synergistic degradation of biomass pretreated by alkaline [26]. However, higher temperature will lead to a good deal of hemicellulose degradation on account of the weak structure of amorphous hemicellulose.
The TSCs and TSYs of SCBs after AAWSSP were significantly higher than that of native SCB (p < 0.05). The TSCs and TSYs significantly elevated with the increment of pretreatment temperature (p < 0.05). The highest TSC of 15.04 g/L and TSY of 0.47 g/g were both gained at 200℃, which probably resulted from the higher delignification efficiency and the destruction of the recalcitrant structure, leading to the higher accessibility of enzyme to cellulose. As the temperature increased to 220℃, the cellulose decreased from 62.38% to 60.70% compared to that of 200℃, resulting in the lower recovery of cellulose. Though the delignification improved at 220℃, the TSC and TSY both decreased. Therefore, 200℃ might be the proper temperature for subsequent investigation of AAWSSP conditions.
Effect of aqueous ammonia concentration on components and enzymatic saccharification of pretreated SCB
Aqueous ammonia pretreatment can partially break the chemical linkages between hemicellulose and lignin, change the physical characteristics of biomass making it more pliable and increase its uptake of water, which plays a crucial part in the digestibility of lignocellulose [27]. Hence, it is likely that the increasing concentration of aqueous ammonia might contribute to higher lignin removal and enzymatic saccharification efficiency.
Fig. 1
The impact of diverse aqueous ammonia concentrations on componential changes and enzymatic saccharification of SCB was shown in Fig. 1. Clear changes were found in the main components of SCBs pretreated with diverse aqueous ammonia concentrations compared to native SCB. Along with the increment of aqueous ammonia concentration from 5% to 25%, the biomass recoveries decreased from 66.57% to 58.24%, while the cellulose contents increased from 57.48% to 67.09%. Over 95% of cellulose was maintained after AAWSSP. Similar to pretreatment temperature, aqueous ammonia concentration has less impact on the hemicellulose contents of SCBs. When aqueous ammonia concentration enhanced from 5% to 15%, the contents of hemicellulose were not significant (p > 0.05) and decreased significantly with 20% and 25% of aqueous ammonia (p < 0.05). The AAWSSP process significantly reduced the lignin content (p < 0.05), which declined from 22.83% of the native SCB to 8.78-4.20% of pretreated SCBs. With a rise in aqueous ammonia concentration from 5% to 20%, the delignification increased significantly (p < 0.05) but no significant difference was shown between 25% and 20% of aqueous ammonia (p > 0.05). When the concentration of aqueous ammonia was 20%, the maximum delignification of 89.17% was obtained, but the lower TSC and TSY were obtained. The highest TSC (15.04 g/L) and highest TSY (0.47 g/g) were achieved with 10% of aqueous ammonia. With the concentration of aqueous ammonia increasing from 10% to 25%, the TSC and TSY decreased slightly. Therefore, 10% (w/w) of aqueous ammonia was appropriate for AAWSSP.
Effect of Na2SO3 concentration on components and enzymatic saccharification of pretreated SCB
It is acknowledged that sulfite can cleave α-alkyl ether bonds, α-benzyl ether bonds, and β-benzyl ether linkages on phenolic lignin groups as well as sulfonate the lignin, thus improving the enzymatic digestibility of substrates [28]. If sulfite was added to pretreatment, the sulfonation of lignin occurred, resulting in the increasing swelling of fiber and higher solubility of lignin.
Fig. 2
The Na2SO3 concentration varied from 4 to 20% (w/w, based on dry SCB) supplemented with 10% (w/w) aqueous ammonia to investigate the impact on AAWSSP at 200℃ for 1 h. As presented in Fig. 2, the maximum biomass recovery of 64.95% was achieved with 4% Na2SO3, followed by that of 63.57% when the concentration of Na2SO3 was 12%, but the Na2SO3 concentration doesn't showed significant influence on the biomass recovery in the range from 8% to 20% (p > 0.05). Previous study reported that high biomass recovery made a sense because the economic feasibility of pretreatment relies extremely on high biomass recovery [29]. When Na2SO3 concentration increased from 8% to 12%, the hemicellulose contents increased from 26.53% to 28.06% although the cellulose content and delignification declined a little.
And the maximum hemicellulose recovery of 77.75% was achieved when the loading of Na2SO3 was 12%. Delignification increased with the chemical loading increasing from 12% to 20% in pretreatment step. The maximum delignification corresponding to 88.03% of AAWSSP with 20% sodium sulfite, indicating that the sulfonation of lignin enhanced with the increasing loading of Na2SO3. The TSCs gradually improved when the loading of Na2SO3 increased from 4% to 12%. The improved TSCs partly resulted from the modification/degradation and the increasing hydrophilicity of lignin [21]. This could result in the easier accessibility of enzyme to carbohydrates, leading to the release of sugar when compared to the native group. When the chemical charge was 12%, the delignification of 81.39% was achieved. The increasing Na2SO3 concentration during AAWSSP resulted in higher delignification since 88.03% of delignification was achieved when the chemical loading was 20%. The highest TSC of 16.48 g/L and TSY of 0.52 g/g were both obtained with 20% Na2SO3 pretreatment, followed by 16.19 g/L for TSC and 0.51 g/g for TSY with 12% Na2SO3. But no significant difference was shown as the concentration of Na2SO3 increased from 12% to 20% (p > 0.05). Therefore, 12% (w/w) sodium sulfite was selected for further investigation of the AAWSSP conditions.
Fig. 3
To evaluate the appropriate pretreatment time, 10% (w/w) aqueous ammonia with 12% (w/w) Na2SO3 was implemented for SCB pretreatment at 200℃ for different time (0.5, 1, 2, 3 h). The components and enzymatic saccharification of the native SCB and pretreated groups were presented in Fig. 3. With the prolongation of pretreatment time, the biomass recovery declined. All the cellulose contents were significantly higher than that of native SCB (p < 0.05), and reached the highest value of 57.59% at 3 h. The contents of cellulose significantly increased from 51.29% to 57.56% with the prolongation of pretreatment time from 0.5h to 2h (p < 0.05), but there was no significant difference between 2 h and 3 h (p > 0.05). The hemicellulose contents of SCBs after pretreatment were also significantly higher than that of native SCB (p < 0.05). The recovery of hemicellulose declined with the increasing of pretreatment time.
Increasing the pretreatment time from 0.5 h to 1 h lowered the content of lignin from 10.92% to 6.68%, and there was no significant difference with the prolongation of pretreatment time from 1 h to 3 h (p > 0.05). As presented in Fig. 3 B, all the TSCs and TSYs of SCBs after AAWSSP were significantly enhanced compared with native SCB (p < 0.05). As the pretreatment time prolonged from 0.5 h to 2 h, the TSC and TSY significantly enhanced from 12.08 g/L to 17.16 g/L for TSC and from 0.41 g/g to 0.53 g/g for TSY (p < 0.05). Both the highest TSC of 17.16 g/L and TSY of 0.53 g/g were obtained at 2 h. With the pretreatment time increasing from 2 h to 3 h, the TSC and TSY showed no significant difference (p > 0.05), which might be caused by the similar lignin content and delignification. Although there was significant difference of the TSC and TSY between 1 h and 2 h (p < 0.05), it was unworthy to double pretreatment time just to obtain more 1 g/L of TSC and 0.02 g/g of TSY. Hence, taking time and energy consumption into account, 1 h might be the more suitable pretreatment time for further investigation of AAWSSP.
Optimization of AAWSSP using RSM
In order to obtain the highest TSC and TSY, the Box-Behnken design (BBD) was then used to optimize the AAWSSP conditions. Four independent contributions, pretreatment temperature (℃, X1) from 180 to 220℃, aqueous ammonia concentration (%, X2) from 5 to 15%, sodium sulfite concentration (%, X3) from 8 to 16% and pretreatment time (h, X4) from 0.5 to 1.5 h, were chosen as the variables, and the TSC and TSY were used as the response values. A set of 29 groups of experiments were conducted according to the software Design-Expert (Additional file 1: Table S2). Based on the experimental data, the quadratic Eqs. (1) and (2) The ANOVA results of the TSC and TSY models (Additional file 1: Table S3 and Table S2). The results of ANOVA implied that pretreatment time affected TSC and TSY most, followed by aqueous ammonia concentration and pretreatment temperature, and sodium sulfite concentration the last (Additional file 1: Table S3 and Table S4). The 3D plots of RSM were utilized to present the interactions between two variables factors. It was found that the interaction between each of the four factors was not significant (p > 0.05), indicating that each factor was independent and would not interact with each other. Based on the regression quadratic Eq. (1) and (2) Confirmatory experiments were carried out for further verification of the predicted results. The actual TSC of 16.92g/L and TSY of 0.51 g/g were obtained (Table 1), which were close to the actual ones and further indicated that the two models fitted well. After RSM optimization, the TSC of SCB pretreated under the optimal pretreatment condition increased by only 4.51% while the TSY was nearly close, but the biomass recovery declined, and the content of cellulose increased to 63.91% and 60.99%, respectively.
The results above revealed that the AAWSSP might be an efficient lignocellulose pretreatment method.
(a) Optimal condition of AAWSSP for the maximum TSC.
(b) Optimal condition of AAWSSP for the maximum TSY.
Ethanol fermentation
After RSM optimization, ethanol fermentation of native SCB, P1 SCB and P2 SCB were performed. The results of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were shown in Fig. 6.
Fig. 6
The results of SHF (Fig. 6 A-B) showed that the glucose of the native SCB was exhausted after 6 h. The concentration of ethanol reached the maximum value of 0.15 g/L, and the corresponding ethanol yield was 7.5 g/kg native SCB. The glucose of P1 SCB and P2 SCB were both exhausted after 15 h. The highest ethanol concentration of 4.71 g/L for P1 SCB and 4.70 g/L for P2 SCB were gained, and the corresponding ethanol yields were 143.30 g/kg and 145.33 g/kg native SCB, respectively, which were both significantly higher than that of native SCB (p < 0.05).
As presented in Fig. 6 C-D, the highest ethanol concentration (0.13 g/L) of native SCB was obtained after 12 h during SSF, and the yield of ethanol was 6.3 g/kg native SCB. The highest ethanol concentration of 4.57 g/L (P1 SCB) and 4.33 g/L (P2 SCB), respectively, were obtained, and the corresponding ethanol yields were 138.92 g/kg and 133.89 g/kg native SCB, respectively. The ethanol concentrations and yields of P1 SCB and P2 SCB were both significantly higher than that of the native one (p < 0.05).
It was also found that xylose cannot be exhausted for ethanol fermentation in both SHF and SSF because saccharomyces cerevisiae CICC 1445 is lack of xylose metabolic pathway [31]. Taking the ethanol yield and fermentation time into consideration, SHF might perform more excellently on bioethanol fermentation than that of SSF.
Conclusion
In this research, we put forward AAWSSP for the first time and the pretreatment conditions were optimized by single factor experiments and RSM optimization to enhance the TSC and TSY of SCB. The maximum TSC (16.92 g/L) and TSY (0.51 g/g) were obtained as followed: 11.06% (w/w) aqueous ammonia with 13.37% (w/w) sodium sulfite at 202.08℃ for 1.22 h and 10.17% (w/w) aqueous ammonia with 13.11% (w/w) sodium sulfite at 199.47℃ for 1.17 h, respectively. The results of ethanol fermentation showed that SHF performed more excellently than that of SSF, and the highest ethanol yields of 143.30 g/kg native SCB for P1 SCB and 145.33 g/kg native SCB for P2 SCB were obtained. This study provided a new pretreatment technology (AAWSSP) for enhancing the enzymatic saccharification and ethanol production of SCB, which provided new guidance for the bio-refinery of lignocellulose.
Biomass and chemicals
Sugarcane bagasse (SCB) was supplied by the Liutang Sugar Factory in Guangxi, China.
The particle of dry SCB was between the sizes of 100 and 200 mesh (75-150 μm). The Table 2.
Calculations and statistical analysis
The components of SCB were measured according to the procedure established by the NREL [32]. Sugars and ethanol concentrations were determined on HPLC system equipped with an Aminex HPX-87H column and refractive index detector at 55℃. The column was operated with 0.6 mL/min H2SO4 (5 mM).
The native SCB, TSC and TSY were calculated as followed: (kg) SCB Native (L) V (g/L) ion concentrat Ethanol (g/kg) yield Ethanol (6) where the native SCB is equal to the mass of the native SCB in enzymatic saccharification; TSC presents the total concentration of glucose and xylose; TSY presents the total yield of glucose and xylose gained from per gram native SCB; V represents the cubage of enzymatic saccharification.
The data was expressed as the mean value ± standard deviation (SD) and analyzed by one-way ANOVA using Origin (Version 8.6, EA, USA). All the tests were implemented in duplicate. Box-Behnken design.
Ethical approval and consent to participate
Not applicable. | v3-fos-license |
2019-05-07T13:49:46.841Z | 2019-04-18T00:00:00.000 | 146005756 | {
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} | pes2o/s2orc | Phytomelatonin Regulates Keratinocytes Homeostasis Counteracting Aging Process
: Phytomelatonin (PM) gained the greatest interest for its application in agriculture and its use to improve human health conditions. PM based supplement has been shown to possess antioxidant capabilities because it functions as a free radical scavenger. Reactive Oxygen Species (ROS), induced by both intrinsic (peroxide production) and extrinsic (UV-radiation) factors are biochemical mediators crucial in skin aging. Skin aging is also regulated by specific microRNAs (miRs). Herein we have shown the e ff ect of PM free radical scavengers on the human keratinocyte cell line HaCat and on ROS formation induced by both extrinsic and intrinsic factors as well as their capability to positively modulate a member of the hsa-miR-29 family linked to aging. Our result highlights the regulatory role of PM for the keratinocytes homeostasis.
Introduction
Phytomelatonin (PM) is a plant derivative, also called N-acetyl-5-methoxytryptamine, known to be synthesized by plants in hard climate conditions. PM plant distribution is ubiquitous and in different parts of plants, including the leaves, roots, fruits, and seeds [1]. At the moment, two different aspects of PM have gained the greatest interest: (i) its application in agriculture; and (ii) its use to improve human health conditions. Indeed, PM and melatonin share both chemical structure and biological properties. Amongst these properties is the capability to act as an antioxidant and to function as a free radical scavenger [1]. Recent data clearly indicate that supplementation in PM with nutraceuticals is related to an increase in serum antioxidant capability [2,3]. Antioxidant defense against Reactive Oxygen Species (ROS) are biochemical mediators crucial in the skin aging. In fact, previous studies have pointed out that continuous exposure to ROS can stimulate through the antioxidant system destruction, melanogenesis, wrinkle formation and skin aging [4]. The skin aging process can be induced by intrinsic and extrinsic factors. This latter is due to the environment, as exposure to ultraviolet (UV) radiation contributes to up to 80% to skin aging [5]. Intrinsic skin aging occurs because of cumulative endogenous damage due to the continual formation of ROS which is also essential for biological functions [6,7]. Therefore, they are generated constantly during normal cellular metabolism and are typically eliminated from the body through the antioxidant defense system [7]. Indeed, antioxidant defense system dysfunction and the imbalance between ROS formation and removal can cause cells damage by free radical reactions. MicroRNAs (miRs) as biomarkers of pathology (mainly in cancer) and regulators of gene expression have been well recognized from end to end of abundant publications over the last 10 years [8]. Their role in the aging process and the oxidative stress is emerging [9]. Amongst these miRs, the physiological role of the hsa-miR-29 family in the control of cell-extracellular matrix and aging is very interesting [9][10][11].
Herein we have shown the effect of PM free radical scavengers on the human keratinocyte cell line HaCat and on ROS formation induced by both extrinsic and intrinsic factors, as well as their capability on a member of hsa-miR-29 family linked to aging.
Phytometonin Exctraction and Cell Culture
Phytomelatonin oil (PM) obtained from EFFEGI LAB Srl was solubilized in several organic solvents e.g., -dimethyl sulfoxide; -ethyl glycol; -acetonitril; -diethyl glycol at 1:1 and 1:10 dilutions. Moreover, -propylene glycol and -glycerol were also tested at at 1:1 and 1:2 dilutions without success. Positive results were achieved by 1,2-dimethoxyethane at both 1:1 and 1:10 dilutions but when it was added to the culture media, after 30 min a phase separation was observed. Therefore, PM oil was extracted in ethyl acetate and then lyophilized and re-dissolved in DMSO and used as weight/volume.
Cell Viability Assay
Cell viability was determined by the MTT assay, measuring the reduction of 3-(4,5-dimethylthiasol-2-yl)-2,4,-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase, as previously described [12]. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored, formazan product. The amount of color produced is directly proportional to the number of viable cells. HaCat cells were incubated with a different amount of PM (from 0 to 200 µg) in 96-well-plates for 24 h. At the time of assay, MTT (10 µL, 5 mg/mL in PBS) was added to each well and incubated for 3 h at 37 • C. The medium was then carefully aspirated, and DMSO (100 µL) was added to solubilize the colored formazan product, agitating the plates for 5 min on a shaker. The absorbance of each well was measured with a microtiter plate reader (Synergy H1 by BioTeck). The optical density (OD) was calculated as the difference between the absorbance at the reference wavelength (690 nm) and the absorbance at the test wavelength (570 nm). Percent viability was calculated as (OD of treated sample/OD of control) ×100.
DAPI and Fluorescent Staining
Change in morphology for nuclei and cytoskeleton remodeling were assessed by 4,6-diamidino-2-phenylindole (DAPI) and phalloidin. HaCat cells were grown on a cover glass lip into 24 well plate and treated with PM at 10 µg for 24 h. The cells were then fixed in 4% paraformaldehyde and the fluorescence was imaged using confocal FV-3000 Olympus microscope (Tokyo, Japan). For measuring intracellular ROS and mitochondrial membrane potential (MMP), PM pre-treatment at 10 µg for 6 h was performed. Then, 5 µM of CellRox Deep Red (λ = ex/em = 640/665) dye was added after H 2 O 2 500 µM exposure and incubated for further 30 min. Then, cells were exposed to UV for 30 min and mitochondrial membrane potential was assessed by adding 2 µM MitoTracker Red CMXRos dye (λ = ex/em = 579/599) for further 15 min (outside the UV lamp). The cells were then fixed in 4% paraformaldehyde and the fluorescence was imaged using confocal FV-3000 Olympus microscope and Leica DVM6 microscope (Wetzlar, Germany), respectively.
MicroRNAs Extraction and Loop Primer Method
MicroRNA was isolated using a PureLink™ miRNA Isolation Kit (K1570-01 Ambion by Life Technologies, Waltham, Massachusetts, U.S.) as already described [13]. The quantification of miRNA was performed with a method termed looped primer RT-PCR, following the protocol. Initially, 10 ng of total RNA was subjected to reverse transcription polymerase chain reaction using the TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.), according to the manufacturer's protocol. The thermocycling conditions were: 30 min at 16 • C, followed by 30 min at 42 • C, 5 min at 85 • C and 5 min at 4 • C.
Quantitative Real Time PCR (qRT-PCR)
The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TaqMan Universal PCR Master Mix Kit (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.) according to the manufacturer's protocol and the equipment QuantumStudio3 TM Real-Time PCR Systems. The thermocycling conditions were: 95 • C for 10 min, and 40 cycles of 15 s at 95 • C, followed by 1 min at 60 • C. After finalization of the qRT-PCR experiments, the average values of the cycle threshold (Ct) of the reactions in triplicate were determined. The relative expression of the miR target was plotted as follows: 40 total qRT-PCR cycle -Ct target miR. This difference (∆ct) was plotted directly as already described [14].
In Silico Analysis for Predicted Target Genes
In order to identify genes as the target of the hsa-miR tested, we performed in silico analysis. The in silico identification of the target genes was performed using miRWalk, miRDB, and miR target link human databases as well as a string database.
Statistical Analysis
Data were analyzed using the ANOVA method followed by the Bonferroni's test. The differences were considered significant for values of P < 0.05 (*) P < 0.01 (**) and P < 0.001 (***).
Results and Discussion
Melatonin is an evolutionary ancient hormone that belongs to both, animals and plants. Although they have the same chemical structure, the hormone present in the latter is indicated as phytomelatonin (PM). At present, in the cosmetic industry phytochemicals are widely used [15], while phyto-stem-cells and phytohormones application is an emerging field [16]. In this view herein we tested the effect of PM on human keratinocytes cell biology. As shown in Figure 1, the exposure of cell culture to five and 200 µg of PM did not affect cell viability compared to the control (DMSO-vehicle). Instead a significant increase of cell viability was observed in HaCaT cell line treated with PM in the range between 10 to 50 µg.
Our results revealed that this compound affected cell viability in a dose-response fashion and strongly suggested the involvement of the receptor mechanisms. Indeed, the higher amount used at 200 µg drastically decreased cell viability. This mechanism was probably due to the melatoninergic receptors (MTRs) desensitization [17]. Likewise, the dose of 5 µg was unable to activate MTRs. Based on our results, the dose chosen for the other in vitro test was 10 µg of PM.
Cytoskeletal remodeling and keratinocytes terminal differentiation are markers of skin regeneration [18]. In this view, evaluation of cell morphology was assessed by fluorescence microscopy. Change in cell morphology gaging cytoskeletal remodeling and nuclear lobe formation was tested by phalloidin-stained F-actin and DAPI, respectively. In Figure 2, keratinocytes cultured without (Panel A) and with PM (Panel B) cytoskeletal remodeling was observed. The yellow arrows in Figure 2 panel A indicate that the F actin filaments show a centromedian cellular localization. In the presence of PM for 24 h, the isoform F of the actin show a cortical localization near to the plasma membrane as indicated with the white arrows (Panel B). Terminal differentiation of keratinocytes was assessed by changes in nuclear morphology. The lobed (half-moon) nucleus are typical of terminal differentiation. In the presence of PM for 24 h, a significant number of lobed nuclei are shown by orange arrows (Panel D) compared to the control (Panel C). Indeed, in our set of experiments PM induced a re-localization of isoform F of the actin near to the plasma membrane as well as lobed (half-moon) nucleus shape typical of terminal differentiation. Our results revealed that this compound affected cell viability in a dose-response fashion and strongly suggested the involvement of the receptor mechanisms. Indeed, the higher amount used at 200 μg drastically decreased cell viability. This mechanism was probably due to the melatoninergic receptors (MTRs) desensitization [17]. Likewise, the dose of 5 μg was unable to activate MTRs. Based on our results, the dose chosen for the other in vitro test was 10 μg of PM.
Cytoskeletal remodeling and keratinocytes terminal differentiation are markers of skin regeneration [18]. In this view, evaluation of cell morphology was assessed by fluorescence microscopy. Change in cell morphology gaging cytoskeletal remodeling and nuclear lobe formation was tested by phalloidin-stained F-actin and DAPI, respectively. In Figure 2, keratinocytes cultured without (Panel A) and with PM (Panel B) cytoskeletal remodeling was observed. The yellow arrows in Figure 2 panel A indicate that the F actin filaments show a centromedian cellular localization. In the presence of PM for 24 hours, the isoform F of the actin show a cortical localization near to the plasma membrane as indicated with the white arrows (Panel B). Terminal differentiation of keratinocytes was assessed by changes in nuclear morphology. The lobed (half-moon) nucleus are typical of terminal differentiation. In the presence of PM for 24 hours, a significant number of lobed nuclei are shown by orange arrows (Panel D) compared to the control (Panel C). Indeed, in our set of experiments PM induced a re-localization of isoform F of the actin near to the plasma membrane as well as lobed (half-moon) nucleus shape typical of terminal differentiation. A crucial aspect of skin aging promotion is the role played by both ROS generation (intrinsic aging factor) and UV exposure which leads to ROS production (extrinsic and intrinsic aging factors) [19]. It is well known that the formation of ROS takes place predominantly on mitochondria and that mitochondrial membrane potential (MMP) upon UV radiation is impaired generating free radicals [20].
In this concern, the estimation of MMP which lead to ROS generation was performed by fluorescence microscopy. The red-fluorescent MitoTracker Red CMXRos dye stains mitochondria in live cells and its accumulation dependent by MMP. The keratinocytes were treated for 6 hours with A crucial aspect of skin aging promotion is the role played by both ROS generation (intrinsic aging factor) and UV exposure which leads to ROS production (extrinsic and intrinsic aging factors) [19].
It is well known that the formation of ROS takes place predominantly on mitochondria and that mitochondrial membrane potential (MMP) upon UV radiation is impaired generating free radicals [20].
In this concern, the estimation of MMP which lead to ROS generation was performed by fluorescence microscopy. The red-fluorescent MitoTracker Red CMXRos dye stains mitochondria in live cells and its accumulation dependent by MMP. The keratinocytes were treated for 6 h with 10 µg PM. As shown in Figure 3, the exposure of the HaCaT cell line to 10 µg PM induced a significant decrease in the mitochondrial membrane potential (Panel B) compared to control cultures (Panel A) when exposed to a UV lamp at 254 nm (UVC spectrum to avoid vitamin D synthesis). In Panel C, graph box plot of red dots pixel analysis (*** P = 0.0002) is shown. Thus, as well as acting on cytoskeletal remodeling and keratinocytes terminal differentiation, which indicate skin regeneration [22], in our set of experiments PM was able to counteract both extrinsic and intrinsic insult acting as a free radical scavenger. The antioxidant defense system dysfunction and imbalance between ROS formation and removal can cause cells to influence gene expression, a process in which microRNAs (miRs) are well recognized to be crucial regulators [8].
The involvement of MiRs in the aging process and oxidative stress is emerging [9] and the role of the hsa-miR-29 family in the control of cell-extracellular matrix, aging and skin regeneration is exciting [9][10][11]22].
In this concern, using qRT-PCR analysis revealed low levels of hsa-miR-29a-3p in the control samples compared to in PM treated ones. As shown in Figure 4 Panel A, the increase of this miR upon PM 24-hour treatment was about 50%.
As a result, we showed the effect of PM on the increase of hsa-miR-29a-3p, which is able to counteract the aging process [11].
In order to predict the target genes of hsa-miR-29a-3p and identify possible genes involved in keratinocytes homeostasis, we used three different tools. Eighty predicted target genes were found for hsa-miR-29a-3p. Of these, the genes linked to keratinocytes homeostasis were chosen, as shown in Table 1 using miR target link. Table 1. Validated target genes with high score interaction linked to keratinocytes homeostasis for hsa-miR-29a-3p according to the miR target link database. The physiological processes due to mitochondrial activity can synergize with intracellular ROS, thus inducing cellular aging [20,21]. Therefore, we further evaluated the intracellular reactive oxygen species (intROS) formation, treating keratinocytes for 6 h with 10 µg of PM and then exposing the cellular layer to H 2 O 2 500 µM for 30 min. The specific dye CellRox Deep Red revealed that PM significantly decreased intROS formation, as shown in Figure 3 Panel E, compared to the control counterpart (Panel D), suggesting a potent scavenging action of PM. In Panel F, a graph box plot of deep-red dots pixel analysis (*** P = 0.0002) is shown.
Thus, as well as acting on cytoskeletal remodeling and keratinocytes terminal differentiation, which indicate skin regeneration [22], in our set of experiments PM was able to counteract both extrinsic and intrinsic insult acting as a free radical scavenger. The antioxidant defense system dysfunction and imbalance between ROS formation and removal can cause cells to influence gene expression, a process in which microRNAs (miRs) are well recognized to be crucial regulators [8].
The involvement of MiRs in the aging process and oxidative stress is emerging [9] and the role of the hsa-miR-29 family in the control of cell-extracellular matrix, aging and skin regeneration is exciting [9][10][11]22].
In this concern, using qRT-PCR analysis revealed low levels of hsa-miR-29a-3p in the control samples compared to in PM treated ones. As shown in Figure 4 Panel A, the increase of this miR upon PM 24-hour treatment was about 50%.
Cosmetics 2018, 5, x FOR PEER REVIEW 7 of 9 The physical interactions between miR and the mRNAs and string protein interactions, both experimentally validated, are depicted in Figure 4 Panels A and B, respectively. The target matrix metalloproteinase (MMP) 2 gene is very interesting. MMPs are collagenase able to degrade collagen and elastin fibers in the connective tissue of the skin. In effect, collagen and elastin degradation has been reported through both, MMP-1 and MMP-2 [23].
Likewise, excessive ROS generation by both intrinsic or extrinsic factors leads to DNA damage in normal cells and an increase of the MMP level, which can promote skin aging [23].
Besides that, this miR is able to target also the lysyl oxidase (LOX) enzyme that strongly impairs terminal differentiation when expressed during the late keratinocytes differentiation process [24].
In conclusion, our study demonstrated that PM affected several aspects of human keratinocytes biology ranging from the promotion of cell survival to a protective effect against oxidative damage. In addition, our results highlight for the time an epigenetic modulation of this compound, paving the way for the use of PM as an advanced cosmetic tool in the improvement of skin performance. As a result, we showed the effect of PM on the increase of hsa-miR-29a-3p, which is able to counteract the aging process [11].
In order to predict the target genes of hsa-miR-29a-3p and identify possible genes involved in keratinocytes homeostasis, we used three different tools. Eighty predicted target genes were found for hsa-miR-29a-3p. Of these, the genes linked to keratinocytes homeostasis were chosen, as shown in Table 1 using miR target link. Table 1. Validated target genes with high score interaction linked to keratinocytes homeostasis for hsa-miR-29a-3p according to the miR target link database.
Target Gene
Gene Name miRTarBase ID
The physical interactions between miR and the mRNAs and string protein interactions, both experimentally validated, are depicted in Figure 4A,B, respectively. The target matrix metalloproteinase (MMP) 2 gene is very interesting.
MMPs are collagenase able to degrade collagen and elastin fibers in the connective tissue of the skin. In effect, collagen and elastin degradation has been reported through both, MMP-1 and MMP-2 [23].
Likewise, excessive ROS generation by both intrinsic or extrinsic factors leads to DNA damage in normal cells and an increase of the MMP level, which can promote skin aging [23].
Besides that, this miR is able to target also the lysyl oxidase (LOX) enzyme that strongly impairs terminal differentiation when expressed during the late keratinocytes differentiation process [24].
In conclusion, our study demonstrated that PM affected several aspects of human keratinocytes biology ranging from the promotion of cell survival to a protective effect against oxidative damage. In addition, our results highlight for the time an epigenetic modulation of this compound, paving the way for the use of PM as an advanced cosmetic tool in the improvement of skin performance. | v3-fos-license |
2021-10-20T15:31:38.053Z | 2021-09-16T00:00:00.000 | 240521330 | {
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} | pes2o/s2orc | Synthesis and Characterization of Super Bulky β -Diketiminato Group 1 Metal Complexes
: Sterically bulky β -diketiminate (or Nacnac) ligand systems have recently shown the ability to kinetically stabilize highly reactive low-oxidation state main group complexes. Metal halide precursors to such systems can be formed via salt metathesis reactions involving alkali metal complexes of these large ligand frameworks. Herein, we report the synthesis and characterization of lithium and potassium complexes of the super bulky anionic β -diketiminate ligands, known [ TCHP Nacnac] − and new [ TCHP/Dip Nacnac] − ( Ar Nacnac = [(ArNCMe) 2 CH] − ) (Ar = 2,4,6-tricyclohexylphenyl (TCHP) or 2,6-diisopropylphenyl (Dip)). The reaction of the proteo-ligands, Ar NacnacH, with n BuLi give the lithium etherate compounds, [( TCHP Nacnac)Li(OEt 2 )] and [( TCHP/Dip Nacnac)Li(OEt 2 )], which were isolated and characterized by multinuclear NMR spectroscopy and X-ray crystallography. The unsolvated potassium salts, [{K( TCHP Nacnac)} 2 ] and [{K( TCHP/Dip Nacnac)} ∞ ], were also synthesized and characterized in solution by NMR spectroscopy. In the solid state, these highly reactive potassium complexes exhibit differing alkali metal coordination modes, depending on the ligand involved. These group 1 complexes have potential as reagents for the transfer of the bulky ligand fragments to metal halides, and for the subsequent stabilization of low-oxidation state metal complexes.
The activation of β-diketiminate coordinated Mg-Mg bonded fragments by the addition of a sub-stoichiometric amount of a Lewis base is known to generate unsymmetrical magnesium(I) compounds with elongated Mg-Mg bonds. These have been shown to increase the reactivity of the magnesium(I) compound towards inert molecules such as CO and ethylene [19,20]. Recently, we showed that a TCHP-substituted magnesium(I) dimer, upon irradiation, was able to reduce benzene to generate "Birch-like" cyclohexadienyl bridged products, e.g., [{( TCHP Nacnac)Mg} 2 (µ-C 6 H 6 )] [21]. Further to this, Harder and co-workers have shown that Nacnac ligands incorporating the DIPeP aryl moiety are able to stabilize molecular magnesium(0) compounds [22,23], as well as calcium complexes, which activate dinitrogen, as in [{( DIPeP Nacnac)Ca} 2 (µ-N 2 )] [24].
Routes to low-oxidation state metal complexes bearing Nacnac ligands typically proceed via the reduction of β-diketiminato metal halide precursors, which can be synthesized by the reaction of an alkali metal salt of the β-diketiminate ligand with a metal halide (Scheme 1). As such, the synthesis of alkali metal complexes of new very bulky ligands is desirable, with the view to using such complexes as ligand transfer reagents in the preparation of metal halide precursors to highly reactive low-valent metal complexes. Potentially, compounds previously inaccessible using the archetypal Dip Nacnac ligand system can be realized using this approach. Herein, we describe the synthesis and characterization of a new unsymmetrical, bulky β-diketimine pro-ligand incorporating the TCHP aryl moiety, as well as four alkali metal complexes derived from this, and a previously reported TCHP-substituted β-diketimine. The activation of β-diketiminate coordinated Mg-Mg bonded fragments by the addition of a sub-stoichiometric amount of a Lewis base is known to generate unsymmetrical magnesium(I) compounds with elongated Mg-Mg bonds. These have been shown to increase the reactivity of the magnesium(I) compound towards inert molecules such as CO and ethylene [19,20]. Recently, we showed that a TCHP-substituted magnesium(I) dimer, upon irradiation, was able to reduce benzene to generate "Birch-like" cyclohexadienyl bridged products, e.g., [{( TCHP Nacnac)Mg}2(µ-C6H6)] [21]. Further to this, Harder and coworkers have shown that Nacnac ligands incorporating the DIPeP aryl moiety are able to stabilize molecular magnesium(0) compounds [22,23], as well as calcium complexes, which activate dinitrogen, as in [{( DIPeP Nacnac)Ca}2(µ-N2)] [24].
Routes to low-oxidation state metal complexes bearing Nacnac ligands typically proceed via the reduction of β-diketiminato metal halide precursors, which can be synthesized by the reaction of an alkali metal salt of the β-diketiminate ligand with a metal halide (Scheme 1). As such, the synthesis of alkali metal complexes of new very bulky ligands is desirable, with the view to using such complexes as ligand transfer reagents in the preparation of metal halide precursors to highly reactive low-valent metal complexes. Potentially, compounds previously inaccessible using the archetypal Dip Nacnac ligand system can be realized using this approach. Herein, we describe the synthesis and characterization of a new unsymmetrical, bulky β-diketimine pro-ligand incorporating the TCHP aryl moiety, as well as four alkali metal complexes derived from this, and a previously reported TCHP-substituted β-diketimine. Scheme 1. Generic reaction of a β-diketiminato alkali metal salt with a metal/metalloid halide.
Results and Discussion
The β-diketimine, TCHP NacnacH, 1, was recently reported to be prepared by a condensation reaction between acacH, H2C(MeC=O)2 and two equivalents of (TCHP)NH2 [17]. Here, the new, unsymmetrical β-diketimine, TCHP/Dip NacnacH 2, was similarly synthesized by the condensation of one equivalent of (TCHP)NH2 with Dip NacacH, {DipN(H)CMe}CHC(O)Me, which gave 2 in a 43% isolated yield [25]. It is of note that, in the current study, during one preparation of 1, a low yield of the β-ketimine, TCHP NacacH, {(TCHP)N(H)CMe}CHC(O)Me 3, was obtained as a by-product. This likely results from the use of slightly less than two equivalents of (TCHP)NH2 in the synthesis. A rational synthesis of 3 was not attempted, but it was spectroscopically characterized. The molecular structure of 2 is shown in Figure 1a, which reveals it to exist as its ene-imine conjugated tautomer in the solid state, as is typical for β-diketimines. The proton on N2 is hydrogen bonded to N1, which leads to the two double bonds within the C3N2 backbone (viz. N1-C1 and C3-C4) adopting a cis-configuration, relative to each other. A similar structure is adopted by β-ketimine, 3 (Figure 1b), in which the double bonds within the C3NO backbone are C1-O1 and C3-C4. Scheme 1. Generic reaction of a β-diketiminato alkali metal salt with a metal/metalloid halide.
Results and Discussion
The β-diketimine, TCHP NacnacH, 1, was recently reported to be prepared by a condensation reaction between acacH, H 2 C(MeC=O) 2 and two equivalents of (TCHP)NH 2 [17]. Here, the new, unsymmetrical β-diketimine, TCHP/Dip NacnacH 2, was similarly synthesized by the condensation of one equivalent of (TCHP)NH 2 with Dip NacacH, {DipN(H)CMe}CHC(O)Me, which gave 2 in a 43% isolated yield [25]. It is of note that, in the current study, during one preparation of 1, a low yield of the β-ketimine, TCHP NacacH, {(TCHP)N(H)CMe}CHC(O)Me 3, was obtained as a by-product. This likely results from the use of slightly less than two equivalents of (TCHP)NH 2 in the synthesis. A rational synthesis of 3 was not attempted, but it was spectroscopically characterized. The molecular structure of 2 is shown in Figure 1a, which reveals it to exist as its ene-imine conjugated tautomer in the solid state, as is typical for β-diketimines. The proton on N2 is hydrogen bonded to N1, which leads to the two double bonds within the C 3 N 2 backbone (viz. N1-C1 and C3-C4) adopting a cis-configuration, relative to each other. A similar structure is adopted by β-ketimine, 3 (Figure 1b), in which the double bonds within the C 3 NO backbone are C1-O1 and C3-C4.
The bulky β-diketimines, 1 and 2, were deprotonated by treating diethyl ether solutions of the compounds with a slight excess of nBuLi at −78 • C. Upon warming to room temperature and stirring the reaction solutions overnight, they were concentrated and stored at −30 • C to yield colorless crystals of lithium β-diketiminate complexes [( TCHP Nacnac)Li(OEt 2 )], 4, and [( TCHP/Dip Nacnac)Li(OEt 2 )], 5, in moderate isolated yields. The solution state NMR spectroscopic data for the compounds are consistent with them retaining their solid-state structures (see below) in solution. However, it is noteworthy that the 13 C{ 1 H} NMR spectrum of compound 4 exhibits 10 cyclohexyl carbon signals (δ = 25-45 ppm), which suggests restricted rotation around one of the sterically more encumbered ortho-cyclohexyl rings of the TCHP substituents.
Compounds 4 and 5 have very similar solid-state structures, as is evident from Figure 2. The molecular structure of these compounds are typical of previously reported β-diketiminate lithium etherate complexes, e.g., [( Dip Nacnac)Li(OEt 2 )] [26]. That is, they are monomeric and their lithium centers possess trigonal planar coordination geometries with one coordinated ether ligand. The β-diketiminate ligand binds as an N,N-chelate to the lithium, with Li-N bond distances between 1.921 and 1.935 Å, within the normal range for such complexes [26]. The Li-O distances are slightly longer when compared to the previously reported [( Dip Nacnac)Li(OEt 2 )] complex (1.953(4) Å for 4 and 1.975(4) Å for 5; cf. 1.911(4) Å for [( Dip Nacnac)Li(OEt 2 )]), which is likely caused by steric repulsion from its bulky TCHP aryl groups. This also leads to a slight narrowing of the N-Li-N angle for 4 and 5 (97.67(18) • and 97.79(17) • , respectively) compared to the angle in [( Dip Nacnac)Li(OEt 2 )] (99.9(2) • ). The bond lengths within the C 3 N 2 backbones of both compounds imply significant electronic delocalizations over those fragments. The bulky β-diketimines, 1 and 2, were deprotonated by treating diethyl ether solutions of the compounds with a slight excess of nBuLi at −78 °C. Upon warming to room temperature and stirring the reaction solutions overnight, they were concentrated and stored at −30 °C to yield colorless crystals of lithium β-diketiminate complexes [( TCHP Nacnac)Li(OEt2)], 4, and [( TCHP/Dip Nacnac)Li(OEt2)], 5, in moderate isolated yields. The solution state NMR spectroscopic data for the compounds are consistent with them retaining their solid-state structures (see below) in solution. However, it is noteworthy that the 13 C{ 1 H} NMR spectrum of compound 4 exhibits 10 cyclohexyl carbon signals (δ = 25-45 ppm), which suggests restricted rotation around one of the sterically more encumbered orthocyclohexyl rings of the TCHP substituents.
Compounds 4 and 5 have very similar solid-state structures, as is evident from Figure 2. The molecular structure of these compounds are typical of previously reported β-diketiminate lithium etherate complexes, e.g., [( Dip Nacnac)Li(OEt2)] [26]. That is, they are monomeric and their lithium centers possess trigonal planar coordination geometries with one coordinated ether ligand. The β-diketiminate ligand binds as an N,N-chelate to the lithium, with Li-N bond distances between 1.921 and 1.935 Å, within the normal range for such complexes [26]. Unsolvated potassium salts of the TCHP-substituted β-diketiminates could also be synthesized using a similar methodology to that previously reported for the synthesis of [K( Dip Nacnac)] [27]. That is, β-diketimine, 1, was combined with benzyl potassium in toluene, pro-ligand 2 was combined with potassium bis(trimethylsilyl)amide (KHMDS) in toluene and both reactions were monitored by NMR spectroscopy. No reaction could be observed after 16 h at room temperature for either pro-ligand, contrary to the synthesis of [K( Dip Nacnac)], which showed good conversion under these conditions. This can be attributed to the increased steric bulk from the TCHP aryl group, perturbing deprotonation Unsolvated potassium salts of the TCHP-substituted β-diketiminates could also be synthesized using a similar methodology to that previously reported for the synthesis of [K( Dip Nacnac)] [27]. That is, β-diketimine, 1, was combined with benzyl potassium in toluene, pro-ligand 2 was combined with potassium bis(trimethylsilyl)amide (KHMDS) in Inorganics 2021, 9, 72 4 of 9 toluene and both reactions were monitored by NMR spectroscopy. No reaction could be observed after 16 h at room temperature for either pro-ligand, contrary to the synthesis of [K( Dip Nacnac)], which showed good conversion under these conditions. This can be attributed to the increased steric bulk from the TCHP aryl group, perturbing deprotonation of 1 and 2 by the base. However, heating these solutions at 60 • C overnight led to clean conversion to the potassium salts [{K( TCHP Nacnac)} 2 ], 6, and [{K( TCHP/Dip Nacnac)} ∞ ], 7, as determined by 1 H NMR spectroscopy. It should be noted that compound 6 could also be formed using KHMDS as the base, though this route gave variable yields of product. Attempts to isolate compound 6 repeatedly led to a degree of decomposition of the complex to 1 upon manipulation of the reaction mixture. Moreover, in the presence of silicone grease, compound 6 decomposed to unknown products in solution over time. This suggests that compound 6 is highly reactive, and as such could only be characterized by NMR spectroscopy after formation in situ. As monitoring the formation of 6 from 1 by NMR spectroscopy showed complete conversion, we believe this compound could be used as a ligand transfer reagent after its formation in situ, and in the absence of silicone grease.
Crystals of 6 suitable for an X-ray diffraction study were formed by the addition of n-hexane to a concentrated toluene solution of 6 at room temperature. The molecular structure (Figure 3) shows the compound crystallizing as a dimer, with the potassium centers having trigonal planar geometries, being N,N-chelated by a β-diketiminate, as well as possessing an intermolecular C···K interaction with a meta-carbon of an opposing TCHP group of the second [K( TCHP Nacnac)] monomer unit. The N-K distances (2.633(3) and 2.619(3) Å) are in the expected range and correspond well to those previously reported for [K( Dip Nacnac)] (2.681 Å mean) [27]. The C···K distance of 3.309(3) Å is outside the sum of the covalent radii for the two elements (2.71 Å [28]), but well within the sum of their van der Waals radii (4.45 Å [29]). Compound 7 crystallized as a colorless material from the toluene reaction solution, upon cooling it to room temperature from 60 °C. However, it should be noted that, like 6, compound 7 undergoes decomposition to the proteo-ligand 2 in the presence of silicone grease. Crystals of 7 suitable for an X-ray diffraction were obtained from a saturated benzene solution, and its molecular structure is shown in Figure 4. It is polymeric, similar to previously reported [K( Dip Nacnac)] [27]. The polymer is propagated via intermolecular C•••K interactions between the potassium centers and para-and meta-carbons of a Dip group on an adjacent monomer unit. The K•••C bond distances are 3.178(4) Å for K-Cpara Compound 7 crystallized as a colorless material from the toluene reaction solution, upon cooling it to room temperature from 60 • C. However, it should be noted that, like 6, compound 7 undergoes decomposition to the proteo-ligand 2 in the presence of silicone grease. Crystals of 7 suitable for an X-ray diffraction were obtained from a saturated benzene solution, and its molecular structure is shown in Figure 4. It is polymeric, similar to previously reported [K( Dip Nacnac)] [27]. The polymer is propagated via intermolecular C···K interactions between the potassium centers and paraand meta-carbons of a Dip group on an adjacent monomer unit. The K···C bond distances are 3.178(4) Å for K-C para Inorganics 2021, 9, 72 5 of 9 and 3.254(3) Å for K-C meta . These separations are slightly shorter than the equivalent C···K interactions in [K( Dip Nacnac)], which are 3.214(3) Å for K-C para , and 3.351(3) Å and 3.276(3) Å for K-C meta interactions. The K-N distances in the compound are comparable to those in 6.
Compound 7 crystallized as a colorless material from the toluene reaction solution, upon cooling it to room temperature from 60 °C. However, it should be noted that, like 6, compound 7 undergoes decomposition to the proteo-ligand 2 in the presence of silicone grease. Crystals of 7 suitable for an X-ray diffraction were obtained from a saturated benzene solution, and its molecular structure is shown in Figure 4. It is polymeric, similar to previously reported [K( Dip Nacnac)] [27]. The polymer is propagated via intermolecular C•••K interactions between the potassium centers and para-and meta-carbons of a Dip group on an adjacent monomer unit. The K•••C bond distances are 3.178(4) Å for K-Cpara and 3.254(3) Å for K-Cmeta. These separations are slightly shorter than the equivalent C•••K interactions in [K( Dip Nacnac)], which are 3.214(3) Å for K-Cpara, and 3.351(3) Å and 3.276(3) Å for K-Cmeta interactions. The K-N distances in the compound are comparable to those in 6.
General Considerations
All manipulations were carried out using standard Schlenk and glove box techniques under an atmosphere of high purity dinitrogen. Toluene and hexane were distilled over molten potassium, while diethyl ether was distilled over 1:1 Na/K alloy. Benzene-d 6 was stored over a mirror of sodium and degassed three times via freeze-pump-thawing before use. The 1 H, 13 C{ 1 H} and 7 Li NMR spectra were recorded on a Bruker AvanceIII 400 spectrometer and were referenced to the residual resonances of the solvent used or external 1 M LiCl. Mass spectra were collected using an Agilent Technologies 5975D inert MSD with a solid-state probe. FTIR spectra were collected as Nujol mulls on an Agilent Cary 630 attenuated total reflectance (ATR) spectrometer. Microanalyses were carried out at the Science Centre, London Metropolitan University or using a PerkinElmer-2400 CHNS/O Series II System. Melting points were determined in sealed glass capillaries under dinitrogen and are uncorrected. The starting materials, TCHP NacnacH 1 [17], Dip NacacH [30] (TCHP)NH 2 [31] and benzyl potassium [32], were prepared by literature procedures. All other reagents were used as received.
Syntehsis of TCHP/Dip NacnacH 2 and Data for TCHP NacacH 3
Preparation of TCHP/Dip NacnacH, 2: 2,4,6-Tricyclohexylaniline (4.57 g, 13.5 mmol), Dip NacacH (3.50 g, 13.5 mmol) and p-toluenesulfonic acid (2.32 g, 13.5 mmol) were dissolved in toluene (100 mL), and the mixture was heated at reflux for 3 days in a Dean-Stark apparatus. Volatiles were then removed in vacuo, the resultant oil was dissolved in dichloromethane (100 mL), the extract washed with a saturated Na 2 CO 3 solution (100 mL, 1 M) and the organic layer was separated. The organic layer was dried over MgSO 4 and volatiles were removed in vacuo. Methanol (5 mL) was added to the oily residue, which was then sonicated and filtered to yield compound 2 as a crude white powder. This was dried and recrystallized from hot ethyl acetate to yield pure TCHP/Dip NacnacH (2.25 g). A further crop of the product could be obtained by concentration of the mother liquor, and letting it stand at room temperature (1.00 g). Total yield: 3.25 g, 42%. X-ray quality crystals could be grown from the slow evaporation of a diethyl ether solution of TCHP/Dip NacnacH. Preparation of [{K( TCHP Nacnac)} 2 ], 6: TCHP NacnacH (400 mg, 0.54 mmol) and benzyl potassium (72 mg, 0.55 mmol) were dissolved in toluene (10 mL) and the mixture stirred overnight at 60 • C. The solution was then cooled to room temperature and volatiles were removed in vacuo. The subsequent solid was dissolved in n-hexane (5 mL) and the extract left to stand at room temperature, yielding colorless crystals of 6 suitable for X-ray diffraction studies. Analysis of the crystalline material by NMR spectroscopy consistently showed contamination of the product with TCHP NacnacH, which could not be removed, despite repeated recrystallizations.
In situ preparation of 6: TCHP NacnacH (10 mg, 0.013 mmol) and benzyl potassium (3 mg, 0.023 mmol) were dissolved in C 6 D 6 (0.6 mL) in a J. Young's NMR tube equipped with a Teflon screw cap and the mixture heated overnight at 60 • C. The subsequent reaction solution was analyzed by NMR spectroscopy, showing near complete conversion to compound 6. 1 (8 mL) and the mixture heated overnight at 60 • C. The solution was cooled to room temperature, after which time micro-crystalline 7 was deposited. The suspension was filtered, and the solid was dried in vacuo giving compound 7 as a spectroscopically near pure colorless solid (500 mg, 91%). Crystals suitable for X-ray diffraction were grown by slow cooling a saturated solution of 7 in C 6 D 6 from 60 • C to room temperature. M.p. > 260 • C: 1 H NMR (400 MHz, C 6 D 6 , 298 K) N.B. integration for cyclohexyl groups are estimated due to complex overlapping signals: δ = 1.14-1.17 (d, 3 J HH = 6.9 Hz, 6H, CH(CH 3 ) 2 ), 1.24-1.30 (m, 2H, Cyc-CH 2 ), 1.32-1.34 (d, 3
Crystallographic Details
Crystals of 2-7 suitable for X-ray structural determination were mounted in silicone oil. Crystallographic measurements were carried out at 123(2) K, and were made using a Rigaku Synergy diffractometer using a graphite monochromator with Cu Kα radiation (λ = 1.54184 Å). The structures were solved by direct methods and refined on F 2 by full matrix least squares (SHELX16) [33] using all unique data. All non-hydrogen atoms were anisotropic with hydrogen atoms typically included in calculated positions (riding model). Crystal data, details of the data collection and refinement are given in Table S1 (in Supplementary Materials). Crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre (CCDC no. 2105059-2105064). Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; email: [email protected] or www: http://www.ccdc.cam.ac.uk).
Conclusions
In summary, the syntheses of four alkali metal complexes of two super bulky βdiketiminate ligand systems are described. These complexes were characterized by X-ray crystallography and multinuclear NMR spectroscopy. The solid-state structures of the lithium etherate complexes are similar to previously reported examples. The molecular structures of the potassium salts show varied coordination environments in potassium, depending on the accompanying ligand framework. That is, [{K( TCHP Nacnac)} 2 ] 6 is dimeric with C···K interactions involving a meta-aryl carbon of the opposing monomer unit. In contrast, [{K{( TCHP/Dip Nacnac)} ∞ ] 7 is polymeric, and shows comparable connectivity between monomer units to that seen in the previously reported complex [K( Dip Nacnac)]. These alkali metal compounds have significant potential for use as ligand transfer reagents in the synthesis of metal complexes incorporating large TCHP-substituted β-diketiminates. We are currently exploring the use of these ligands for the kinetic stabilization of lowoxidation state metal complexes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/inorganics9090072/s1, Table S1: Summary of crystallographic data for 2-7; Figures S1-S14: 1 H, 13 Data Availability Statement: NMR spectra and crystal data are given in the supporting information. Crystal data, details of the data collection and refinement are given in Table S1. Crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre (CCDC no. 2105059-2105064). Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; email: [email protected] or http://www.ccdc.cam.ac.uk). | v3-fos-license |
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} | pes2o/s2orc | Structural Origins for the Loss of Catalytic Activities of Bifunctional Human LTA4H Revealed through Molecular Dynamics Simulations
Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents.
Introduction
Leukotriene cascade is associated with the biosynthesis of variety of leukotrienes (LT) from the phospholipids of the nuclear membrane of the leukocytes [1]. The LTs are a group of lipid mediators associated with acute and chronic inflammatory diseases such as asthma, rhinitis, psoriasis, chronic obstructive pulmonary disease, and atherosclerosis [2][3][4][5][6][7][8][9][10][11][12][13]. Cytosolic phospholipase A2 (cPLA2) hydrolyzes the ester bond present in sn-2 position of phospholipids and yields lysophospholipids and free fatty acid, arachidonic acid (AA) [1,14]. This increases the level of free AA available for the synthesis of inflammatory leukotrienes upon the action of more enzymes. The enzyme 5-lipoxygenase (5-LO) assisted by file-lipoxygenase-activating protein (FLAP) converts the AA into the highly unstable allylic epoxide, leukotriene A4 (LTA4) [15][16][17][18][19][20][21]. This unstable intermediate is converted into two different products LTB4 and LTC4 by the action of two different enzymes LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), respectively [1,[22][23][24][25]. The LTC4 is subsequently converted to LTD4 and LTE4 substances by the action of different enzymes. All of these LTB4, LTC4, LTD4, and LTE4 are powerful proinflammatory mediators [1,26]. The LTA4H, which catalyzes the conversion of LTA4 to the chemotactic agent LTB4, was identified as a bi-functional enzyme capable of processing two highly diverse substrates such as LTA4 (a fatty acid) and peptide through its epoxide hydrolase and aminopeptidase activities [27,28]. This enzyme was first discovered for its epoxide hydrolase activity and later for its aminopeptidase activity based on the presence of consensus Zn binding motif (HEXXH-X 18 -E), which was found in M1 family of Zn containing aminopeptidases [29][30][31][32]. The natural peptide substrate for this enzyme is still not known but preference is shown over arginyl di-and tripeptide and can selectively be blocked by the mutation of either E296 or Y383 residues [33][34][35][36]. Upon the determination of LTA4H crystal structures it was revealed that this enzyme is composed of three domains, a fully beta N-terminal domain, a mixed alpha/beta catalytic domain, and a fully alpha-helical C-terminal domain ( Figure 1) [37][38][39][40][41][42]. In terms of the hydrolase activity of the enzyme, D375 from a narrow hydrophobic pocket is specifically required as it is involved in the nucleophilic attack targeting C12 atom of LTA4 [43]. In addition, this residue belongs to the peptide K21 (L365-K385) segment identified by Lys-specific peptide mapping of suicide inactivated LTA4H. The carboxylate moiety of LTA4 was observed to form direct electrostatic interactions with the two positively charged conserved R563 and K565 residues present at the entrance of the active center [28,44]. These interactions are very much essential in aligning LTA4 along with the catalytic elements of the active site. Based on the mutagenic experiments, E271 residue from another conserved GXMEN motif in the family of zinc peptidases was found to be important for both the functions of the enzyme [14] as the mutagenic replacements abrogated both the activities. A crystal structure of LTA4H with E271Q mutation has revealed only minimal conformational changes and did not explain the loss of enzyme function [14]. It was also suggested that the carboxylate of E271 participates in an acid-induced opening of the epoxide moiety of LTA4 and as N-terminal recognition site in terms of peptide substrates [14,26,45]. Some mutagenic experiments have also reported the critical role of R563 residue in epoxide hydrolase reaction by positioning the carboxylate tail along the catalytic elements of the active site [28,34]. In aminopeptidase reaction, both R563 and K565 residues co-operate each other to ensure the necessary binding strength and productive alignment of the substrate. Altogether, R563 plays important roles in both the functions of the enzyme whereas K565 residue assists R563 in catalyzing the peptide substrate but not in hydrolase reaction, which catalyzes the fatty acid substrate [27]. These residues were reported to be the common carboxylate recognition site for both lipid and peptide substrates in the active site of LTA4H [27].
Mutations of R563 to any other amino acid including a conservative replacement of R by K preserving the positive charge abolished the enzyme function but exhibited a significant residual aminopeptidase activity [27]. Crystal structure with R563A mutant could not reveal any structural changes explaining the complete loss of catalytic activity. It was also reported that esterified LTA4 cannot be the substrate of the enzyme and this phenomenon was explained with the steric hindrance [46], which also proved that a free carboxylate group of LTA4 is critical for the hydrolase function. The other carboxylate recognition residue K565 located in a way that it can also involve in carboxylate recognition but its mutagenic replacements have not decreased the epoxide hydrolase activity [27]. The difference in observed aminopeptidase activities between the wild type and K565 mutants have suggested that K565 is a carboxylate binding site for peptide substrates also [27]. The information of a binding pocket for its ligand is very important for drug design, particularly for conducting mutagenesis studies [47]. In the literature, the binding pocket of a protein receptor to a ligand is usually defined by those residues that have at least one heavy atom (i.e., an atom other than hydrogen) with a distance from a heavy atom of the ligand. Such a criterion was originally used to define the binding pocket of ATP in the Cdk5-Nck5a* complex [48] that has later proved quite useful in identifying functional domains and stimulating the relevant truncation experiments [49]. The similar approach has also been used to define the binding pockets of many other receptor-ligand interactions important for drug design [50][51][52][53][54].
This study has focused on structural changes and binding mode differences between wild types and mutant forms of hLTA4H-fatty acid and -tripeptide substrate complexes. Molecular dynamics (MD) simulations of two wild types of the enzyme-substrate complexes and three mutations including E272Q, R563A, and K565A were studied with LTA4 (fatty acid) and Arg-Ala-Arg (RAR, tripeptide) substrate ( Figure 2). The binding mode comparison of enzyme-substrate complexes revealed essential structural differences, which were not shown in X-ray structures, explaining the loss of catalytic functions of the enzyme. The overlay of binding modes of LTA4 and RAR substrates has proved the previous assumption of similar but overlapped active site is used in both the functions of the enzyme. The results from this study provide valuable information over the way the two functions of the enzyme are exerted over two substrates of diverse nature. In addition, the structural information obtained from this study can be utilized in structure-based hLTA4H inhibitor design as inhibition of the hydrolase and aminopeptidase functions will lead to the development of anticancer and anti-inflammatory drugs, respectively.
Preparation of complex structures
As this study was aimed at investigating the binding of fatty acid and peptide substrates of the enzyme, independent hLTA4H-LTA4 (L-LTA4) and hLTA4H-RAR (L-RAR) complexes were prepared. The LTA4 of AA cascade is the natural substrate of the enzyme but no 3D structural information was available for its binding from X-ray studies. Some previous studies have reported the proposed binding of this fatty acid substrate at the active site of the enzyme. In this study, molecular docking methodology using GOLD 5.0.1 program was employed to obtain the reliable binding mode of LTA4. The GOLD program from Cambridge Crystallographic Data Centre, UK uses a genetic algorithm to dock the small molecules into the protein active site [46,55]. The GOLD allows for a full range of flexibility of the ligands and partial flexibility of the protein. One of the crystal structures of hLTA4H bound with most active inhibitor (PDB ID: 3FUN) solved at high resolution was used in molecular docking. The bound inhibitor of this crystal structure was removed and the apoform of this enzyme was subjected to MD simulations with the parameters discussed below. The representative structure with a RMSD value close to the average structure of last 3 ns of the 5 ns MD simulations was selected and utilized in molecular docking experiments. The active site was defined with a 10 Å radius around the bound inhibitor. The ten top-scoring conformations of every ligand were saved at the end of the calculation. Early termination option was used to skip the genetic optimization calculation when any five conformations of a particular compound were predicted within an RMS deviation value of 1.5 Å . The GOLD fitness score is calculated from the contributions of hydrogen bond and van der Waals interactions between the protein and ligand, intramolecular hydrogen bonds and strains of the ligand [56]. Protein-ligand interactions were analyzed using DS and Molegro Molecule Viewer [57]. The best pose was selected based on the molecular interactions and the distance between epoxy group of LTA4 and metal ion (Zn 2+ ) present in the active site as well as the location of its carboxylate group which interacts with the carboxylate recognition residues R563 and K565. Finally, the enzyme-LTA4 complex was prepared to be used in further steps in this study.
In terms of preparing the enzyme-peptide complex to investigate the aminopeptidase function of LTA4H enzyme, 3D coordinates of the bound tripeptide Arg-Ala-Arg (RAR) in a solved X-ray structure of hLTA4H with a mutation E271Q (PDB ID: 3B7T) was utilized. The Superimpose Structures protocol as available in Accelrys Discovery Studio 3.0 (DS) was employed to copy the 3D coordinates of this tripeptide into the representative structure of LTA4H picked from the 5 ns MD simulation by superimposition. This complex was subjected to energy minimization using Energy Minimization protocol of DS before considered further in this study.
Molecular dynamics simulations
Many marvelous biological functions in proteins and DNA and their profound dynamic mechanisms, such as switch between active and inactive states [58,59], cooperative effects [60], allosteric transition [61,62], intercalation of drugs into DNA [63], and assembly of microtubules [64], can be revealed by studying their internal motions [65]. Likewise, to really in-depth understand the action mechanism of receptor-ligand binding, we should consider not only the static structures concerned but also the dynamical information obtained by simulating their internal motions or dynamic process. To realize this, the MD simulation is one of the feasible tools. Initial coordinates for the protein atoms were taken from the wild type (WT) and mutant forms of both L-LTA4, L-RAR complex structures. Mutations were introduced at E271Q, R563A, and K565A of the enzyme based on the previous experimental reports to investigate the single active site that catalyzes two different functions upon diverse substrates [14,45]. The protonation states of all ionizable residues were set to their normal states at pH 7. Eight MD simulations were performed for systems including WT and mutant forms of L-LTA4 and L-RAR complexes ( Table 1). All MD simulations were performed with GROMOS96 forcefield using GROMACS 4.5.3 package running on a high performance linux cluster computer [66,67]. During the MD simulations, all the protein atoms including divalent metal ion (Zn 2+ ) were surrounded by a cubic water box of SPC3 water molecules that extended 10 Å from the protein and periodic boundary conditions were applied in all directions. The systems were neutralized with Na + and Cl 2 counter ions replacing the water molecules and energy minimization was performed using steepest descent algorithm for 10,000 steps. A 100 ps position restrained MD simulations were performed for every system followed by 5 ns production MD simulations with a time step of 2 fs at constant pressure (1 atm), temperature (300 K). The electrostatic interactions were calculated by the PME algorithm and all bonds were constrained using LINCS algorithm. A twin range cutoff was used for long-range interactions including 0.9 nm for van der Waals and 1.4 nm for electrostatic interactions. The snapshots were collected at every 1 ps and stored for further analyses of MD simulations. The system stability and behavior of the catalytic structural components present in every system were analyzed using the tools available with GROMACS 4.0.5 and PyMol programs.
Molecular mechanisms and enzyme-substrate complexes
Surprisingly, the LTA4H enzyme catalyzes both hydrolase and aminopeptidase functions over fatty acid and peptide substrates utilizing the same active site. In order to obtain deeper insight upon this unique characteristic of the enzyme, a set of MD simulations were performed with WT and mutated enzymesubstrate complex structures. The natural epoxy substrate LTA4 of arachidonic acid pathway, which is converted to LTB4 upon the action of the enzyme, was selected as fatty acid substrate to investigate the hydrolase function of the enzyme. In the other hand, RAR tripeptide that is reported to be the preferred peptide substrate of the enzyme [14] was selected to investigate the aminopeptidase function of the enzyme. The L-LTA4 complex was prepared through the molecular docking methodology whereas L-RAR complex was prepared by copying the 3D coordinates of RAR from the X-ray crystal structure of LTA4 ( Figure 3). The representative structure obtained from the MD simulation of LTA4H-apoform was used in preparing both the complex structures to compare the structural changes effectively with no artifacts. From the reported site-directed mutagenesis experiments, three amino acid residues from the catalytic active site of the enzyme were predicted to be very important for the enzymatic activities of the enzyme. These residues include one negatively charged E271 residue from the central catalytic domain and two positively charged R563 and K565 residues from the Cterminal domain. Studies mentioned that mutation of this negatively charged residue to a neutral glutamine (E271Q) has completely abrogated both the catalytic activities of the enzyme. But the crystal structure solved with this mutation (PDB ID: 1H19) could not report any structural or conformational changes causing this drastic change in the catalytic activity [14]. It was also proposed that metal (Zn 2+ ) ion present close to the epoxide moiety of LTA4 acts as a weak Lewis acid to activate and open the epoxide ring. It continued to explain that the E271 located in proximity to Zn 2+ also participates in this acid-induced opening of the epoxide ring of LTA4. It was also reported that E271 acts as the N-terminal recognition point in stabilizing the peptide substrates in terms of aminopeptidase reaction of the enzyme. The positively charged residues R563 and K565 were predicted as the carboxylate recognition sites for both the substrates of the enzyme. All mutations of R563 including R563K, which preserved the positive charge, have resulted in complete loss of catalytic functions of the enzyme. The R563K mutant has shown a significant aminopeptidase activity. All these mutations of R563 leading to tremendous change in enzyme functions did not reveal any structural changes explaining the loss of activities. Especially in epoxide hydrolase reaction, the role of R563 was presumed to position the carboxylate tail of the substrates along the catalytic components of the active site [45]. In terms of aminopeptidase reaction, both the positively charged (R563 and K565) residues help each other in aligning the substrate with the catalytic elements and maintaining the binding strength. The mutations K565A and K565M lacking the positive charge have reduced the aminopeptidase and revealed that this positively charged residue assists R563 in carboxylate recognition in aminopeptidase reaction [27]. Despite of this information over the importance of E271, R563, and R565, the structural changes explaining the catalytic activities of the enzyme are lacking. The results of MD simulations of WT and mutant enzyme-substrate complexes discussed in this study will provide a deeper insight from the structural perspective.
Overall structural stability of the systems The overall stability analyses are considered important to note that the systems did not undergo any unusual changes during the time scale of simulation because of erratic system preparation. In this study, root mean square deviation (rmsd), root mean square fluctuation (rmsf), and intra-molecular hydrogen bonds were used in analyzing and comparing the stability of the systems under study (Figure 4).
In terms of L-LTA4 systems, the calculated average rmsd value of K565A system was 0.158 nm which is lower than the rmsd values of WT (0.189 nm), E272Q (0.186 nm), and R563A (0.202 nm) systems ( Figure 4A). The R563A system has shown the higher rmsd value and thereby indicating the additional effect of this particular mutation. Despite of these small differences in the rmsd values between the L-LTA4 systems, the systems were stabilized well throughout the timescale of simulation. As another method to investigate the stability of the systems, rmsf values of all systems were calculated during the simulation and plotted. From the plot, it was observed that none of the active site residues were fluctuating higher than 0.2 nm and explained the stable nature of the systems over the time scale of simulation. In addition, the number of intramolecular hydrogen bonds were calculated for all the systems and plotted. The average number of hydrogen bonds revealed that R563A system has formed more number of hydrogen bonds (466.9) compared to WT (452.8), E272Q (451.7) and R565A (450.6) systems which displayed the reduced number of hydrogen bonds. This result also has confirmed the stability of the systems despite of small differences between systems ( Figure 4A).
In L-RAR systems, the average rmsd value of K565A (0.212 nm) was higher than other systems, which is completely contrasted to the rmsd value of equivalent L-LTA4 system whereas the R563A system has shown the average lower rmsd value (0.168 nm). The other two systems, WT and E272Q, have shown the same average rmsd value of 0.189 nm from last 3 ns of the simulation time ( Figure 4B). In terms of rmsf calculations, the rmsf plot has shown that except D375 and Y378 residues of the active site all other important active site components were stable throughout the simulation. This high fluctuation of these two residues was mainly observed in K565A system. The average number of intramolecular hydrogen bonds during last 3 ns of the simulation was very similar in all the systems. At the end of the simulation time, the R563A and K565A systems started losing their hydrogen bonds and thereby became less intact compared to other systems. All the systems were investigated for the stability and found to be well stabilized during the simulation. Thus the representative structures close to the least rmsd value of each system was obtained and used in structural comparison.
Distance and hydrogen bond analyses
The distance between the most important metal (Zn 2+ ) ion and oxygen atom of the epoxy ring in case of L-LTA4 systems is very important for the hydrolase function of the enzyme. The average distance value observed in WT system (0.56 nm) was lower compared to the mutant systems. Among mutant systems R563A system has shown the higher average distance value of 0.78 nm whereas E271Q and K565A systems have shown similar average distance value of 0.69 nm and 0.63 nm, respectively. During the end of the simulations, this distance in E271Q and K565A systems has reduced close to the distance of WT but R563A has maintained higher distance until the end ( Figure 5A). In terms of L-RAR systems, the distance between the same metal ion and carbonyl oxygen atom of N-terminal peptide bond was measured and compared between the L-RAR WT and mutant systems. As observed in L-LTA4 systems, WT of L-RAR systems has maintained the lower average distance value of 0.59 nm whereas Figure 5B). The hydrogen bonds formed between the protein and bound ligands were also calculated for all the systems under study to investigate the molecular interactions that are lost during the mutations. In L-LTA4 systems, WT system has formed high number of average hydrogen bonds (5.7) compared to the mutant systems. The K565A system has shown an average number of hydrogen bonds of 5.0 whereas E271Q and R563A systems have formed only 1.7 and 1.8 average number of hydrogen bonds with the bound ligand ( Figure 6A). This reduced number of hydrogen bonds correlate well with previously reported loss of hydrolase activity of the enzyme in E271Q and R563A mutated systems [14,45]. In terms of L-RAR systems, the E271Q system has formed more number of average hydrogen bonds (8.7) with the bound RAR. Whereas the WT system has shown an average hydrogen bond value of 8.0, the other mutant systems R563A and K565A have shown the average hydrogen bond values of 4.3 and 3.9, respectively ( Figure 6B). This result of number of hydrogen bond values observed between protein and RAR has proved the importance of both R564 and K565 to maintain the substrate alignment in the active site and the binding strength of the substrate. But the high number of observed hydrogen bonds in E271Q system does not correlate with the observed loss of catalytic activity due to the mutation of E271. This indicated that other structural disturbances script the loss of catalytic function of the enzyme.
Binding mode analyses
L-LTA4 systems. In L-LTA4 systems, the binding mode of LTA4 in WT system has formed hydrogen bonds with Y383 through the oxygen atom of epoxy ring and the distance between this oxygen atom and the metal ion was maintained in a closer distance compared to that of mutant systems. The carboxylate group of LTA4 in WT system was well recognized by both the positively charged residues R563 and K565 which are reported to be the carboxylate recognition sites. Because of this recognition the carboxylate group has formed strong hydrogen bond interactions with R563 and K565. The distance between the carboxylate of E271 and metal (Zn 2+ ) ion was maintained in close vicinity for the reported acid-induced catalytic reaction. The other residue Y378 has also formed a hydrogen bond interaction with the carboxylate group of LTA4 ( Figure 7A). In E271Q system, the binding mode of LTA4 is so different from that of WT system. Because of the uncharged nature of the mutation (E271Q) the metal ion has slightly moved towards the carboxylate group of LTA4, which has also mutually moved towards the metal ion. This movement of carboxylate group of LTA4 has moved the central epoxy group of LTA4 further down and made it inaccessible by the catalytic metal ion ( Figure 7B). This change in the E271 and distance between Zn 2+ and epoxy group (0.69 nm) can be directly correlated with the loss of activity ( Figure 5A). The hydrogen bonds formed with R563 and K565 residues were completely lost because of this mutation. In R563A system, though E271 residue has maintained its hold on the metal ion because of the absence of R563, the important carboxylate recognition site, the LTA4 has moved backwards into the hydrophobic cavity formed by hydrophobic residues such as W311, F362, K364, L365, V366, V367, and V381 ( Figure 7C). This change has not only brought the distance between Zn 2+ and the epoxy ring of LTA4 higher (0.78 nm) compared to any other L-LTA4 systems but also made hydrogen bonding with K565 impossible. In terms of K565A system, the binding mode of LTA4 was similar to that of WT system. Regardless of mutated K565 the hydrogen bonds were maintained with R563 but still the distance between Zn 2+ and epoxy ring of LTA4 was higher (0.68 nm) in this mutant system as well. The hydrogen bond between LTA4 and Y378 was also maintained as observed in WT system ( Figure 7D). This observation also correlated the experimental observation that K565A mutation reduces the activity but does not abolish it.
L-RAR systems. The binding modes of the tripeptide RAR in all systems were observed and compared to investigate the changes due to the mutated residues. More number of hydrogen bonds was observed between the protein and bound substrate compared to the L-LTA4 systems because of the high number of polar hydrogen in the peptide substrate. In the WT system, strong molecular interactions were observed through the hydrogen bonds and p-cation interactions formed between protein and substrate ( Figure 8A). The C-terminal carboxylate which is equivalent to the carboxylate of LTA4 has formed strong hydrogen bond interactions with both positively charged residues R563 and K565, the carboxylate recognition sites. These interactions mainly hold the RAR at the active site and improve its binding strength. As reported, the N-terminal amino group interacted with the E271 which is the N-terminal recognition site for the peptide substrates.
These interactions altogether brings the carbonyl oxygen atom of N-terminal peptide bond close to the catalytic metal ion. A pcation interaction was formed between the side chain of Cterminal Arg residue and Y378, which was found highly fluctuating in rmsf analysis. This p-cation interaction between the same atoms was observed in R563A and K565A systems as well whereas it was between Y383 and C-terminal Arg residue in E271Q system (Figure 8). The binding mode of the substrate in E271Q system was different to that of WT system. The strength of the hydrogen bonds with R563 and K565 has become weak in this system because of the conformational changes of both carboxylate of substrate and R565 residue ( Figure 8B). The metal ion located in the active center was thrown away from its initial position because of the absence of negatively charged E271. This behavior observed in this system clearly reveals that E271 acts as a hook to hold the Zn 2+ ion in the active site. In terms of R563A system, the observed binding mode of this system is similar to that of E271Q system ( Figure 8C). The metal ion was hooked by the presence of E271 residue but still the distance between Zn 2+ and the carbonyl oxygen atom of N-terminal peptide bond was high compared to that of WT. The hydrogen bonds were formed between Y378, Y383, and G269 residues. Surprisingly, two hydrogen bonds were formed with K565 residue in absence of R563. This is different compared to that of the equivalent L-LTA4 system where the hydrogen bonds with both the positively charge residues were completely lost. This observation indicates the importance of K565 in assisting R563 in carboxylate recognition during peptide binding. In K565A system, the binding mode of the substrate is folded and completely different to the other systems. The hydrogen bonds were observed only with Y383 and A565 residues along with an additional p-s interaction between Y378 and C- terminal Arg residue ( Figure 8D). No hydrogen bonds were formed with R563 residue, which is one of the positively charged carboxylate recognition site residues. The molecular interactions observed in R563A and K565A systems revealed that both the residues are important for recognizing the carboxylate group and aligning the peptide substrate along the catalytic elements as reported.
Active site structural changes L-LTA4 system. The overlay of each mutant system with the WT system has allowed observing the structural changes occurred because of the mutations (Figure 9). In E271Q system, the loop of G269 was fluctuating and moved into the active site when compared to other systems. This change observed in this loop could be because of the newly formed hydrogen bond between G269 and the tilted carboxylate group of LTA4, which was found to be a response to the metal ion that lost the interaction with mutated E271. The carboxylate group of LTA4 and terminal NH 2 of R563 moved away from each other (2.7 to 8.4 Å ) because of the missing E271. A short beta sheet formed by the residues V306-N308 of HEXXH-(X) 18 -E motif that possess two catalytically conserved histidine residues coordinating with Zn 2+ ion disappeared in E271Q system. Another helix followed by this short beta sheet was extended by four amino acids W311-F314 making the important F314 slightly backward from the active center ( Figure 9A). Two tyrosine residues Y378 and Y383 located opposite to each other in the active site were highly fluctuating in this system to adjust the binding of the LTA4. The K565 residue present in the loop has become a part of the long helix originally formed by T567-A575 residues during the simulation of E271Q system. This change slightly drew back the K565 residue from the active site. The other important positively charged residue did not show any structural changes during the simulation. The R563A system has shown different structural changes compared to E271Q system. The loop containing G269 has shown slight fluctuation only at the location of G269 because of the hydrogen bonds formed between the carboxylate of LTA4 and G269 but the lower part of the loop was stable unlike E271Q system ( Figure 9B). The short helix formed by V306-N308 residues was maintained in this system and the helix containing F314 was extended but kept for the same length during the simulation. The region (W311-F314) that turned an extended helix was highly fluctuating in this system because of the moving alkyl part of LTA4. This backward movement buried the alkyl part into the hydrophobic pocket, formed by a mixture of aliphatic and aromatic hydrophobic residues (W311, F362, K364, L365, V366, V367, and V381), was observed because of the missing interactions with R563 residue (not shown in figure). Unlike E271Q system, Y378 residue has shown only slight side chain movement as a response to the moving LTA4 whereas Y383 did not show any fluctuations from its initial position. The same helix extension was observed as in E271Q system and thus K565 was included in helix formed by T567-A575 residues. The missing R563 led to the loss of correct alignment of LTA4 along the catalytic elements and severe instability of the binding mode of LTA4. In the final mutant (K565A) system, the G269 loop was completely stable and no hydrogen bond interaction was observed between LTA4 and G269 residue. The short helix of V306-N308 disappeared during the simulation of K565A system as observed in E271Q system ( Figure 9C). The helix of F314 was extended as seen in R563A system and thus has shown the mixed characteristics of E271Q and R563A systems. The Y378, one of the oppositely located pair of tyrosine residues, has fluctuated highly in this system. The binding mode of LTA4 was quite similar to that of WT except its carboxylate group, which moved back because of the missing hydrogen bonds from K565 residue. But the hydrogen bonds with R563 were maintained and thus kept the alignment of LTA4 along with the catalytic elements. The overlay of active sites of all the systems have made clear about the structural changes where Y378 was observed to be fluctuating differently in each system maintaining a close distance with the substrate ( Figure 9D). Thus Y378 residue, along with the carboxylate recognition site residues R563 and K565, can play a key role in aligning the substrate at the active site.
L-RAR systems. Comparison of active site residues of WT and E271Q systems using the representative structures obtained from the simulations revealed the structural changes led to the differences in the catalytic activity of the enzyme ( Figure 10). The overlaid WT and E271Q structures have shown the difference in the locations of catalytically important Zn 2+ ion in the catalytic center ( Figure 10A). The uncharged nature of the mutant residue Q271 the metal ion has lost the important interaction and moved far away from its original location. The distance between the Zn 2+ ion and the oxygen atom of the N-terminal peptide bond was so high compared to the WT system. Thus the catalytic aminopeptidase reaction becomes impossible in E271Q system. The helix containing Y383 was extended during the simulation E271Q system moving Y383 backward from the active site. This movement of Y383 has formed p-cation and a hydrogen bond interactions with the C-terminal part of RAR whereas in WT system this residue has formed a hydrogen bond interaction with the N-terminal amino group. The other tyrosine residue Y378, which was found to be guiding the substrate along with the carboxylate recognition site residues, has formed hydrogen bond with the carboxylate of RAR. The binding mode of RAR observed in E271Q system has shown only weak hydrogen bonding interactions with R563 as it was moving away from it. The helix formed by K565-A575 residues was shortened slightly leaving K565, one of the carboxylate recognition residues, as a part of loop making it more flexible. But this residue has maintained hydrogen bond interactions with the peptide substrate through its carboxylate group. This change observed in helix containing K565 is different from that of L-LTA4 systems. The K565 is the key residue that assists the other positively charged residue R563 to maintain the proper alignment of RAR in the active site whereas in LTA4 binding K565 is not required to assist R563 residue [27]. Moreover, the absence of negatively charged E271 residue also played a major role in observed loss of catalytic activity. The R563A mutation has caused a high fluctuation of E271 that moved far from the N-terminal amino group of RAR and makes it impossible to act as N-terminal recognition site ( Figure 10B). The interacting distance between Zn 2+ and E271 was maintained in this mutation. The helix extension was observed near Y383 as displayed in E271Q system and this changed the flexibility of Y383 in the active site. The other tyrosine residue Y378 was highly fluctuating in this mutant system compared to any other systems and formed strong p-cation interaction than it is in WT system. The C-terminal part of RAR substrate has slightly went back as it missed the strong interactions from R563 but the alignment was almost maintained as observed in WT system except the side Cterminal side chain of RAR. Interestingly, K565 has taken the location of R563 in this mutant system to maintain the hydrogen bonds with the carboxylate of RAR and there was no change observed in the K565-A575 helix as observed in E271Q system. The K565A system also has shown some structural changes that were not observed in other L-RAR systems ( Figure 10C). The short helix (V306-N308) that has shown structural changes in L-LTA4 system disappeared in K565A system and the F314 helix was extended including W311-F314 residues. These changes were observed in other L-RAR systems. The extension of helix has drawn F314 residue back from the active site center. A folded binding mode of RAR was observed in K565 system much different from that of WT and other mutant L-RAR systems. Though R563 is present, the carboxylate group of RAR has moved back from its original position and formed p-interactions with Y378 and completely lost interactions with R563. This observation completely correlates with the observed activity and the reported statement that K565 assists R563 to act as carboxylate recognition site in aligning the substrate along the catalytic elements of the enzyme. The overlay of all L-RAR systems revealed that along with E271, R563, K565 residues, Y378 and Y383 were also important in keeping the peptide substrate aligned within the active site ( Figure 10D). As observed in L-LTA4 systems, Y378 residue has acted as a baffle to control the binding modes of the substrates. This part of the study has documented various structural changes explaining the differences in activities between WT and mutated forms of the enzyme bound to its two different substrates which were not determined by the X-ray crystallography so far (Table S1).
Binding modes of LTA4 and RAR substrates
The binding modes of fatty acid and peptide substrates that are catalyzed by hydrolase and aminopeptidase functions of the enzyme using same active site were compared. The overlay of two WT systems has given the overview of which parts of the active site were occupied by these two highly diverse substrates. Both the substrates bind perpendicular to each other occupying majorly the different portion of the active site and sharing the carboxylate recognition sites in common ( Figure 11A). The long alkyl part of LTA4 was snuggly bound into the hydrophobic pocket formed by a mixture of aliphatic and aromatic residues including W311, F314, K364, L365, V367, Y378, and V381. The side chain of Cterminal Arg residue of RAR was fit into the small cavity formed by F356, Y378, S379, M564, K565, and R568 residues. The overlay of active site residues has shown very few structural changes between the WT systems of L-LTA4 and L-RAR systems including the side chain movements of Y378, Y383, and K565 residues ( Figure 11B). The K565 residue was present in the loop in WT of L-LTA4 system and in the extended helix in WT of L-RAR system and thereby changing the flexibility and interacting behavior of K565. This adds explanation to the importance of K565 residue in assisting R563 residue in aligning the peptide substrate whereas this residue is not required in aligning fatty acid substrate.
Conclusion
In this study MD simulation methodology was used to simulate hLTA4H enzyme complexed with its two diverse substrates along with mutated key residues. The aim of this study was to investigate the structural and conformational changes in the bi-functional active site of the enzyme reflecting on the catalytic activity. This was considered very important and necessary to script the reasons for the observed loss of activity due to particular mutations as the solved X-ray structures failed to show the structural changes. Eight systems including two WT, enzyme-LTA4 and enzyme-RAR complexes along with three independent mutations (E271Q, R563A, and K565A) in each complex were simulated in this study. The observed hydrogen bond interaction network and distance between the catalytically important atoms have correlated well with the experimental results. The E271 residue which is considered very important for both functions of the enzyme and E271Q systems have revealed from our study that this residue acts as a hook to hold the catalytic metal ion at its location and also plays a role of N-terminal recognition point for the aminopeptidase function. Both the E271Q systems have lost the expected binding mode of the substrates for the successful catalysis. The other mutant R563A and K565A systems have also revealed the structural changes and binding mode differences explaining the loss of activity in mutant systems. In L-LTA4 systems, the substrate binding mode in R563A system has changed completely that the long alkyl chain of LTA4 was completely buried into the hydrophobic pocket. This difference in binding mode of LTA4 was completely because of the loss of hydrogen bond interaction with R563 residue. In terms of L-RAR systems, the same mutation R563A has affected the binding mode of RAR and N-terminal recognition through E271 residue in peptide binding. Because of this missing N-terminal recognition the catalytic distance between the metal ion and the carbonyl group of the N-terminal peptide bond was high in this system. The K565A systems in both the substrate complexes have shown different structural changes. In L-LTA4 system the binding mode of the substrate was very much similar to the WT explaining the less importance of K565 in LTA4 binding whereas in L-RAR system the binding mode has lost both the N-terminal and C-terminal recognitions leading to the loss of activity. These results obtained from this study can be effectively used in designing future hLTA4H inhibitors as anti-inflammatory and anti-cancer therapeutics. Table S1 The structural changes which were not seen in experimental studies observed through the MD simulation studies. | v3-fos-license |
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} | pes2o/s2orc | Quaternized Chitosan as an Antimicrobial Agent: Antimicrobial Activity, Mechanism of Action and Biomedical Applications in Orthopedics
Chitosan (CS) is a linear polysaccharide with good biodegradability, biocompatibility and antimicrobial activity, which makes it potentially useful for biomedical applications, including an antimicrobial agent either alone or blended with other polymers. However, the poor solubility of CS in most solvents at neutral or high pH substantially limits its use. Quaternary ammonium CS, which was prepared by introducing a quaternary ammonium group on a dissociative hydroxyl group or amino group of the CS, exhibited improved water solubility and stronger antibacterial activity relative to CS over an entire range of pH values; thus, this quaternary modification increases the potential biomedical applications of CS in the field of anti-infection. This review discusses the current findings on the antimicrobial properties of quaternized CS synthesized using different methods and the mechanisms of its antimicrobial actions. The potential antimicrobial applications in the orthopedic field and perspectives regarding future studies in this field are also considered.
Introduction
Chitosan (CS), as a polycationic polymer, is obtained from crustacean shells by partial and full alkaline deacetylation [1]. As a result of its biodegradability, nontoxicity and antimicrobial activity, CS has been widely used for biomedical applications, such as tissue engineering scaffolds, drug delivery, wound dressings and antibacterial coatings [1,2]. However, CS is insoluble in most solvents at neutral or high pH, except in organic acids, which substantially limits its usefulness [1]. To address this limitation, CS derivatives by chemical modifications have recently been studied. One purpose of the chemical modifications of CS, which have included saccharization, alkylation, acylation, quaternization and metallization, has been to improve its water solubility and increase its antimicrobial activity. As one example, quaternary ammonium CS can be prepared by introducing a quaternary ammonium group on a dissociative hydroxyl group or amino group. In some studies, quaternized CS derivatives exhibited stronger antibacterial activity, a broader spectrum and higher killing rates compared to unmodified CS [3][4][5][6][7].
Based on the current state of research and progress in corresponding areas, this review attempts to summarize the antimicrobial properties of quaternized CS synthesized with different methods and modes of action as antimicrobial compounds. Subsequently, the present and potential future applications of this material in the orthopedic field are also discussed in detail.
N-substituted CS was quaternized using N-(3-chloro-2-hydroxy-propyl) trimethylammonium chloride (GTMAC) to increase its water solubility. The minimum inhibitor concentration (MIC) experiment was performed on E. coli and S. aureus to explore the impact of the extent of N-substitution (ES) on the antibacterial activities. The results showed that when the ES is higher than 20%, MIC values are also higher. The antibacterial activities ranged from 8 to 64 µg/mL for S. aureus and from 16 to 64 µg/mL for E. coli [13]. Another study from the same research group presented N-methylation of N-arylated CS derivatives containing N,N-dimethylaminophenyl and pyridyl substituents, which produced quaternary ammonium salts in the presence of sodium iodide and iodomethane. The methylated products were water soluble over all pH ranges and displayed antibacterial activity against S. aureus and E. coli. Their MIC values were in the range of 32-128 µg/mL against both bacteria [14]. A di-quaternary group can contribute to the antibacterial activity of CS; an obvious inhibition against the G + strains of S. aureus and Staphylococcus pneumoniae was found by a CS derivative with an N- [1-carboxymethyl-2-(1,4,4-trimethylpiperazine-1,4-diium)] substituent, which was generally more active at pH 7.2 than at pH 5.5 [15]. Quaternary ammonium and disaccharide CS were successfully synthesized by using N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (GTMAC) as a quaternizing agent to modify residual free primary amino groups and some hydroxyl groups of CS derivatives. All quaternary ammonium CS derivatives were water-soluble over an entire range of pH values and displayed antibacterial activity against S. aureus and E. coli, as observed by using the MIC method. The results implied that N-benzyl chitosans GTMAC derivatives would be useful as potential new antibacterial agents [5]. The permanent positive charges in the form of quaternary ammonium groups were introduced to the surface of pre-fabricated CS particles under heterogeneous conditions via either a direct methylation or a reductive N-alkylation using two different aldehydes-propionaldehyde and benzaldehyde-followed by methylation with methyl iodide. It was found that all quaternized CS particles exhibited a higher antibacterial activity against S. aureus than the CS particles did in a neutral pH media. The results obtained from this research suggest that the surface-quaternized CS particles may potentially be used as an effective antibacterial material for biomedical applications [16].
Numerous studies support the essential importance of a polycationic structure in antimicrobial activity. The positive charge is associated with the degree of substitution (DS) of CS derivatives, which affect positive charge density [17]. With regard to CS derivatives, antimicrobial activity mostly depends on the DS of the grafting groups. With a different degree of substitution of the quaternary ammonium, quaternized CS exhibits different antibacterial activities. N,N,N-trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl CS (TMC) with 3-chloro-2-hydroxy-propyl trimethylammonium chloride (CHPTMAC). This quaternized CS exhibited enhanced antibacterial activity compared with TMC alone, and the activity increased with an increase in the degree of substitution [7]. Hydroxypropyltrimethylammonium chloride CS (HACC) was synthesized with differing degrees of substitution (6%, 18% and 44%) of quaternary ammonium by reacting CS with glycidyl trimethylammonium chloride. The antibacterial activities of these polymers were tested in vitro against Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. The results showed that the antibacterial activities of the HACC with 18% or 44% substitution were significantly higher than the HACC with 6% substitution or CS alone against all three bacteria [6].
However, there exist discrepancies among different reports on the antibacterial activity of CS and its quaternary derivatives. N,N,N-diethylmethyl CS exhibits enhanced antibacterial activity against E. coli in comparison with CS and a decrease in pH results in stronger activity [18]. HTCC, which was prepared by the reaction of CS with GTMAC, displays an increased antibacterial efficiency against both E. coli and S. aureus relative to CS [19,20]. In contrast, Chi et al. [21] found that HTCC had no notable activity against E. coli. Qin et al. [22] reported that the antibacterial activity of HTCC was stronger under alkaline conditions than under weak acidic conditions. In a previous report, modification of the free amino group of the CS backbone decreased its antibacterial activity [23]. These results showed that quaternization does not always enhance the antibacterial activity of CS and that the effect of pH on the antibacterial activity of quaternary ammonium CS is uncertain. The discrepancies among different reports on the antibacterial activity of CS and its quaternary derivatives are most likely caused by various intrinsic and extrinsic factors that are related to the CS itself (e.g., type, MW, DD, viscosity solvent and concentration) and the environmental conditions (e.g., test strain, its physiological state and the bacterial culture medium, pH, temperature, ionic strength, metal ions and organic matter), respectively [24].
In addition, the biocompatibility of the quaternized CS should be considered. Mouse fibroblasts and bone-marrow-derived stromal cells (hMSCs) were used to investigate the biocompatibility of the HACC. The results showed that the antibacterial activities of the HACC with a higher degree of substitution of the quaternary ammonium were significantly higher than the lower degree of substitution against bacteria. However, they also exhibited interference in the proliferation and osteogenic differentiation of hMSCs and a cytotoxic effect on the activities of the mouse fibroblasts. Conversely, HACC with a lower substitution was highly biocompatible with osteogenic cells [6].
Based on the above analysis, the antibacterial activity of quaternized CS has been shown to be stronger than that of CS, and the antibacterial activity increases accordingly with the increasing DS of the quaternary ammonium. However, the biocompatibility of the quaternized CS was affected with the different DS, with higher DS exhibiting a cytotoxic effect on the cell activity. In addition, there existed discrepancies among the antibacterial activities of CS and its quaternary derivatives. Therefore, further investigation should be performed.
Antifungal Activity of Quaternised CS
The modification of CS to improve its activity is a promising approach to achieving effective bio-fungicides [15,25]. With the development of the science of CS, it has been found that CS has antifungal activities [1,26]. Based on the available evidence, bacteria appear to be generally less sensitive to the antimicrobial action of CS than fungi [17]. EI Ghaouth reported that CS could inhibit the growth of Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides and Rhizopus stolonifer [27]. The growth of fungi, such as F. oxysporum, R. stolonifer, Penicillium digitatum and C. gloeosporioides can be completely inhibited by CS at a concentration of 3% [28][29][30]. Four CS types with different MWs were tested against fungal pathogens. The most bioactive type of CS that inhibited the growth of Candida albicans had the lowest molecular weight (32 kDa) and the highest degree of deacetylation (94%). The MIC of this CS type towards C. albicans strains were 2.0, 1.75 and 1.25 mg/mL against C. albicans-A, C. albicans-H and C. albicans-C, respectively [31]. However, relatively little work has been reported on the antifungal activities of quaternized CS derivatives. The results indicated that all the quaternized CS derivatives have better antifungal activities compared with the activity of unmodified CS [32]. In another study, quaternized CS derivatives with different molecular weights were synthesized, their antifungal activities against Botrytis cinerea Pers. (B. cinerea Pers.) and Colletotrichum lagenarium (Pass) Ell. et Halst (C. lagenarium (Pass) Ell. et Halst) were conducted. The results indicated that quaternized chitosan derivatives have stronger antifungal activities than chitosan. Furthermore, quaternized chitosan derivatives with high molecular weight were shown to have even stronger antifungal activities than those with low molecular weight [33].
Two series of new quaternized CS derivatives were synthesized by the reaction of deacetylated chitosan (CH) with propyl (CH-Propyl) and pentyl (CH-Pentyl) trimethylammonium bromides to obtain derivatives with increasing degrees of substitution (DS). The antifungal activities of these derivatives on the mycelial growth of Aspergillus flavus were investigated in vitro. The results showed that the antifungal activities increased with DS and that the more substituted derivatives of both series, CH-Propyl and CH-Pentyl, exhibited antifungal activities three and six times higher, respectively, than those obtained with commercial and deacetylated CS. The results showed that the quaternary derivatives inhibited the fungus growth at one-fourth the polymer concentration of deacetylated chitosan (CH). The antifungal activity of CS can be improved by increasing the degree of substitution (DS) of alkyltrimethylammonium groups on the polymer chain. The inhibition indexes for both synthesized series (propyl and pentyltrimethylammonium) increased with the DS, and the most substituted derivatives (CH-Propyl-40 and CH-Pentyl-65) exhibited inhibition values three and six times higher, respectively, than those obtained with CS [34].
Five water-soluble chitosan derivatives were recently carried out by quaternizing either iodomethane or GTMAC as a quaternizing agent under basic condition [35]. The degree of quaternization (DQ) ranged between 28% ± 2% and 90% ± 2%. The antifungal activity was evaluated by using the disc diffusion method, MIC and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte) and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives were shown to be more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 g/mL and 500-4000 g/mL, respectively, against all fungi. The results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl)chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was dependent on type of fungus, as well as the chemical structure of the quaternized chitosan derivatives.
In summary, quaternized CS derivatives have better antifungal activities than that of unmodified CS. The antifungal activity of CS can be improved by increasing the degree of substitution (DS) of the quaternary ammonium of the CS. However, the investigation of the antifungal activity of the quaternized CS was not performed in clinically derived fungi.
Mechanism of Antibacterial Action
The exact mechanisms of the antibacterial activities of CS or quaternized CS are still unknown. The polycationic structure of quaternized CS is a prerequisite for antibacterial activity. Electrostatic interaction between the polycationic structure and the predominantly anionic components of the microorganisms play a fundamental role in antibacterial activity. The number of ammonium groups linking to the CS backbone is important in electrostatic interaction for the antibacterial activity of quaternized CS. It has been reported that quaternized CS with a higher degree of substitution of the quaternary ammonium exhibited a strong interaction with negative charges on the bacterial cell surface and showed better antibacterial activity than CS [5,7,17,36].
The additional effect derived from the hydrophobic-hydrophobic interactions between the aryl substituent and the hydrophobic interior of the bacterial cell wall is proposed to explain the mechanisms of the antibacterial activities, because alkyl substituents with an increased chain length on the quaternary ammonium CS salt also displayed higher antibacterial activities [13,37]. The hydrophobicity and cationic charge density of the introduced substituent play important roles in determining the antibacterial activity of quaternized CS derivatives. In addition, under neutral or higher pH, quaternized CS modified with lipophilic groups showed higher activity than native CS, and the hydrophobic and chelating effects may be responsible for antibacterial activity in addition to the electrostatic effect [17].
The antibacterial action of the CS derivatives is also based on the physical states and molecular weight. In general, high-molecular-weight CS derivatives and solid particles cannot pass through cell membranes and only interact with the cell surface to alter cell permeability [38] or form a film around the cell that protects cells against nutrient transport through the microbial cell membrane [39]. However, low-molecular-weight, water-soluble CS derivatives or nanoparticles could penetrate the cell walls of bacteria, incorporate with DNA and inhibit the synthesis of mRNA and DNA transcription [40].
The microorganism may also affect the antimicrobial activity of the quaternized CS. The quaternized N-aryl CS derivatives were not as effective against E. coli bacteria as against S. aureus bacteria [41], because the outer membrane (OM) of Gram-negative bacteria functions as an efficient barrier against macromolecules, such as CS derivatives. The OM, which contains lipopolysaccharide (LPS), provides the bacterium with a hydrophilic surface. The lipid components and the inner core of the LPS molecules contain anionic groups that contribute to the stability of the LPS layer through electrostatic interactions with divalent cations [42]. Therefore, overcoming the OM is a prerequisite for any material to exert bactericidal activity towards Gram-negative bacteria [43,44]. There exists, however, a converse viewpoint. Polyanions on the cell surface take part in the electrostatic interactions with CS derivatives. The negative charge on the cell surface of the tested Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli) was higher than on the tested Gram-positive bacteria (Staphylococcus aureus and Streptococcus faecalis), leading to more CS derivatives adsorbed and higher inhibitory effects against the Gram-negative bacteria [45].
Despite the distinction between Gram-negative and Gram-positive bacterial cell walls, antibacterial modes both begin with interactions at the cell surface and compromise the cell wall or OM first. For Gram-positive bacteria, lipoteichoic acids may provide a molecular linkage for CS derivatives at the cell surface, allowing it to disturb membrane functions [46]. LPS and proteins in the Gram-negative bacteria OM are held together by electrostatic interactions with divalent cations that are required to stabilize the OM. Polycations may compete with divalent metals, such as Mg 2+ and Ca 2+ ions present in the cell wall, which will disrupt the integrity of the cell wall or influence the activity of degradative enzymes [17]. Contact between CS derivatives and the cell membrane, which is essentially a negatively charged phospholipid bilayer, may slightly change the membrane permeability. Further interactions may denature membrane proteins and initiate penetration into the phospholipid bilayer. The increased membrane permeability leads to destabilization of the cell membrane, leakage of intracellular substances and, ultimately, the death of cells [17,47,48].
Mechanism of Antifungal Action
Similar to the antibacterial action, the antifungal activity of CS derivatives is believed to occur from the interaction between the cationic chain and the negatively charged residues of macromolecules exposed on the fungal cell surface, leading to a leakage of intracellular electrolytes and other constituents [49][50][51][52][53]. It is believed that CS may affect the morphogenesis of the cell wall, interfering directly with the activity of enzymes responsible for growth of the fungi [27]. Recently, Li et al., based on confocal laser scanning microscopy of fluorescein-labeled chitosans, showed that low-molecular-weight chitosans could enter into the hypha of Fulvia fulva, suggesting that the growth of F. fulva may be inhibited by chitosans from inside the cell [54]. The target site of the cation is the negatively charged cell surface membrane, which prevents nutrients from entering the cell, and the antifungal activity of quaternized CS is also likely the result of this type of activity [32].
Another possibility for the antifungal activity of CS is based on its chains crossing the cell membrane, inhibiting the cell from growing from the inside [32]. However, for deacetylated chitosan (CH) and the derivatives with low degrees of substitution, the mechanism on A. flavus and the interaction with the cell surface may form an impermeable layer around the cell, thus blocking the transport of essential solutes into the cell [39]. The modification of CS by introducing permanently charged quaternary groups may improve the antifungal activity of CS. The addition of quaternized CS to the BDA medium inhibited the mycelial growth of A. flavus significantly at all concentrations tested. The results obtained in microbiological assays showed that the capability to inhibit fungus growth in vitro was clearly increased for the higher degrees of substitution for the two series tested [39,54].
Mechanism of Anti-Biofilm Formation
Implant-associated infection is primarily caused by bacterial growth in biofilms [55]. Biofilm formation is considered to be an important virulence mechanism, because the biofilm impairs the activity of antibiotics, prevents normal immune responses and complicates the eradication of infections [56,57]. Once an infection has been established and a well-organized biofilm has formed on the implant surface, antibiotic therapies are less efficacious and removal and substitution of the implant are often the only way to eradicate the problem [58,59]. Bacterial adherence to orthopedic implant surfaces occurs in two essential steps [60]. Its adherence to the implanted surface is followed by an accumulation process and the production of extracellular substances, such as polysaccharide intercellular adhesin (PIA) [61,62]. The production of PIA is mediated by the intercellular adhesin (ica) locus, which comprises four core genes (icaA, icaB, icaC and icaD) and a regulatory gene (icaR) [63][64][65]. Peng ZX, et al. [66,67] assessed icaA transcription as an index of biofilm formation on a titanium surface by RT-PCR. The results showed that HACC can inhibit icaA expression and the level of inhibition increased with higher HACC concentrations in the biofilm prevention and susceptibility assays. This effect was more significant for HACC concentrations of 18%, 26% and 44%, which blocked the transcription of icaA at concentrations of 128 µg/mL and 256 µg/mL in the biofilm prevention assay and at 256 µg/mL in the biofilm susceptibility assay. The authors postulated that this inhibition of icaA transcription results in reduced biofilm formation and increased susceptibility to HACC, because the biofilm protects and supports the growth of bacteria on the surface of implants and inhibition of biofilm formation further impairs bacterial viability. This transcriptional response data may provide indirect evidence that quaternized CS treatment interferes with cellular energy metabolism.
Application in the Orthopedic Surgery Field
Biomaterial-associated infections remain a serious complication in orthopedic surgery. Antimicrobial prophylaxis, including the systemic and local use of antibiotics, has proven to be valuable in the prevention of infection in both experimental and clinical research [68][69][70]. Using bone cement as the carrier for antibiotics is a way of delivering high levels of antibiotics locally without causing systemic toxicity. PMMA bone cements and beads loaded with gentamicin are becoming the standard practice for preventing infection in joint arthroplasty and for treating infection in osteomyelitis [71][72][73][74]. However, the overuse of antibiotics leads to the evolution of antibiotic-resistant bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) [70,75]. In addition, to succeed in orthopedic surgery, implant materials must be anti-infective (discouraging bacterial adhesion), as well as habitable by bone-forming cells (favoring the activity of osteogenic cells) [76,77]. According to previous studies, gentamicin at high local concentrations reduced the viability, proliferation and alkaline phosphatase activity of the osteoblasts [78][79][80][81][82][83] and inhibited the proliferation and differentiation of human bone marrow mesenchymal stem cells in vitro and in vivo [78,84,85], which compromised the bone-healing process. To overcome these disadvantages derived from antibiotics, a new quaternized CS derivative (hydroxypropyltrimethylammonium chloride CS, HACC) with a 26% degree of substitution was synthesized and loaded at a 20% by weight ratio into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE 287) and two standard strains, S. epidermidis (ATCC 35984) and methicillin-resistant Staphylococcus aureus (ATCC 43300), were selected. The results showed that HACC-loaded PMMA inhibited biofilm formation on its surface compared to other control groups, providing a promising new strategy for combating implant infections and osteomyelitis [66]. The same research team simultaneously found better stem cell proliferation, osteogenic differentiation and osteogenesis-associated gene expression on the surface of the HACC-loaded PMMA compared to the gentamicin-loaded PMMA. Therefore, this new anti-infective bone cement also exhibited improved physical properties and osteogenic activity, which may lead to better osseointegration of the bone cement in cemented arthroplasty [86]. Shi ZL et al. produced similar results. In their research, the use of CS nanoparticles (CS NP) and quaternary ammonium CS derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl methacrylate) (PMMA) bone cement with and without gentamicin were investigated. The antibacterial activity was tested against S. aureus and S. epidermidis. This in vitro study demonstrated that the incorporation of nanoparticles of CS and quaternary ammonium CS derivative in bone cements can provide effective antibacterial action against S. aureus and S. epidermidis. These nanoparticles also enhance the antibacterial efficacy of gentamicin-loaded bone cements, and this property is retained even after an extended period of immersion of the modified bone cement in an aqueous medium [87].
Titanium-based biomaterials are currently the best and most widely used materials in the manufacture of orthopedic and dental implants, because of their high strength, low weight, excellent corrosion resistance and good biocompatibility [88][89][90]. The biocompatibility of titanium implants can be attributed to a surface protein layer formed under physiological conditions that actually makes the surface suitable for bacterial colonization and biofilm formation [91][92][93]. The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. To overcome these two major problems of Ti implants, an effective approach to minimizing implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time [94][95][96][97][98]. In one study, the efficacy of hydroxypropyltrimethylammonium chloride CS (HACC) with different degrees of substitution (DS; referred to as HACC 6%, 18% and 44%) in preventing biofilm formation on a titanium surface was evaluated [67]. The results showed that HACC, especially HACC with DS of 18% and 44%, significantly inhibited biofilm formation compared to the CS control, even at concentrations far below their MICs. Therefore, HACC may serve as a new antibacterial agent to inhibit biofilm formation and prevent orthopedic implant-related infections. In addition, HACC with DS of 18% exhibited good biocompatibility with osteogenic cells, which is beneficial to the osseointegration of the implants [6].
In orthopedic surgery, open fractures with bacterial infections are often seen and the treatment of these injuries is challenging for surgeons. Biomaterials with properties that promote wound healing and simultaneously eliminate infections have attracted the interest of scientists. Chitosans, which have hydrogel-forming properties, have been considered to be advantageous in their application as a wound dressing material, and CS-based materials have received attention in this regard [99][100][101][102][103]. A majority of micro-and nano-fiber materials derived from antimicrobial products are suitable for preparing wound dressings. Electrospinning is a favorable technique for producing continuous polymer fibers with diameters down to the nanoscale range [104]. Because of unique properties, such as their high surface-to-volume ratio, high porosity and diameters at the nanoscale, electrospun mats made from ultrafine polymer fibers have been drawing great interest. Quaternized CS has shown high antibacterial activity against Gram-positive and Gram-negative bacteria. In one study, quaternized CS-containing nanofibers were successfully prepared by the electrospinning of mixed aqueous solutions of QCh and PVA, and the electrospun QCh/PVA (quaternized chitosan/poly vinyl pyrrolidone) nanofibrous mats were efficient in inhibiting the growth of Gram-positive bacteria and Gram-negative bacteria [4]. In addition, in this authors' previous work [3], the antibacterial activity of cross-linked electrospun QCh/PVA (polyvinyl alcohol) mats made of quaternized CS derivatives was observed to be bactericidal rather than bacteriostatic. PVA are nontoxic, biocompatible and highly hydrophilic, while possessing good complexation properties and good film-forming abilities. Therefore, the antibacterial activity of electrospun QCh/PVA mats is an important property for wound-healing applications, because it has the potential to contribute to the prevention of secondary infections in wounds by S. aureus, resulting in limited scar formation [3,4,105].
Perspectives and Areas for Future Research
Although quaternized CS derivatives have been regarded as effective antimicrobial agents, their modes of action need to be further studied in depth. Investigations have recently focused on the morphological changes of the microorganism with respect to its antimicrobial property and mechanism. The methods used to evaluate the antimicrobial phenomena have been limited to the biological conception only. Therefore, future work should aim at demonstrating the molecular details of the underlying mechanisms and their relevance to the antimicrobial activity of quaternized CS. Additionally, whether this compound induces bacterial resistance and its mechanisms should also be considered.
According to previous reports, with the increasing DS of the ammonium of the quaternized CS, the antimicrobial activities were enhanced, but the cytotoxicity also increased. Thus, the toxicity and biocompatibility of this CS derivative should be the main focus of further studies. Finding the DS with a balance between good antimicrobial activity and low mammalian toxicity is important.
Studies about this compound have largely been performed in in vitro experiments. It is important to clarify its potential use as an antimicrobial agent in vivo. In that sense, researchers should emphasize in vivo studies to establish the efficacy of the antimicrobial activity of quaternized CS. Furthermore, quaternized CS would be valuable as an effective antibacterial coating or antimicrobial dressing in orthopedic surgery. Therefore, a significant increase in the number of scientific studies to obtain evidence to support this use can be expected. | v3-fos-license |
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} | pes2o/s2orc | Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase
The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochromec heme edge is within 4 Å of the indole ring of subunit II residue Trp104, indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys13 with Asp119 and Lys72 with Gln103 and Asp158 are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp104 in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover.
The determination of structures of bacterial and mitochondrial cytochrome c oxidases (CcO) 1 by x-ray crystallography (1)(2)(3)(4)(5) has opened the door to understanding the structural basis for its function in respiratory chain activity. One step of the respiratory chain involves the transfer of electrons from cytochrome bc 1 complex to CcO, both membrane-bound complexes, via the water-soluble protein cytochrome c (Cc). Efficient electron transfer requires both rapid complex formation and rapid product dissociation. Determination of the structure of the complex formed between CcO and Cc would reveal the interactions promoting this transient interaction as well as possible paths for intermolecular electron transfer. The complex structure, however, has yet to be determined, in part due to the difficulties in crystallizing membrane-bound proteins and possibly also due to the transient nature of the interaction. On the other hand, this redox system may be particularly amenable to computational search methods based on static crystallographic structures because of the fast kinetics of these interactions, which suggest that large conformational changes do not occur upon complex formation. In addition, computational techniques may shed light on long range electrostatic guidance between the two partners and on the dynamic nature of the interaction.
The docking program DOT (6) provides a complete search of all orientations between two rigid molecules by systematically rotating and translating one molecule about the other, a procedure considered computationally unfeasible just a few years ago (7). At that time, the extent of the search was limited to, at most, energy calculations for a few million configurations, as in the programs TURNIP (8) and DOCK (9). Alternately, the representation of the moving molecule was greatly simplified to a few point charges (10,11). DOT systematically samples configurations for two rigid molecules over all space and expresses the interaction energies as correlation functions, which are rapidly computed using fast Fourier transforms. The electrostatic potentials used by DOT are calculated by Poisson-Boltzmann methods (12)(13)(14), which take both solvent and ionic strength effects into account. Because DOT performs a complete search, both long range and short range interactions can be examined.
Here, we describe the application of DOT to the interaction of bovine CcO with horse Cc. The calculated complex identifies the CcO Trp 104 (Trp 143 ) 2 side chain as the point of electron entry into CcO and implicates specific charged side chains in protein-protein association, in agreement with mutagenesis, binding, and time-resolved kinetics studies (15)(16)(17). Properties of the calculated interface resemble those found in the crystallographic structure of the electron transfer complex of yeast cytochrome c peroxidase (CcP) with yeast Cc (18), suggesting a general structural motif for transient protein-protein interactions.
EXPERIMENTAL PROCEDURES
Coordinate Preparation-Coordinates for bovine CcO (1OCC) and horse Cc (1HRC) were obtained from the Protein Data Bank (19). Rhodobacter sphaeroides cytochrome c 2 was also investigated because its interactions with CcO have been examined (16,17). Polar hydrogen atoms were added with the computer graphics program Insight (MSI Inc., San Diego). Partial atomic charges for protein atoms and heme groups were based on the AMBER library that includes polar hydrogen atoms only (20). Histidine residues were neutral with a single proton on N-⑀ unless metal ligation or salt-bridge formation indicated otherwise. For horse Cc, the C terminus was negatively charged, the N terminus was neutral because of acetylation, and the heme group had an overall charge of Ϫ2, resulting in a total charge of ϩ6. Of the three available coordinate sets for R. sphaeroides cytochrome c 2 (1CXA, 2CXB, and 1CXC), 1CXC was used because it is the most complete and determined at the highest resolution (1.6 Å). Missing atoms for residues 10,32,33,35,74,78,85,89,95,97,105, and 106 were added with Insight while using other two structures as guides. Both termini were charged, and both His 75 and His 111 were doubly protonated to satisfy intramolecular salt bridges, giving a total charge of 0. An overall charge of Ϫ2 was previously used (21), possibly because all His residues were left neutral.
CcO presents a challenging computational problem because of its large size (ϳ204,000 kDa). Since Cc interacts with CcO in the mitochondrial intermembrane space, only this region of CcO was used in the calculation. The bovine CcO complex was cut by a plane placed below the ring of positively charged residues that presumably lie at the inner mitochondrial membrane surface (See Fig. 1C). All residues with any atom above this plane were retained. The selected coordinates included all of subunit II, except the transmembrane helical segment, and parts of subunits I, IV, VIa, VIb, VIc, VIIb, VIIc, and VIII. The heme groups and the other bound metal ions lie in the membrane-bound portion of the molecule and were not included. Polypeptide termini resulting from cutting out the membrane-bound region were kept neutral, while standard N termini and C termini were charged. Each copper atom of the dicopper site Cu A in subunit II was assigned a charge of ϩ1.5, giving the metal cluster a total charge of ϩ3 (22). The total charge on the selected bovine CcO coordinates was Ϫ7.
Electrostatic Potentials-Electrostatic potentials were calculated with the program UHBD (13), which uses finite difference methods to solve the Poisson-Boltzmann equations for electrostatic potential. Potentials were calculated on a grid of the same size and spacing used in the DOT calculation: 128 Å on a side with 1-Å spacing. A dielectric of 3 for the protein, a dielectric of 80 for the surrounding environment, an ion exclusion radius of 1.4 Å, and an ionic strength of 50 mM were used.
Molecular Descriptions Used in the DOT Calculation-In the DOT calculation, Cc was assigned as the moving molecule and CcO as the stationary molecule. The shape distribution of Cc was represented by a value of 1 at each atomic position. Note that with this description, the volume of the moving molecule is represented by its atomic coordinates rather than by its full van der Waals volume. The charge distribution of Cc was represented by partial charges at the atomic positions of all nonhydrogen and polar hydrogen atoms, with charges taken from the AMBER library.
The selected coordinates of CcO were centered in the x and y directions on a cubical grid 128 Å on a side with 1-Å grid spacing (about 2.1 million total grid points). The coordinates were moved in the z direction to position the approximate plane of the membrane near the bottom of the box, increasing the region representing the surrounding intermembrane space that Cc can occupy. The shape of CcO was represented by an excluded van der Waals volume surrounded by a 3.0 Å layer of favorable potential. To map the shape potential of CcO onto the grid, grid points within 4.5 Å of all nonhydrogen atoms were assigned a value of Ϫ1, and then grid points within 1.5 Å of all nonhydrogen atoms were assigned a highly unfavorable value of 1000. All other grid points were assigned a value of 0.
Since the electrostatic potential is calculated only once for the stationary molecule, sophisticated, computationally expensive methods can be used. The Poisson-Boltzmann electrostatic potential for CcO was calculated with UHBD. It was assumed that an aqueous environment surrounded the selected CcO coordinates; the low dielectric of the membrane was not taken into account. Since the stationary molecule is represented by its van der Waals volume but the moving molecule is represented by its atomic centers, some atoms of the moving molecule can approach within 1.5 Å of the stationary molecule atom centers. This accommodates small conformational changes induced upon complex formation. Unfortunately, too close an approach can also result in an artificially large electrostatic contribution when a moving molecule atom lies between the molecular and the solvent-accessible surfaces of the stationary molecule. Large changes in magnitude and distribution of electrostatic potential occur in the region between the two surfaces. The most intense potential on the molecular surface is concentrated on protuberances (termini of Lys, Arg, Asp, and Glu side chains), whereas the potential on the solvent-accessible surface is more delocalized, with the most intense potential generally concentrated in concave regions of the protein (see Fig. 1, A, B, and D). To alleviate this problem, the electrostatic potential grid values of CcO were clamped based on the extreme potentials found at the solvent-accessible surface, which represents the closest approach of the center of a water molecule (ϳ2.9 Å between atomic centers). For CcO, the upper limit was ϩ4 kcal/mol/e and the lower limit was Ϫ6.0 kcal/mol/e. We initially used limits of Ϯ 15 kcal/mol/e, the extremes found on protein molecular surfaces, but these limits gave a much larger number of favorable-energy false positives in DOT calculations.
Energy Computation by Correlation Functions-All energies computed by DOT are derived by placing the moving molecule in a potential field generated by the fixed molecule. The total energy for each configuration of the two molecules is the sum of the electrostatic and van der Waals energy terms, each of which can be described as a correlation function. Given a potential field S(r) describing the stationary molecule and a probe function M(r) describing the moving molecule, the energy is given by the following.
If the moving molecule is rotated by an angle and displaced from the origin by a vector r 0 , the energy of the system is given by the following.
Mathematically, this is the correlation of the potential field S(r) and the rotated function M(r). For the electrostatic energy term, S(r) is the electrostatic potential field of CcO and M(r) is the set of partial charges for Cc. For the van der Waals term, S(r) is the shape potential for CcO described above. Each Cc atom within the favorable layer surrounding CcO contributes Ϫ0.1 kcal/mol to the interaction energy. A Cc atom lying within the van der Waals volume of CcO contributes a large unfavorable value to the interaction energy. To allow for side chain rearrangement induced upon complex formation, a user-specified number of moving molecule atoms can penetrate the volume of the stationary molecule without incurring an energy penalty, but no penetrations were allowed in these runs. This method for computing overlap is similar to that previously described (23,24) except that here S(r) has been constructed to return a composite number containing both a count of collisions and a count of favorable interactions.
Correlations can be calculated very efficiently through the use of the Convolution Theorem. The electrostatic and nonbonded energy functions are described on a grid of N points (128 3 ). Evaluating the correlation directly requires N ϫ N multiplications, in our case 128 6 . Evaluating the correlation using fast Fourier transforms costs three fast Fourier transforms, each proportional to NlogN, and N multiplications, so the computational cost is proportional to N(3logN ϩ 1), in our case 128 3 ϫ 20, or a savings of about 10 5 .
Results from DOT: The Minimum-energy and Free-energy Grids-In the systematic search performed by DOT, Cc is centered at all grid points and then rotated, and the calculation is repeated. The full search performed here uses a grid of 128 3 points and 17,354 distinct rotational orientations, resulting in ϳ36 billion configurations between Cc and CcO. DOT maintains two 128 3 interaction grids. In the minimumenergy grid, the rotational orientation with the most favorable energy at each grid point is kept. The second grid, the free-energy grid, represents a free-energy landscape of intermolecular energies. To obtain the free-energy grid, the Boltzmann-weighted sum of the energies over all orientations at each grid point is calculated to give the partition sum as follows, where j is a grid point, R is the number of angles through which the moving molecule is rotated, T is the temperature in degrees Kelvin, and k B is the Boltzmann constant. Q j is then converted to the Helmholtz free energy as follows.
RESULTS
Calculation of Electrostatic Potentials-Electrostatic potentials for CcO and Cc were derived by integration of the Poisson-Boltzmann equation, which accurately takes into account the ionic strength of the surrounding medium and the dielectric boundaries between protein and solvent. Accurate representation of electrostatic forces is particularly important for electron transfer systems because of the role of electrostatics in both short range orientation and long range guidance, as suggested by the strong dependence of the reactions on ionic strength. Electrostatic potentials were calculated at a low ionic strength (50 mM), where Cc and CcO show strong binding (17).
Electrostatic Potentials of Horse Cc and R. sphaeroides Cytochrome c 2 -R. sphaeroides cytochrome c 2 has a slower electron transfer rate with R. sphaeroides CcO than does horse Cc (17). To investigate the source of this difference, we calculated the electrostatic potential for both molecules, as has been done previously (21). In horse Cc, the Lys residues surrounding the exposed heme edge create patches of strong positive potential (Fig. 1A). The opposite face of Cc is generally neutral, creating a dipole (21). The sequence of R. sphaeroides cytochrome c 2 is very different from that of horse Cc, including the loss of lysine at positions 13 and 72, resulting in a very different distribution of positive potential on the face of the molecule surrounding the exposed heme edge (Fig. 1B).
Electrostatic Potential of Bovine CcO-Since Cc and CcO interact in the intermembrane space, only that region of CcO along with the adjacent membrane-embedded portion (Fig. 1C) was used (see "Experimental Procedures"). The selected coordinates include the subunit II dicopper Cu A site, the initial site of electron transfer from Cc (25)(26)(27). The most significant electrostatic feature is a large patch of negative potential formed by a concentration of Asp and Glu side chains in subunit II (Fig. 1D). The greatest concentration of negative potential lies in a shallow pocket between Asp 119 (Glu 157 ) and Glu 109 (Glu 148 ). Within this negative patch is a hydrophobic surface loop that lies over the Cu A site and consists of subunit II residues His 102 (Tyr 141 ), Gly 103 (Gly 142 ), Trp 104 (Trp 143 ), and Tyr 105 (Tyr 144 ). The rest of the CcO surface is generally neutral with small regions of positive and negative potential.
DOT: Calculation of Intermolecular Energies-DOT centers each rotational orientation of one molecule, the moving molecule, at each position on a grid that surrounds a second molecule, the stationary molecule. For each configuration of the two molecules, the intermolecular energy, a sum of electrostatic and van der Waals terms, is calculated (see "Experimental Procedures"). The time required for the calculation is independent of the number of atoms in either molecule and instead depends on the size of the grid and the number of rotational orientations of the moving molecule. The mathematics of the DOT calculation requires periodic boundary conditions; the grid is repeated in all directions over all space. Therefore, the grid must be large enough that the stationary molecule potentials are close to zero at the grid boundary. In addition, at least one diameter of the moving molecule about the stationary molecule must fit within the grid. We have found that a grid spacing of 1 Å provides a sufficiently accurate description of the stationary molecule and a reasonable fineness for the translation search. A cubical grid of 128 Å on each side fulfilled the size requirements for the interaction of the CcO fragment with Cc.
The larger molecule, CcO, was assigned as the stationary molecule, and the smaller Cc was assigned as the moving molecule. This assignment provided two advantages. First, it allowed use of a finer spacing for the 128 ϫ 128 ϫ 128 grid, since the minimum dimension of the grid must be at least 2M ϩ S, where M and S represent the diameters of the moving and stationary molecules. Second, a smaller molecule will be sampled more finely over its surface for a given set of rotations. Our set of 17,354 rotational orientations provides ϳ9°sampling, which corresponds to about a 2-Å spacing on the Cc surface.
Storage requirements make it impractical to retain the energies and positions of all 36 billion configurations (128 3 ϫ 17,354) calculated. Instead, two types of interaction summary grids are maintained (see "Experimental Procedures"). The minimum-energy grid contains the energy, position, and orientation for the configuration (out of a possible 17,354) with the most favorable energy at each grid point. The free-energy grid provides a free-energy landscape combining information from all energy calculations. This grid displays a smoother change in energy across the grid than the minimum-energy grid, but rotational information is not retained. Both grids can be calculated simultaneously in about 7 h using 20 Sun Ultra-1 workstations running in parallel.
Application of DOT to CcO-A preliminary test with a single proton as the moving molecule demonstrated that the CcO shape and electrostatic potentials were properly aligned on the grid. A complete rotational search with 17,354 rotational orientations was then performed with DOT. Although our description of CcO allows Cc to move into the region of the grid that should be occupied by the membrane and membrane-bound portions of CcO, this area was electrostatically neutral, making favorable-energy interactions here unlikely. The top 2000 so- is the largest tight cluster of solutions with five (black and yellow) particularly close in both spatial and rotational orientation and two outliers (blue). From these seven solutions, the yellow one, which is in the middle of the cluster, showed the shortest distance from heme edge to CcO and was selected for investigating specific interactions in the interface. C, the free-energy landscape of the interactions between bovine CcO and horse Cc. Each point represents an energy-weighted sum over all 17,354 rotations centered at that grid point. The green sphere is the center of the Cc solution selected to represent the complex; its C-␣ trace (yellow) is also shown. The brown surface, which represents the free-energy grid points with the 2000 most favorable energies, consists of two connected lobes. One lobe is centered over the selected Cc coordinates. The second lobe is above the first, farther from the membrane, and may indicate a second Cc-binding site. D, the heme rings of the top 30 solutions from the fine rotational search. The selected Cc configuration from the full DOT search (yellow) almost superposes the best-energy configuration from the fine search (red). The most exposed atom (CBC, orange) of the heme is well clustered, showing the tight spatial and rotational alignment among these solutions.
The top 30 configurations of the minimum-energy grid show a more localized spatial distribution and had interaction energies ranging from Ϫ27.2 to Ϫ24.5 kcal/mol. Of these 30 solutions, 29 lie over the patch of negative potential on the CcO surface, with 25 having their exposed heme edge generally oriented toward the CcO molecule (Fig. 2B). Within the top 30 configurations, there is a single, tight cluster consisting of five solutions with two nearby outliers (Fig. 2B) having energies ranging from Ϫ26.5 to Ϫ24.9 kcal/mol. Among the closest five, rotations vary by 8 -30°, and root mean square (r.m.s.) deviations for all nonhydrogen atoms range from 2.8 to 5.5 Å (average r.m.s. deviation ϭ 4.1 Å). The two outliers have an r.m.s. deviation of less than 6 Å with at least one of the other five solutions. The r.m.s. deviations of the heme atoms are generally smaller, ranging from 1.8 to 5.5 Å (average r.m.s. deviation ϭ 3.4 Å) for the closest five solutions, with the two outliers having an r.m.s. deviation of less than 4.5 Å with one of the five. The tighter clustering of the heme rings relative to the whole molecule indicates that the fit is best at the interface. All seven Cc solutions in this cluster have their exposed heme edge near the indole ring of CcO residue Trp 104 (Trp 143 ).
In the free-energy grid, Cc solutions having the most favorable energies (Fig. 2C) are concentrated over the negative patch of CcO, forming a two-lobed grouping. One lobe is centered about the best-energy cluster found in the top 30 configurations of the minimum-energy grid, and the other lobe is further from the membrane. This second lobe may indicate a weaker binding site for Cc. In both the free-energy and minimum-energy grids, favorable interactions extend from the large surface patch of negative potential on CcO out into solution, showing the effect of long range electrostatic guidance.
The CcO⅐Cc Interface-The five configurations in the single, large cluster (Fig. 2B) are energetically equivalent (within 1.0 kcal/mol in energy). From this group, the Cc configuration with the shortest distance from its exposed heme edge to CcO (ranked 19th in energy) was examined for specific intermolecular interactions. Distances are approximate, since rigid structures from individually determined structures were used and complex formation will cause some local rearrangement. The interface shows a central, relatively hydrophobic region surrounded by polar interactions. The exposed Cc heme edge is within 4 Å of the indole ring of CcO residue Trp 104 (Trp 143 ) ( Table I, Fig. 3A), supporting the role of Trp 104 (Trp 143 ) as the site of electron entry (15)(16)(17). The hydrophobic region of Cc consists of the exposed heme edge, the adjacent Gln 16 and Cys 17 side chains, and residues 81-83, which form a -strand paralleling one heme face (Fig. 1A). This region contacts a corresponding hydrophobic region of CcO (Fig. 3, A and B) consisting of residues 102-105 (R. sphaeroides residues 141-144), which form a loop over the Cu A site, the adjacent residues Tyr 121 (Tyr 159 ) and Asn 203 (Ser 259 ), and backbone atoms of CcO Glu 157 (Ala 213 ) and Asp 158 (Asp 214 ) (Table II).
Asp 158 (Asp 214 ) is central to the interface (Fig. 3B), lying adjacent to the backbone of the hydrophobic loop. Unlike all other CcO acidic residues in the interface, the Asp 158 (Asp 214 ) side chain does not extend out into solvent. Instead it forms two intramolecular hydrogen bonds, one with Cu A ligand His 161 (His 217 ) and a second with the backbone amide nitrogen atom of Gln 103 (Gln 142 ) (Fig. 3C), indicating that Asp 158 (Asp 214 ) is unlikely to undergo conformational rearrangement upon complex formation. The closest positively charged Cc residue, Lys 72 , could interact directly with Asp 158 (Asp 214 ) by undergoing substantial conformational change. Intermolecular distances, however, suggest that Lys 72 forms a hydrogen bond with the conserved CcO Gln 103 (Gln 142 ) side chain (Table I) and has a more delocalized electrostatic interaction with CcO Asp 158 (Asp 214 ). One of the Asp 158 (Asp 214 ) carboxylate atoms is close enough to the backbone nitrogen atom of Cc Ala 83 to form a hydrogen bond without side chain rearrangement (Table I, Fig. 3C).
Immediately adjacent to the hydrophobic region of the interface is CcO residue Asp 119 (Glu 157 ) with its carboxylate group very close to Cc Lys 13 (Table I, (Table I). At the edge of the interface, Asp 139 (Asp 195 ) and subunit I residue Asp 221 (Gln 265 ) probably contribute to local orientation, perhaps through water-mediated interactions with Cc (Table I). The five bovine subunit II acidic side chains that contribute to the interface with Cc correspond to acidic side chains in R. sphaeroides CcO, except for Glu 157 (Ala 213 ).
A Fine Rotational Search about the Best-energy Cluster-The tight cluster of seven configurations in the 30 best-energy solutions was the basis of a finer rotational search. For each configuration, 1000 rotational orientations were generated within 30°. This provided a set of 7000 rotations with better than a 3°spacing, which corresponds to a distance of less than 0.6 Å on the Cc surface. Because of the DOT methodology, each rotation of Cc must still undergo a full translational search. The top 30 minimum-energy grid solutions from this fine rotational search were close in both position and rotational orientation (Fig. 2D). The best-energy solution from this search closely overlays the solution selected from the full search used to determine specific interface interactions (Fig. 2D), with an r.m.s. deviation for all nonhydrogen atoms of 1.4 Å. The fit between these two solutions is best at the interface, with distances between corresponding atoms of Lys 13 , Lys 72 , and Phe 82 ranging from 0.4 to 0.7 Å. Both solutions display the same interactions with CcO, validating the choice of the selected configuration from the full search. DISCUSSION Given the difficulties in determining crystallographic structures of electron transfer protein complexes, computational techniques may provide the best method for predicting these transient interactions and, moreover, may suggest pathways of approach. Recent applications of computational docking to pro- 3. The interface for the transient electron transfer interaction. A, stereo pair of the CcO⅐Cc interface. The hydrophobic patch of horse Cc (right, yellow C-␣ backbone and selected side chains (black labels), with blue Lys side chains and green heme) consisting of the exposed heme tein systems (28,29) demonstrate that these methods are now sufficiently developed to successfully predict intermolecular interactions. A major problem with computational docking is distinguishing correct solutions from false positives (9), which are favorable-energy incorrect solutions. This problem is exacerbated when coordinates from individually determined proteins rather than from crystallographic complexes are used. Visual inspection of hundreds of possible solutions along with use of biochemical knowledge has been necessary to separate correct answers from false positives (28), but this is a timeconsuming and subjective task.
The efficient, systematic search generated by DOT allows us to investigate new techniques for distinguishing correct solutions from false positives. We have tested DOT on systems for which complex and individual structures are available, including dimer interfaces, strongly bound protein-protein complexes, and electron transfer partners. 3 DOT partially allows for induced fit by letting atomic centers of the moving molecule approach to within one atomic radius of the atomic centers of the stationary molecule. Unfortunately, this can result in a few artificially large electrostatic energy terms that may dominate the interaction energy. To solve this problem, the electrostatic potential of the stationary molecule was clamped to the maximum values found on its solvent-accessible surface. This has greatly reduced the number of false positives and allowed the same relative weighting of electrostatics and van der Waals terms for all systems we have examined. Because DOT retains the most favorable energy configuration at every grid point (the minimum-energy grid), adjacent configurations can be examined and compared. We expect that correct solutions should have nearby configurations with closely related rotational orientations and similarly favorable energetics.
Both the minimum-energy and the free-energy grids generated by DOT showed one distinct, energetically favored region for the interaction of Cc with CcO. This region predominates in the top 2000 and in the top 30 solutions in the minimum-energy grid (Fig. 2, A and B). Only one of the top 30 solutions lies far from this localized region (Fig. 2B) and was immediately distinguishable as a false positive because of the lack of surrounding, favorable-energy solutions. Calculation of r.m.s. deviations among the top 30 configurations revealed a single, tight cluster of seven solutions. Just these seven Cc positions were examined in detail by computer graphics. All positioned their exposed heme edge near CcO residue Trp 104 (Trp 143 ). We selected the Cc configuration with its heme edge closest to CcO as representative of the docked complex for examining specific intermolecular contacts. This selection was further justified by its very close alignment with the best-energy solution from a finer rotational search (Fig. 2D). Thus, data from the DOT calculation itself were sufficient to predict a docked complex; no screening using biochemical data was required. The consistency of the predicted CcO⅐Cc complex to concurrent mutagenesis, binding, and kinetics studies (15)(16)(17)30) shows that DOT can be a successful predictive tool.
Structural Similarity of Bovine and R. sphaeroides CcO-The principal peptide chain of CcO involved in the interaction with Cc, subunit II, has high sequence similarity in the bovine and R. sphaeroides enzymes (Fig. 3E). Sequence identity is highest surrounding the Cu A site and on the Cc-binding surface, with only one conservative difference in the hydrophobic loop overlaying the Cu A site, residues 101-105 (R. be applicable to the bovine enzyme. Differential Activity for Horse Cc and R. sphaeroides Cytochrome c 2 -In contrast, horse Cc and R. sphaeroides cytochrome c 2, the two cytochrome c molecules examined in experimental studies on R. sphaeroides CcO (16,17), have significantly different sequences, structures, and electrostatic environments (Fig. 1, A and B). Both have a small, solventexposed hydrophobic region surrounding the exposed heme edge that includes the Cys residue bound to the heme and a -strand running parallel to the heme edge with a Phe (position 82 in horse Cc) side chain packed against the heme. The distribution of positive electrostatic potential around the hydrophobic patch appears to be critical for alignment of cytochrome c with electron transfer partners (21). This distribution is very different in the two cytochrome c molecules (Fig. 1, A and B), explaining their different activities and differential sensitivity to specific mutations in R. sphaeroides CcO (17). Thus, even though R. sphaeroides cytochrome c 2 and horse Cc share some Lys side chains, it is unlikely that they have the same intermolecular contacts with CcO. In a related system, crystallographic structures of yeast cytochrome c peroxidase show different orientations for bound yeast and horse Cc (18), despite their Ͼ60% sequence identity and similar structures.
Trp 104 (Trp 143 ) and Its Hydrophobic Loop: The Electron Entry Site into CcO-Our calculated complex of horse Cc with bovine CcO indicates specific intermolecular contacts that are consistent with mutagenesis data on both Cc and CcO and suggests new interactions to be tested. In the tightly oriented, favorable-energy cluster of Cc solutions, the exposed heme edge is close to Trp 104 (Trp 143 ) (Fig. 2B), strongly implicating this side chain in electron transfer. In the calculated complex, the Trp 104 (Trp 143 ) indole ring has two contacts under 4 Å with the exposed heme edge (Table I), which is remarkable given the coarseness of the search (1-Å translation and 9°rotation) and the use of rigid, uncomplexed protein structures. The hydrophobic loop of CcO, residues 102-105 (R. sphaeroides residues 141-144), is packed against the -strand paralleling the Cc heme (Fig. 3A, Table II). These hydrophobic contacts may facilitate rapid electron transfer across the interface, explaining their conserved structures in both CcO and Cc. The position of Trp 104 (Trp 143 ) in the interface explains the severe drop in intrinsic electron transfer rate upon mutation to Phe or Ala (16). Mutation of residues 102 (141) and 105 (144), whose side chains contribute to the hydrophobic patch, should also decrease the electron transfer rate if the overall hydrophobic environment is an important factor in intrinsic electron transfer rates. Although mutation of Tyr 105 (Tyr 144 ) has little effect on the turnover rate (30), 4 the effect of this mutation on the rate of intrinsic electron transfer has not yet been examined.
The Position of Charged Side Chains in the Interface Parallels Their Influence on Binding Affinity-The closer a charged side chain is to the hydrophobic region central to the interface of the calculated complex, the more important it is to binding affinity. Chemical modification of Lys residues of horse Cc showed decreased activities with the order Lys 13 Ͻ Lys 72 Ͻ Lys 87 Ͻ Lys 8 (31,32), which follows their distance from the heme and adjacent -strands (Fig. 1A). Similarly, mutation of the R. sphaeroides CcO side chains Asp 158 (Asp 214 ) or Asp 119 (Glu 157 ), which are close to the hydrophobic loop residues 102-105 (R. sphaeroides 141-144) (Fig. 3B), decreases binding to horse Cc more than mutation of Glu 109 (Glu 148 ) or Asp 139 (Asp 195 ), which are farther from the hydrophobic loop (16).
In the interface, the two most influential horse Cc Lys side chains, Lys 13 and Lys 72 , are paired with the two most influen-tial CcO acidic side chains, Asp 119 (Glu 157 ) and Asp 158 (Asp 214 ) (17). Close contact (Table I) and good orientation (Fig. 3A) of Lys 13 and Asp 119 (Glu 157 ) are consistent with salt bridge formation, which may be important for aligning the hydrophobic patches of the two proteins. This pairing brings together the greatest concentration of positive electrostatic potential on horse Cc (between Lys 13 , Lys 86 , and Lys 87 ; Fig. 1A) and the greatest concentration of negative electrostatic potential on bovine CcO (between Asp 119 (Glu 157 ) and Glu 109 (Glu 148 ); Fig. 1D). Lys 72 lies near Asp 158 (Asp 214 ) (Fig. 3A), but constraints on the Asp 158 geometry (see below) suggest that electrostatic interactions between the two charged side chains are indirect. Lys 72 may, however, form a hydrogen bond with conserved CcO residue Gln 103 (Gln 142 ), explaining the reduced binding upon mutation of Gln 103 (Gln 142 ) (33).
Further from the hydrophobic contact region are interactions across the interface that may be direct or mediated by water molecules (Table I) (16), consistent with their greater distance from the interface. These side chains lie on the Cc-binding face of CcO, and therefore may have a small role in long range guidance.
Neutralization of a single charge in the CcO⅐Cc interface only modestly affects binding affinity (15,17). In contrast, neutralization of a charged side chain in tightly bound protein-protein complexes such as dimer interfaces or antibody-antigen complexes can decrease binding by orders of magnitude. In these stable complexes, pairs of oppositely charged side chains are often buried within a large hydrophobic interface. The surrounding low dielectric and separation from ions and solvent creates a strong, highly directional interaction. In the CcO⅐Cc interface, complementary charge-charge interactions lie outside the small hydrophobic contact region, allowing interaction with bulk solvent and ions and decreasing their contribution to binding affinity.
Despite the weak contribution of each charged side chain to binding affinity, their combined electrostatic interactions appear to dominate the binding interaction, with the total interaction energy of the complex having a greater electrostatic energy term (Ϫ14 kcal/mol) than van der Waals energy term (Ϫ11 kcal/mol). Consistent with the small contribution of van der Waals energy, mutating Trp 104 (Trp 143 ), which is centered in the hydrophobic region, to Phe or Ala has little or no effect on the equilibrium dissociation constant, despite its large effect on intrinsic electron transfer rate (16). In contrast, DOT calculations show that the van der Waals energy term in strongly bound complexes is 3-4-fold greater than the electrostatic energy contribution. 3 The closely oriented, favorable-energy configurations from the restricted, finely sampled rotational search (Fig. 2D) suggest that electrostatic forces align the electron transfer partners but are not sufficiently directional to provide a single, precise "lock-and-key" fit. These computational results fit well with the idea of a pseudospecific docking surface for electron transfer proteins that allows a single protein to recognize multiple partners (34).
CcO Residue Asp 158 (Asp 214 ) Has a Unique Role-Asp 158 (Asp 214 ) is unique among the acidic CcO side chains in the interface for two reasons. First, it lies along the protein surface instead of extending into solution. Second, its mutation to Asn significantly decreases the rate of intrinsic electron transfer (16). Asp 158 (Asp 214 ) is a second-sphere ligand of the Cu A site, forming an Asp-His-copper triad (Fig. 3C). Such triads are common in protein metal sites and may align metal-binding ligands or modulate metal site properties. In the metalloenzyme carbonic anhydrase, mutation of Glu 117 in the Glu-Hiszinc triad to Gln caused a Ͼ1000-fold reduction of catalytic activity and altered the kinetics of metal binding (35). Surprisingly, no discrete structural changes were found in the crystallographic structure of the mutant. Like Asp 158 (Asp 214 ) in CcO, carbonic anhydrase Glu 117 forms hydrogen bonds with both a metal-binding His ligand and a backbone amide nitrogen atom. The Gln mutant maintains these bonds, resulting in the stabilization of the His ligand as an imidazolate anion, thereby causing an electrostatic rather than a structural change (35). Analogously, mutation of CcO Asp 158 (Asp 214 ) to Asn neutralizes residue 158 (214) but may stabilize His 161 (His 217 ) as an imidazolate anion. Given the sensitivity of the electron transfer reaction to charge distribution and the central position of His 161 (His 217 ) between the interface and the Cu A center (Fig. 3C), a shift in charge distribution could have a more profound effect on electron transfer than neutralization of a single charge within the interface. The shift in charge distribution could lock the complex into an unproductive configuration, resulting in the observed decrease in the rate of intrinsic electron transfer at low ionic strength (16).
A Second Binding Site for Cc-Experimental evidence strongly supports a second, weaker Cc-binding site on CcO. This second Cc may also interact directly with Cc bound in the primary binding site (36). The less well oriented group of solutions lying in the upper portion of the CcO negative patch (Fig. 2, A-C) may indicate the second site. Cc binding could be more disordered than the high affinity site or could require a bound Cc at the high affinity site to define its orientation. If the latter is true, then adding our bound Cc molecule to the stationary complex and searching with a second Cc moving molecule would resolve this question.
Comparison of the CcO⅐Cc Interface with That of CcP⅐Cc, a General Motif for Transient Interactions-The similarity of yeast Cc to horse Cc (Ͼ60% sequence identity) prompted us to compare our predicted complex of bovine CcO and horse Cc with the complex of yeast CcP and yeast Cc, which has been determined by x-ray crystallography (18). The crystallographic structure of CcP⅐Cc (2PCC in the Protein Data Bank) shows close contacts between CcP residues Ala 193 and Ala 194 and the exposed Cc heme edge, consistent with mutagenesis experiments indicating that the surface loop containing residues Ala 193 and Ala 194 is the point of electron entry (37,38). The CcP⅐Cc interface is unusual in having a small van der Waals contact region and only one specific hydrogen bond and a single salt bridge, despite the large number of charged side chains in the interface. All other interface interactions are mediated by a layer of water molecules.
To compare the calculated complex of bovine CcO and horse Cc with the yeast CcP⅐Cc complex, the heme rings of the two Cc molecules were superposed. This superposition puts the two electron entry sites, the CcO Trp 104 (Trp 143 ) indole ring and CcP residue Ala 194 , on top of each other (Fig. 3D) despite the very different structures of CcO and CcP. The hydrophobic contact surface of CcP residues Ala 194 -Val 197 overlays the CcO conserved side chains Gln 103 (Gln 142 ) and Trp 104 (Trp 143 ). Differences between the distribution of Lys side chains in yeast and horse Cc correspond to differences between the acidic environments in CcP and CcO (Table III). Horse Cc residue Lys 13 , which interacts with CcO Asp 119 (Glu 157 ), is replaced by the more charge-delocalized Arg 13 in yeast Cc, which interacts with CcP Tyr 39 . Yeast Cc Lys 72 is trimethylated on the terminal amine, preventing hydrogen bond formation. The polar environment in CcO that interacts with Lys 72 (Gln 103 (Gln 142 ) and Asp 158 (Asp 214 )) is replaced by less polar side chains in CcP (Val 197 and Gln 120 ) (Fig. 3D). There are equivalent chargecharge interactions for two Lys residues shared by both Cc molecules: Cc Lys 87 interacts with Glu 109 (Glu 148 ) in CcO and with Glu 32 and Asp 34 in CcP; Cc Lys 73 interacts with subunit I Asp 221 (Gln 265 ) in CcO and Glu 290 in CcP. The similarities of the two interfaces also extend to the energetics. A preliminary DOT calculation of the complex between CcP and Cc 3 shows that the electrostatic energy term dominates, as found for the CcO⅐Cc interaction.
The similarities in the two complexes suggest that an interface consisting of a small hydrophobic region surrounded by a highly charged, electrostatically complementary surface is a general motif for electron transfer partners involved in transient interactions. The delocalized distribution of charge over the surface of the electron transfer partners, rather than the formation of many specific salt bridge or hydrogen bonding interactions, appears to control local orientation while allowing rapid product dissociation. Maximizing the electrostatic complementarity concurrently brings the electron transfer groups into close proximity. Thus, the most energetically favored complex is also optimized for electron transfer. This is consistent with time-resolved kinetics studies showing that the intrinsic electron transfer rate is the same at low and intermediate ionic strengths, indicating that slow product dissociation is responsible for the slow rate of turnover at low ionic strength (16). | v3-fos-license |
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} | pes2o/s2orc | The extracellular matrix of mycobacterial biofilms: could we shorten the treatment of mycobacterial infections?
A number of non-tuberculous mycobacterium species are opportunistic pathogens and ubiquitously form biofilms. These infections are often recalcitrant to treatment and require therapy with multiple drugs for long duration. The biofilm resident bacteria also display phenotypic drug tolerance and thus it has been hypothesized that the drug unresponsiveness in vivo could be due to formation of biofilms inside the host. We have discussed the biofilms of several pathogenic non-tuberculous mycobacterium (NTM) species in context to the in vivo pathologies. Besides pathogenic NTMs, Mycobacterium smegmatis is often used as a model organism for understanding mycobacterial physiology and has been studied extensively for understanding the mycobacterial biofilms. A number of components of the mycobacterial cell wall such as glycopeptidolipids, short chain mycolic acids, monomeromycolyl diacylglycerol, etc. have been shown to play an important role in formation of pellicle biofilms. It shall be noted that these components impart a hydrophobic character to the mycobacterial cell surface that facilitates cell to cell interaction. However, these components are not necessarily the constituents of the extracellular matrix of mycobacterial biofilms. In the end, we have described the biofilms of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Three models of Mtb biofilm formation have been proposed to study the factors regulating biofilm formation, the physiology of the resident bacteria, and the nature of the biomaterial that holds these bacterial masses together. These models include pellicle biofilms formed at the liquid-air interface of cultures, leukocyte lysate-induced biofilms, and thiol reductive stressinduced biofilms. All the three models offer their own advantages in the study of Mtb biofilms. Interestingly, lipids (mainly keto-mycolic acids) are proposed to be the primary component of extracellular polymeric substance (EPS) in the pellicle biofilm, whereas the leukocyte lysate-induced and thiol reductive stress-induced biofilms possess polysaccharides as the primary component of EPS. Both models also contain extracellular DNA in the EPS. Interestingly, thiol reductive stressinduced Mtb biofilms are held together by cellulose and yet unidentified structural proteins. We believe that a better understanding of the EPS of Mtb biofilms and the physiology of the resident bacteria will facilitate the development of shorter regimen for TB treatment.
INTRODUCTION
Bacteria are generally studied in the research laboratories as single cell suspensions called as planktonic cultures, however, in nature, bacteria primarily exist as a community encased in a self-produced extracellular matrix called as biofilms. There are many advantages of studying bacteria in the planktonic cultures such as development of a homogenous population of bacterial cells having similar tran-scriptomic, proteomic and metabolomic profile etc. But the bacterial growth in biofilms requires a varied but coordinated transcriptional, proteomic and metabolomic profile. The bacterial cells residing in biofilms exhibit quite different phenotypic properties compared with their planktonic counterparts [1]. Formation of bacterial biofilms requires cooperation, differentiation and division of labor, capturing and sharing of resources such as nutrients. Microbial biofilms ensure improved survival following exposure to antimicrobials and physicochemical stresses. Bacteria residing in biofilms are highly heterogeneous and understanding their physiology is challenging. Given the physiological heterogeneity, the biofilm resident bacteria depict phenotypic drug tolerance that is of relevance for a number of infections. Bacterial biofilms are associated with a number of infections such as endocarditis, cystic fibrosis, pneumonia, infectious kidney stones, inner ear infections and many hospital-acquired infections from catheters and ports [2][3][4]. Biofilm resident bacteria display 100-1000 folds higher minimal inhibitory concentration (MIC) as compared to planktonic bacteria making their treatment a challenging task [5]. It is believed that the extracellular polymeric substance (EPS) could act as a barrier for antibiotic penetration and thus may contribute to the drug tolerance observed in biofilms. The basic ultrastructure of the bacterial biofilms largely depends on the extracellular matrix produced by the cells within the biofilms. The matrix of biofilms is composed of different types of biopolymers known as EPS. In most of the bacterial biofilms, most of the dry mass is due to EPS, while bacteria contribute only to a small fraction of the total dry mass [6]. EPS provides mechanical stability to biofilms through physiochemical interactions that involve electrostatic forces, hydrogen bonds and van der Waals interactions [6,7]. Although the composition of EPS varies significantly among different bacterial species, extracellular polysaccharides, proteins and lipids remain as the key components of EPS [8].
A number of Mycobacterial species are known to form biofilms including Mycobacterium tuberculosis (Mtb), Mycobacterium smegmatis (Msm), Mycobacterium avium, Mycobacterium marinum and Mycobacterium ulcerans [9][10][11][12]. Schulze et al. described the capability of Mycobacterium to form biofilms in 1989 [13]. In this pioneering work, they analyzed the capability of Mycobacterium kansasii and Mycobacterium flavescens to form biofilms in water drainage systems. They demonstrated densely packed mycobacterial colonies in the silicone tube constantly perfused by the water from the distribution system. The same group further reported the occurrence of ~ 4.5×10 5 CFU/L of Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium fortuitum, M. kansasii and M. flavescens in the biofilms formed in domestic water supply systems using specific biochemical reactions and thin layer chromatography for mycolic acids [14,15]. A number of studies have shown the presence of non-tuberculous mycobacteria (NTMs) in the cooling water distribution systems, dental sprays [16], potable drinking waters [17,18], water filters in contaminated hospital bronchoscopes [19], and other environmental sources. These sources could act as a reservoir for NTMs that could infect humans and animals through swallowing, inhalation or inoculation and subsequent colonization in oral, respiratory or gastric wounds. In the last two decades, understanding the mycobacterial biofilms has evolved into a niche area for research. It must be noted that both pathogenic and non-pathogenic species of mycobacteria are capable of forming biofilms and this capability is not essentially a virulence mechanism.
However, biofilms could protect the pathogenic mycobacterial species from the immune-system of the host and could help bacteria to persist during treatment with antibiotics. Given these observations, studying biofilms of pathogenic mycobacterial species is important. In this review, we will describe the pertinent information on mycobacterial biofilms with emphasis to their clinical relevance and the nature of EPS. The biofilm formation occurs through a series of steps involving the initial attachment of the bacterial cells to substratum which is followed by the aggregation of the cells and irreversible binding. This step is followed by maturation of the biofilm cells which is formed by layering of the aggregates, which upon reaching an ultimate thickness starts to disperse only to start aggregating at a new site (as depicted in Figure 1). Current understanding of the mechanisms and characteristic features of mycobacterial biofilms are described in this review.
BIOFILM FORMATION BY MYCOBACTERIUM SMEGMATIS
Msm is a rapidly growing non-pathogenic mycobacterial species that is often used as a model organism for studying the mycobacterial physiology [20]. Since it is a model organism, it is quite well studied for the biofilm formation. Msm is known to form well organized colonies and microcolonies which have been described by Danese et al. as a type of biofilm. These colonies are composed of microbial cells encapsulated by a large amount of exopolysaccharides [21]. Importantly, sliding motility plays a critical role in the formation of colonies on plate [22]. Recht et al. demonstrated that transposon mutants in the glycopeptidolipids (GPL) biosynthesis pathway are attenuated for colony formation and lack the capability to form biofilms on PVC plates [22]. GPLs are an important component of the mycobacterial cell wall, and it was observed that they play an important role in the initial attachment of mycobacterial cells to the substratum like PVC. The same group later on demonstrated that acetylation of GPL is also important in determining the colony morphology and formation of biofilms on PVC plates [23]. Subsequently, mycolic acids, another major component of the mycobacterial cell wall, was implicated in pellicle biofilm formation. Pellicle is a bacterial growth at the media-air interface. This mode of growth is primarily seen in aerobic bacteria wherein the bacterial cells have access to both air and nutrients of media. Some of the recent studies have demonstrated that in the pellicle, the mycobacterial cells are encapsulated in self-produced EPS [24]. Thus, mycobacterial pellicles are considered to be a form of biofilms. In an exciting discovery, Ojha et al. demonstrated that mycolic acids play a critical role in maturation of pellicle biofilms [25]. Importantly, the mycolic acids produced during maturation of pellicle growth are shorter (C 56 -C 68 ) compared to the regular mycolic acids of the cell wall of Mycobacteria (C 70 -C 70 ). Apparently, chaperone GroEL1 regulates this transition in the type of mycolic acid and thus plays an important role in pellicle biofilms formation [25]. Ojha and colleagues also suggested that the short chain free mycolic OPEN ACCESS | www.microbialcell.com acids are released through hydrolysis of trehalose dimycolate (TDM) by serine esterase [26]. Interestingly, Msm rpoZ gene (encoding for the ω subunit of the RNA polymerase), deletion mutant displayed an altered colony morphology. Analysis of this mutant revealed that it is defective in sliding motility and biofilm formation [27]. Importantly, this mutant has equal quantities of GPL compared with wild type Msm. Mass spectroscopy-based analysis of mycolic acids suggested that it possessed very low levels of short chain mycolic acids. SEM analysis demonstrated the absence of ECM in the pellicle growth of the mutant. These findings strongly support that short chain mycolic acids are a component of ECM [27]. The role of lipids in biofilm formation is also supported by the observation that a Msm mutant in mmpL11 (required for the transport of monomeromycolyl diacylglycerol (MMDAG) and mycolate ester wax to the bacterial surface) has a delay in biofilm formation [28]. However, it must be noted that this mutant forms quite mature pellicle biofilms sometime later. The role of MMDAG in cell-to cell attachment and biofilm formation was also independently demonstrated in a separate study [29]. In this study a transposon mutant library was created and analyzed for defects in colony morphology. It was observed that transposon insertion in the Lsr2 (a histone like protein in mycobacteria) leads to smooth, wet, and round colonies, opposed to the dry, rough, and rugose colonies of the parent Msm strain. This strain was also compromised in biofilm formation. Further analysis suggested that this transposon mutant strain has equivalent levels of GPLs and mycolic acid, but is deficient in the MMDAGs [30]. It was further suggested that the presence of MMDAGs on the cell surface may increase its hydrophobicity to facilitate cell-to-cell interaction. The role of Lsr2 in biofilm formation was confirmed by another study, which demonstrated that Lsr2 mutant of Msm has increased sliding motility, reduced surface hydrophobicity and is attenuated for pellicle growth [31]. The role of Lsr2 in the different genetically programmed stages of biofilm formation in Msm was further illustrated by Yang et al. in 2017, where they monitored the participation of different genes in the individual stages of biofilm formation -from attachment with the substratum to intercellular aggregation followed by maturation of the pellicle architecture [32]. The group presented a model of biofilm formation where the planktonic cells, with the help of Lsr2, start forming aggregates which in turn triggers upregulation of GroEL and GroEL dependent free mycolate synthesis. It is worth noting that aggregation may impart benefits like drug tolerance, but it is not equivalent of biofilms. However, it shall be noted that bacterial aggregates may play a role in biofilm formation in bacterial species such as P. aeruginosa, and that variant strains that readily make aggregates also form strongly adherent biofilms [33][34][35]. It further induces iron sequestration pathways, which mark the onset of biofilm maturation [32]. The role of the cell wall in pellicle biofilm formation is also FIGURE 1: The biofilm formation and dispersal cycle. The planktonic bacteria form biofilms through a series of steps, which involve the initial attachment of the cells to a substratum followed by biofilm maturation and proliferation of bacteria within the matrix and finally a part of the matured biofilm dispersing to another site for subsequent localization and attachment. During this process, bacteria undergo phenotypic changes. Several genes playing roles in virulence and redox sensing are upregulated. Biofilms formation is associated with upregulation in cellulose synthesis during maturation of the biofilms; however, localized expression of cellulases and proteases degrades the extracellular the matrix of the biofilm thereby leading to bacterial dispersal followed by start of a new cycle of biofilm formation. supported by the observation that the ΔhadC mutant with a defect in dehydratase activity of fatty acid synthase type II (FAS-II) takes longer time for pellicle formation and has altered colony morphology [36]. On the contrary, a mutant of the mammalian cell entry (mce) 1 operon that accumulates free mycolic acids in its cell wall forms normal colonies [37]. Whether these mutants form better pellicle or make it rapidly remain to be analyzed. However, the mce operon mutant of Msm, wherein all the six operons are deleted, is attenuated for pellicle formation and has altered colony morphology [38]. It will be interesting to see if this mutant also accumulates free mycolic acids in the cell wall. Intriguingly, ectopic expression of a putative peptidoglycan amidase (Rv0024) in Msm induces biofilm formation and leads to an increase in drug resistance [39]. Importantly, overexpression of Rv0024 was associated with increased hydrophobicity of mycobacterial cells. These observations suggest that abundant amounts of free mycolic acids may still be the part of the cell wall, but lead to increased surface hydrophobicity that may help the mycobacterial cells to associate more with each other. However, if this hypothesis is true, then the free mycolic acids are not really part of the extracellular matrix (since they are part of the cell wall, rather than the extracellular matrix). This hypothesis is supported by the observation that incubation of Msm cells with secretory antigen MTC 28 (encoded by Rv0040c) increases hydrophobicity of the mycobacterial cells and induces cellular aggregation [40]. Furthermore, inhibition of peptidoglycan biosynthesis by knockdown of phosphoglucosamine mutase (glmM) also reduces biofilm formation [41]. These observations suggest that composition of the cell wall could greatly affect the mycobacterial biofilm formation through modulation of initial cell-to-cell interaction. We believe that further research is required to study the effect of changes in cell-tocell contact on the biofilm formation.
How does the mycobacterial biofilms form and what induces their formation have remained important questions in the field. In the recent years, answers to these questions are emerging, but the entire picture is not as clear as is desired. A number of redox stresses are known in other bacteria to induce biofilms [42]. Bhat et al. have earlier demonstrated that redox stress in culture and/or inside macrophages leads to accumulation of NADH levels in mycobacterial cells [43,44]. Interestingly, intracellular NADH levels are sensed by the PknG. PknG, along with ribosomal proteins L13 and Nudix hydrolase RenU, constitutes a redox homeostatic system responsive to cellular NADH levels named as RHOCS. Wolff et al. demonstrated that upon sensing higher cellular levels of NADH, PknG phosphorylates L13 protein and thus increases its association with RenU. L13 with RenU leads to NADH hydrolysis thereby balancing redox homeostasis in the cells. Interestingly, PknG, L13 and RenU all are required for biofilm formation by Msm [45]. These findings suggest that the metabolic state of the mycobacterial cells regulates the biofilm formation in Mycobacteria. On one hand, the NADH:NAD + redox couple along with the ATP:ADP depicts the metabolic state of the cell, on the other hand, mycothiol along with the antioxidant ergothioneine constitutes the thiol buffering system of the mycobacterial cells [46]. Interestingly, Msm mutants in mycothiol biosynthesis (ΔmshC) or mycothiol dependent metabolism of nitrosothiols (ΔmscR) are compromised for pellicle biofilm formation [47]. These observations suggest a critical role of the redox state in the biofilm formation. Along these lines, Trivedi et al. demonstrated that intracellular thiol reductive stress induces biofilm formation in Mtb cells. It remains to be analyzed whether thiol reductive stress also induces biofilm formation in Msm or other mycobacterial species. Besides the redox stress, marker of the stringent stress response (p)ppGpp and cyclic nucleotide c-di-GMP play a critical role in biofilm formation. (p)ppGpp is synthesized by Rel Msm while the c-di-GMP is synthesized by DcpA. Msm knockout strains of these second messengers (ΔrelA and ∆dcpA) are compromised for biofilm formation [48]. Furthermore, Kuldeep et al. have demonstrated that low levels of these second messengers assist in bacterial growth, while higher intracellular concentration promotes biofilm formation [49]. However, the mechanisms through which the altered redox and metabolic state modulates these second messengers to promote biofilm formation needs to be delineated.
It shall be noted that biofilm formation is an active process that is tightly regulated at translational and transcriptional levels. In order to understand the genes involved in biofilm formation, Ojha et al. studied the transcription profiling of Msm biofilms [50]. They demonstrated that a 3day old Msm biofilm had 1.5% differentially regulated genes, whereas 4.5% of the total genes are modulated in the 4-day old biofilm and 4.9% in the stationary phase Msm cultures. There was an increase in the expression of mycobactin biosynthesis genes, exochelin biosynthetic genes and the putative iron ABC transporter in the 3-day and 4-day old biofilm cultures, suggesting an importance of iron uptake in the development of Msm biofilms. But more transcriptomics experiments need to be performed to generate a transcriptional map of the important regulatory network that plays an important role in the biofilm formation.
BIOFILM FORMATION BY THE NON-TUBERCULOUS MYCOBACTERIA (NTMS)
NTMs include all the mycobacterial species (~178 different species listed at http://www.bacterio.net/ mycobacterium.html) besides the ones classified under the "Mycobacterium tuberculosis complex" and those known to cause leprosy (Mycobacterium leprae). These are also known as "mycobacteria other than tuberculosis" and "atypical mycobacteria" [17]. NTMs are ubiquitous and are found in diverse environments such as soil and water. These mycobacterial species could infect and cause skin and soft tissue infections (SSTIs) in animals and humans [17]. A number of the NTMs make biofilms naturally in the environment [51]. NTMs such as species belonging to the Mycobacterium avium complex form biofilms [12] and also cause infections in humans and animals [52]. Given that many of the NTM infections are chronic and require long treatment, studying their biofilms could be relevant to reduce the course of treatment. Although a number of NTMs form biofilms in the environment, here we have focused on the biofilms of medically relevant species of NTM.
Mycobacterium avium
M. avium is capable of infecting humans [53], domestic animals [54] and birds [55] and is found abundantly in different environmental niches such as water bodies and soil. Being an opportunistic pathogen, it mostly infects immunocompromised patients, especially those suffering from AIDS [53], cystic fibrosis [56], or pulmonary alveolar proteinosis [57]. Falkinham et al. demonstrated the presence of M. avium biofilms in the drinking water distribution system [58]. Following that, many studies have confirmed that M. avium forms detectable biofilms in potable drinking water as well as household plumbing water pipes [59][60][61]. Importantly, several human, avian and porcine isolates of M. avium are capable of forming biofilms in vitro when incubated for 7 days as a suspension in 7H9 medium with O-ADC and Tween in the 96-well flat bottom polystyrene microtiter plate [62]. M. avium infections are difficult to treat and require prolonged treatment with multiple drugs. Formation of M. avium biofilms in vivo could explain the requirement of such a prolonged treatment. Formation of colony biofilms has been studied extensively for M. avium. Sliding motility is dependent on the presence of glycopeptidolipids (or GPLs are a class of amphiphilic molecules localized in the cell envelope) and plays a critical role in colony biofilm formation in M. avium [22,63]. Interestingly, Yamazaki et al. observed a correlation between M. avium biofilm formation and its capability to colonize the bronchial and bronchiolar mucosa [64]. M. avium strains, namely MAC104, MAC101 and MAC A5 could invade and infect the bronchiolar epithelial cells. Incidentally, these strains are also capable of forming biofilms in vitro. However isogenic mutant clones of MAC A5 (namely 5G4, 6H9 and 9B5), that are attenuated to form biofilms were unable to invade and infect the bronchiolar epithelial cells. Additionally, it was observed that these mutant strains were also compromised in their ability to infect mice lung while the MAC A5 was capable of infecting mice lungs, spleen and liver. Furthermore, M. avium biofilms or their supernatants were capable of inducing TNF-α production when compared with their planktonic counterparts. This excessive TNF-α production in response to M. avium biofilms leads to apoptotic cell death of the macrophages [65]. Investigations on biofilm formation suggested a requirement of divalent cations like Ca 2+ , Mg 2+ , Zn 2+ for biofilm formation [12]. These might act as stabilizing agents for the negatively charged nucleic acids present in the biofilms. Besides these agents, the presence of glucose and peptone as carbon sources enhanced the biofilm formation. On the contrary, humic acid could inhibit biofilm formation. Interestingly, the supernatant from the M. avium biofilm culture induced biofilm formation in planktonic cells suggesting some kind of quorum sensing could assist biofilm formation [12]. Also, oxidative stress induced by Autoinducer-2 (AI-2) activates biofilm formation in M. avium [66]. Apart from GPLs, extracellular DNA (eDNA) has also been found to be a part of the M. avium biofilm matrix. Importantly, exposure to Dnase disrupts M. avium biofilms suggesting that DNA is an integral component of the EPS of M. avium biofilms [67]. Generally, eDNA is produced in biofilms through (i) autolysis, (ii) active secretion, (iii) and via membrane vesicles [68]. The release of eDNA is governed through quorum sensing. Interestingly, an unbiased transposon mutant screening identified the FtsK/SpoIIIE DNA transport system and carbonic anhydrase as sufficient components for DNA export in M. avium, and these genes were induced by bicarbonate [69]. These observations point to a quorum sensing based mechanism for production of eDNA in M. avium biofilms. Another study identified 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein) to play an important role in biofilm formation [70]. However, a detailed analysis of the extracellular polymeric substance of M. avium biofilms remains to be done.
Mycobacterium abscessus
M. abscessus (Mab) is a fast-growing NTM that causes a wide variety of human infections, including those of lung, skin, soft tissue, ocular and central nervous system, etc. These infections are recalcitrant to treatment with a multitude of antibacterial drugs [71]. Mab is emerging as a major pathogen associated with cystic fibrosis [72]. In an elegant study, Qvist et al. demonstrated the presence of Mab microcolonies surrounded by extracellular matrix in sparse intra-alveolar walls [73]. Microcolonies are microscopic communities of ~50 cells that spontaneously aggregate and could nucleate the growth of a biofilm. In the abovementioned study, the aggregates/microcolonies were mostly observed embedded deep in the alveolar wall and only occasionally observed in the phagocytosed Mabs [73]. Importantly, these microcolonies were equated to biofilms in the absence of any evidence for the self-produced extracellular matrix. In our view, more studies are required to demonstrate the presence of extracellular matrix surrounding the bacterial communities to conclude the presence of mycobacterial biofilms in the lungs. Such studies could prove to be milestones in the current understanding of the pathogenesis of diseases caused by mycobacteria. Interestingly, another study by Fennelly et al. demonstrated the presence of Mab biofilms in the lung cavity of a patient with chronic obstructive pulmonary disease using M. abscessus specific PNA-FISH probes. They also demonstrated that ~2% of Mabs in the lung cavity were in the biofilms and were embedded in the extracellular matrix [74]. Interestingly, bacteria residing in biofilms exhibit drug tolerance [75]. A few other reports also suggest that Mab biofilms formed on implants can lead to post-operative surgical site contamination [76,77]. It is worth mentioning that Mab grows on LJ slants and Middlebrook 7H10 agar to produce two different colony morphologies, namely rough (Mab-R) and smooth (Mab-S). Importantly, rough and smooth morphotypes exhibit different virulence phenotypes [11].
Interestingly, Mab-R forms extensively corded microcolonies and is virulent, on the other hand, Mab-S forms rounded, smooth, smaller microcolonies and is less virulent [11,78]. Further studies on the morphology switching revealed that the gene mab_3168c, encoding for a putative acetyltransferase, regulates this morphotypic switching and the mab_3168c deletion mutant grows in smooth colonies. Importantly, Δmab_3168c is unable to revert back to rough colonies and is deficient in biofilm formation and intracellular survival [79]. Although much information is not available about the nature of EPS of this NTM, few reports suggest that the matrix of Mab biofilms contains glycopeptidolipids as well as extracellular DNA [67]. The secretion of eDNA is regulated by bicarbonate and is important for structural integrity of the biofilm [69]. The role of glycopeptidolipids in Mab biofilms has been worked out using genetic approaches. It was observed that deletion of genes like mmpL4b [80] or mab_3168c [79], that play important roles in the glycopeptidolipid biosynthetic pathway, results in impaired biofilm formation. However, more studies are required for characterization of the nature of extracellular matrix of this pathogen.
Mycobacterium fortuitum and Mycobacterium chelonae
Other important fast-growing human pathogens are M. fortuitum and M. chelonae. These are opportunistic human pathogens that primarily infect people with compromised immune system or those suffering with chronic diseases [81]. These species are also known to lead to post-surgical infections and could form biofilms in eye or skin tissues [82]. These species quickly form biofilms within 48 hrs [83]. The biofilm formation is robust and does not depend upon the nature of the substrate [84]. Importantly, these biofilms are biocide resistant [85]. The biofilms of M. fortuitum are impermeable to several antibiotics, including ciprofloxacin, thereby suggesting that biofilm permeability of antibiotics might be an important reason behind antimicrobial drug resistance. Interestingly, these biofilms contain eDNA and a combination of antibiotics with DNase was more effective in disrupting the biofilms and killing biofilm residents than antibiotics alone [9].
Mycobacterium ulcerans
M. ulcerans causes buruli ulcer in humans. Buruli ulcer is the third most common infection caused by a mycobacterial pathogen after tuberculosis and leprosy. These infections are most commonly reported from sub-saharan Africa [86]. A unique feature of M. ulcerans infection is the development of necrotic cutaneous lesions caused by polyketide toxin -Mycolactone [87]. Importantly, Mycolactone is among only few of the virulence toxins identified for mycobacterial species. M. ulcerans is a slow-growing environmental bacterium that is capable of forming in vivo biofilms on the salivary glands of the aquatic insect Naucoris cimicoides [88] and on aquatic plants [89]. In an elegant study, Marsollier et al. [90] characterized the biofilms of M. ulcerans. SEM analysis also revealed that in the biopsy samples from Buruli ulcers patients and those isolated from infected mice mycobacterial cells were organized into discrete bacterial clusters enveloped in the extracellular matrix suggesting the formation of biofilms in vivo. Importantly, these biofilm-like structures also contained vesicles between 50 and 200 nm in diameter. Similar vesicles were also detected in the ECM from M. ulcerans biofilms. They further showed that these vesicles contain Mycolactone and its biosynthesis machinery. This group was able to separate the ECM from the bacterial cells using mechanical disruption (with glass beads) along with treatment with Tween 80 detergent. They demonstrated that the ECM was capable of protecting the mycobacterial cells from antimycobacterials such as Rifampicin. They further demonstrated that more than 80 proteins are present in the ECM and that these proteins play roles in stress responses, respiration and intermediary metabolism. They also revealed that M. ulcerans biofilms are rich in carbohydrates, with glucose being the most abundant monosaccharide unit, relating it structurally to the D-glucan of Mtb. More research work is required to fully understand the role of polysaccharides in the ECM of M. ulcerans biofilms. Other ECM components include lipids such as phosphatidylinositol mannosides (PIM2, PIM5 and PIM6), phospholipids (phosphatidylethanolamine, phosphatidylinositol, cardiolipin), triacylglycerol, phthiodiolone diphthioceranates, etc [78]. The bacterial adherence and attachment to the surface are enhanced by the small 18-kDa-heat shock protein (Hsp18) [91] suggesting an important role for this chaperon in the biofilm formation.
Mycobacterium marinum
M. marinum is a slow-growing bacterium that causes infection in fish and occasionally infects humans. Its infection in Zebrafish has been used as an important model system for teasing out the molecular events associated with TB pathogenesis [92]. Importantly, M. marinum could form biofilms within 14 days on a variety of abiotic surfaces. However, silicon surface yielded the highest levels of biofilm production. Hall-Stoodly also studied the ultra-structure of these biofilms and observed that the cording of mycobacterial cells progressed during the later phase of biofilm development [93]. This cording phenotype was suppressed by OADC supplement [93]. Lipooligosaccharides are cell wall components and play an important role in cell motility. M. marinum mutants incapable of forming lipooligosaccharides were defective in biofilm formation [94]. The role of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) in biofilm formation by M. marinum was analyzed by Mohandas et al. [95]. They demonstrated that genetic mutants defective in the PDIM/PGL biosynthetic pathway are attenuated for biofilm formation. These mutations also affect cell-surface properties but not sliding motility. These mutants also display increased antibiotic susceptibility [95]. However, the precise nature of the extracellular matrix of M. marinum biofilm remains to be deciphered.
BIOFILMS OF MYCOBACTERIUM TUBERCULOSIS
Mtb causes tuberculosis (TB) and is the leading cause of human deaths due to a single pathogen. The currently used treatment of TB involves usage of multiple drugs namely isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol for at least 6-9 months. Such lengthy treatment results in non-compliance and emergence of multi-drugresistant TB (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). However, it must be emphasized that such a combination can efficiently eliminate Mtb in culture in significantly shorter duration of time. These differences in the killing of Mtb cells under laboratory conditions and in infected hosts point towards a disconnect between the current understandings of Mtb physiology emerging from labs and its actual physiological state in humans. Current literature suggests two plausible hypotheses to explain the phenotypic drug tolerance displayed by the genetically drug susceptible Mtb inside the host. The first hypothesis suggests that Mtb senses the host environment and a fraction of Mtb cells transits into non-replicating persistent (NRP) state. During the NRP state, Mtb cells are believed to be metabolically quiescent, but generating sufficient energy to keep the membrane energized [96,97]. Since most of the antimycobacterial drugs (besides bedaquiline) target components of active cell growth, these metabolically quiescent cells are drug tolerant. It is well documented that the NRP state could be induced by hypoxia, nitric oxide and starvation [46,[98][99][100][101] and could be reversed by resumption of ambient oxygen and nutrients, declining nitric oxide levels [102]. Another hypothesis to explain the phenotypic drug tolerance is the formation of Mtb biofilms inside the host. Mtb forms biofilms harbouring drug-tolerant bacteria in vitro [10,103] however, the factors controlling the biofilm formation and the properties of the extracellular material are poorly known. The Mtb biofilm hypothesis arose from the work of Ojha et al., wherein Mtb pellicle growth was equated to the biofilms as pellicles contain selfproduced EPS which holds the cells together. Interestingly, this study also demonstrated that Mtb cells residing in the pellicle exhibit drug tolerance and harbour significantly higher number of persister Mtb cells. In the following section, we have described the most pertinent information regarding the Mtb biofilms.
Mtb biofilm models
Three models of Mtb biofilm formation have been proposed to study the factors regulating biofilm formation, the physiology of the resident bacteria, and the nature of biomaterial that holds these bacterial masses together. These models include pellicle biofilms formed at the liquidair interface of cultures, leukocyte lysate-induced biofilms, and thiol reductive stress-induced biofilms (depicted in the Figure 2).
Pellicle biofilms
Mtb has a propensity to grow as pellicles at the liquid-air interface in vitro cultures. The Mtb pellicles are formed through several stages of development over a period of 5-7 weeks [104]. Keto-mycolic acids play an important role in pellicle formation and has been proposed to be a structural component of ECM in pellicle biofilms [24]. The usage of this model of biofilms for the screening of antimycobacterial compounds resulted in the identification of the potential antimycobacterial drug candidate TCA1 [105]. Major advantage of the pellicle model of Mtb biofilms is that it allows for tracking of the different developmental stages during biofilm maturation. Given the simplicity of this model, it remains one of the most studied models of Mtb biofilm formation.
Leukocyte lysate-induced biofilms
Recently, Ackart et al. demonstrated that mycobacterial cells organize into substratum-attached drug tolerant microbial communities in culture media (RPMI 1640) supplemented with the lysate of leukocytes over a period of 7 days [106]. Importantly, the drug tolerant phenotype of these mycobacterial communities could be reverted through the disintegration of the communities, using DNase or Tween 80. These studies clearly depict that the drug tolerance of mycobacteria could be explained solely through their capability to form biofilms. It is important to note that inside caseous necrotic granulomas, extracellular mycobacteria are exposed to leucocyte lysate. Thus, this model, in a way mimics the in vivo environment. Intriguingly, this model was employed in the discovery of molecules capable of dispersing Mtb communities and, thus, aiding killing by first-line anti-TB drugs [107]. Based on these findings, the classical anti-TB therapy could be shortened in the future through the use of biofilm-dispersing adjunct therapy with similar anti-biofilm agents.
Thiol reductive stress-induced biofilms
Recently, another model of Mtb biofilms was established by Kumar and coworkers [108]. In this model, upon exposure to thiol-reductive stress (TRS), Mtb cells organize into microbial communities strongly attached to the substratum. The architecture of the microbial community depends on the prevailing culture conditions, i.e., submerged biofilms form in standing cultures while biofilms at the liquidair interface form in shake flask cultures. One of the biggest advantages of this model is that these biofilms require only 29-30 hrs for biofilm formation in comparison to the 7 days required for biofilms induced by host cell-derived complex macromolecules and ~35 days required for the development of pellicle biofilms [108]. Owing to the short duration required for biofilm formation in this model, the processes of bacterial cell attachment, cellular differentiation and the synthesis of EPS are amenable to tracking through microscopy, transcriptomic, proteomic and metabolomic profiling. Furthermore, these biofilms are strongly attached to the substratum similar to conventional biofilms, such that a simple treatment with the detergent Tween 80 or manual shaking does not disrupt these biofilms.
TRANSCRIPTIONAL CHANGES ASSOCIATED WITH BIOFILM FORMATION
In order to understand the tight regulation of genes at the transcriptional levels in TRS induced biofilm formation, Trivedi et al. analyzed the transcription kinetics, both when the Mtb cells were subjected to sub-optimal TRS as well as optimal TRS for biofilm formation. Upon milder treatment, the genes involved in protein synthesis are downregulated suggesting that the cells go into energy conserving mode. Genes responsible for iron uptake, aerobic respiration and lipid degradation are, however, upregulated. Upon treatment leading to high TRS, DNA replication, RNA biosynthesis as well as protein synthesis machinery come to a halt, as all the genes involved in these central processes are downregulated, which is again suggestive of the fact that the bacterial cells stop replicating. The formation of TRS-induced Mtb biofilms is associated with induction of sigE, sigB and whiB3 expression. However, the precise role of sigE, sigB and whiB3 in the formation of mycobacterial biofilms remains to be defined. Importantly, the type VII secretion system ESX-3 is upregulated in the Mtb biofilm which indicates a requirement of iron uptake in biofilm formation. In agreement furA, responsible for iron uptake, is highly upregulated in Mtb biofilms. Other upregulated genes involve those playing a role in cysteine and arginine metabolism. It must also be noted that the SenX3/RegX3 system which is induced upon mild TRS is also overexpressed in the Mtb cells residing in the biofilms [108]. The SenX3/RegX3 two component system is involved in growth in response to resumption of ambient oxygen levels [102]. These data suggest that oxygen is not evenly distributed in biofilms. Interestingly, PknA is also induced in the biofilm resident Mtb cells. Since PknA is involved in the maintenance of bacterial cell growth [109], these data suggest that some bacterial cells may be growing in the biofilm. However that the transcriptomic data obtained through microarray experiments do not reflect upon the differential expression of genes in the bacterial cells localized at different spatial location in the biofilms and hence such data should be analyzed with caution. We believe that development of tools having spatial resolution for monitoring gene expression could shed light on the differential expression in biofilms.
PHENOTYPIC DRUG TOLERANCE IN BIOFILMS
Phenotypic drug tolerance is the ability of genetically drug susceptible bacterial cells to evade killing by the antimicrobial agents [110]. It is worth noting that Mtb biofilms, developed using either of the three models, harbor phenotypically drug-tolerant Mtb cells. However, the mechanisms and molecular events that dictate the drug tolerance of the biofilm-resident cells have not been deciphered. Several mechanisms have been proposed to explain the phenotypic drug tolerance displayed by biofilm-resident bacteria [111], i.e., metabolic heterogeneity of biofilm residents, increased persister population, induction of reactive oxygen species scavengers, increased expression of efflux pumps, extracellular drug inactivation, protection by polysaccharides that acts as a physical barrier resulting in low penetrance of antibiotics, etc. Currently, we don't know which of these mechanism/s contribute more for the phenotypic drug tolerance but the study from Ackart et al. and Trivedi et al. suggest that the formation of bacterial communities is important for the observed phenotypic drug tolerance [106,108]. Understanding such mechanisms for Mtb will be helpful in designing new therapeutic agents that may reduce the duration of TB therapy. In this direction, Ojha et al. demonstrated that the pellicle biofilms of Mtb harbor a significantly larger number of persister cells compared to planktonic cells [104]. It is commonly believed that bacterial cells residing in the biofilms are metabolically quiescent and, thus, display drug tolerance. However, Trivedi et al. reported that the ATP/ADP, NADH/NAD + , NADPH/NADP + ratios of Mtb cells residing in TRS-induced biofilms were only slightly lower than those of planktonic bacteria [108]. However, such studies presume that the metabolic states of all residents of the biofilm are similar while bacterial cells in different regions of the biofilms have differences in terms of access to nutrients and thus are expected to have a different metabolic state. To dissect the metabolic heterogeneity of biofilms resident bacteria, new tools with spatial resolution (and preferably temporal resolution as well) are urgently required. Recently, genetically encoded biosensors for the measurement of the metabolic [43,44], energy [112] and redox state [113] of Mtb cells were developed. The application of these biosensors to understand the metabolic flux and redox state of the residents of Mtb biofilms represents a new research opportunity. Furthermore, knowing absolute concentration of the metabolites with spatiotemporal details will be desirable. It is worth noting that alterations of the redox homeostatic system that controls the cellular levels of FADH and NADH modulate biofilm formation [45]. Importantly, accumulation of intracellular thiol also facilitates the formation of Mtb biofilms [98]. Redox homeostasis is also known to play an important role in the persistence of Mtb [98,99]. Furthermore, Trivedi et al. performed transcriptome kinetic analysis during the biofilm formation. This analysis suggested that biofilm-resident bacterial cells utilize alternative metabolic pathways for the generation of energy [108]. However, we believe that such a transcriptomic analysis only provides a bird's eye view of the transcriptional changes associated with the biofilms. New tools having spatiotemporal resolution could provide details of the differential transcriptional regulation in different regions of the biofilms are needed to understand the transcriptional profile of biofilm resident bacteria. Further analysis of metabolic profiles, using other techniques such as quantitative mass spectroscopy, could help us to understand the metabolic networks that play critical roles in the biofilm formation, maintenance, and disruption. Once these alternative metabolic pathways are delineated, they could be targeted to kill the biofilm-resident mycobacterial cells.
Extracellular matrix of biofilms
The extracellular matrix consists of EPS, produced by cells present in the biofilm. Self-produced EPS is also considered to be the hallmark of a microbial biofilm [114]. Bacterial cells, along with nutrients and enzymes, are embedded in this EPS. EPS acts as the glue that keeps the bacterial cells together in microcolonies and attaches them to the substratum. EPS allows cell to cell interaction, communication and synergy within the microcolonies [114]. EPS represents a wide array of polymers, including proteins, nucleic acids, polysaccharides and lipids that serve as carbon and energy reserves. EPS could also be accumulated on the cell surface to protect the cells against the external environment. In most types of microbial biofilms, the EPS is composed of polysaccharides, structural proteins, extracellular DNA (eDNA), and lipids (as depicted in Figure 3). In the following section, we have described each of the components of the extracellular matrix of Mtb biofilms.
Lipids
One of the most studied models of Mtb biofilms are pellicle biofilms. Importantly, mycobacterial pellicles contain large quantities of free mycolic acids [25,104]. This free mycolic acid is produced by cleavage of trehalose dimycolate using a TDM specific esterase [26]. An inability in keto-mycolic acid biosynthesis or in cleaving TDM leads to the inability to form pellicle biofilms or retarded biofilm growth [24]. Besides mycolic acids, meromycolyl diacylglycerol [29,30] and glycopeptidolipids [23] also contribute to biofilm formation in mycobacteria. These studies suggested that mycobacterial biofilms are uniquely held together by a waxy EPS [115]. However, another hypothesis is that these cell wall components increase the cell-surface hydrophobicity to increase the cell-to-cell interaction and thus are required for biofilm formation, but are not the components of the EPS of Mtb biofilms.
Polysaccharides
The view that mycolic acids/lipids are primary components of Mtb biofilms was recently challenged by the observation that large quantities of polysaccharides are present in TRSinduced Mtb biofilms [108]. Importantly, by staining with carbohydrate-specific stains, Texas Red, lectin Concanavalin A, and Calcofluor-white, this group of researchers demonstrated that polysaccharides are present in the extracellular space of the biofilm. Using Nile red to stain lipids, they also suggested that lipids primarily localize on mycobacterial cells and not in the extracellular space of the Mtb biofilms. This observation is also suggestive of mycolic acids being a component of the cell wall rather than EPS. On the other hand, polysaccharides were detected at the base of the microcolony stalk and between the microcolonies. These findings suggested that polysaccharides are the major component of the EPS. Basaraba and coworkers have previously demonstrated the presence of complex polysaccharides in the leukocyte lysate-induced substratum-attached biofilms of Mtb [106]. The presence of extracellular polysaccharides in mycobacterial biofilms is also supported by studies in which abundant Texas Red staining was observed in the extracellular matrix of M. ulcerans biofilms [90] and the observation that aggregation of the mycobacterial cell is influenced by sugars [116]. Importantly, Kumar and coworkers have further characterized these polysaccharides by several biochemical methods and demonstrated that TRS-induced Mtb biofilms employ cellulose as a major structural component [108]. Intriguingly, cellulose was detected primarily in the spaces between the microcolonies and some cellulose was also detected at the stalks of the microcolonies. Cellulose is a polymer of glucose linked through β(1→4)-glycosidic bonds. It is hydrophilic, but water-insoluble and has strength comparable or more to that of steel [117]. Given the strength, cellulose has been shown to be an important component of several bacterial biofilms [114,118]. Importantly, cellulase can disintegrate TRS-induced Mtb biofilms, suggesting that cellulose is critical for Mtb biofilm formation. The role of cellulose as an integral component is also supported by the observation the Mtb genome encodes for cellulases [119,120] that could facilitate the dispersal of Mtb biofilms. The role of cellulase in mycobacterial pellicle biofilms was also analyzed by Wyk et al. [121], through over-expression of Rv0062 homologue MSMEG_6752 in Msm. They observed that upon overexpression of MSMEG_6752, Msm cells are not able to form pellicle biofilms. Furthermore, treatment of Mtb biofilms with cellulase MSMEG_6752 leads to disruption of the biofilms and release of glucose subunits (28168306). However, it remains to be determined whether cellulose is present in other models of Mtb biofilms, namely leukocyte-induced biofilms and pellicle biofilms. Additionally, the presence of cellulose should be analyzed in the biofilms of other mycobacterial species as besides Mtb and Msm. Another important type of biofilm that has not been studied in mycobacteria is the macrocolony. Cellulose has been shown to play a critical role in the architecture of the macrocolony morphology for Escherichia coli [122], but the role of cellulose in the macrocolony morphology of mycobacterium species remains to be established. Cellulose is currently detected in biofilms using dyes such as Congo red and Calcofluor-White. Although these methods are suitable, but more specific biologically encoded sensors of cellulose (such as a cellulose-binding domain coupled with a fluorescent tag) could help in the analysis of the architectural role of cellulose in biofilms. The demonstration of cellulose as a critical component of Mtb biofilms has opened new avenues of research. The genetic pathway used by Mtb for the synthesis of cellulose and their regulations are yet to be identified. Cellulose is synthesized in bacterial cells using a multiprotein complex called cellulose synthase with the core catalytic activity residing in the BcsA and BcsB proteins [123,124]. Although a number of mycobacterial species, such as M. neoaurum (Uniprot ID -A0A024QK68), M. cosmeticum (Uniprot ID -W9B851), etc., contain genes encoding for putative components of the cellulose synthase, but the Mtb genome does not seem to encode either BcsA or BcsB. Interestingly, the Mtb genome encodes few cellulases [120], which could be expressed and secreted in a spatiotemporal manner to facilitate the regulated dispersal of biofilm residents. It is noteworthy that the Mtb genome encodes for a large number of glycosyltransferases [125], including many uncharacterized ones that may function as non-canonical cellulose synthase. Previously a number of approaches besides the sequence similaritybased method were utilized for identification of components of the cellulose synthase in other bacterial species. These include the use of activity-based enrichment and purification of the cellulose synthase complex [126] using the photo-affinity probe 5-azido-UDP-Glc [127], proteinprotein interaction-based screening [128], and transposon insertion mutagenesis screens [129]. We believe that such approaches could lead to the identification of the cellulose synthase of Mtb as well. Identification of the genetic pathway/s involved in cellulose biosynthesis could further facilitate the analysis of the role of Mtb biofilms in TB pathogenesis. Interestingly, cellulose synthesis is regulated posttranslationally via cyclic-di-GMP (c-di-GMP) [130]. C-di-GMP is synthesized by the diguanylate cyclase (dgc) enzymes with a characteristic GGDEF motif and is degraded by phosphodiesterase (pde) enzymes with an EAL or HD-GYP motif [131]. Mtb possesses both a dgc (Rv1354c) and pde (Rv1357c). Interestingly, Rv1354c has the GAF, GGDEF, and EAL domains organized in tandem and are able to synthesize and degrade c-di-GMP, whereas Rv1357c contains only the EAL domain and degrades c-di-GMP to pGpG [132]. Intriguingly, the pde deletion mutant of M. bovis BCG forms highly matured pellicle biofilms and colonies with higher levels of cording. In line with these findings, the pde deletion mutant survived better in immunocompetent mice [133]. On the contrary, a pde mutant of Mtb has decreased survival in THP-1 cells and in a mouse model [134]. Despite these exciting studies, the role of c-di-GMP in the regulation of cellulose synthesis and in vivo biofilm formation still needs to be explored. Importantly, TRSinduced biofilms seem to contain other polysaccharides, in addition to cellulose [108]. Further research is required for the isolation, purification, identification, and characterization of these polysaccharides. We believe that our understanding of mycobacterial biofilms will rapidly expand with the characterization of these polysaccharides. Additionally, the identification of the genetic pathways that contribute to polysaccharide synthesis and their regulation in vitro and in vivo represent exciting research opportunities.
Proteins
A number of structural proteins play an important role in microbial biofilms. Structural proteins are known to facilitate the interaction of the bacterial cells with the substratum, the EPS of the biofilm, and with other bacterial cells. Mtb cells produce a number of adhesins such as fibronectin-binding proteins, heparin-binding hemagglutinin adhesin (HBHA), and pili. A few of these proteins have been shown to play important roles in biofilm formation or aggregation of Mtb cells. HBHA is a bacterial cell surfaceassociated protein that can also be secreted. Importantly, HBHA can induce auto-aggregation of Mtb cells at a concentration of 0.5 µg/ml [135]. Mtb pili encoded by Rv3312A are involved in the formation of pellicle biofilms of Mtb [136]. TRS-induced Mtb biofilms can be disintegrated by the use of proteases [108] suggesting that some yetunidentified structural proteins play a critical role in maintaining biofilm integrity. Efforts towards the identification of such proteins should be made, as this holds the key to our capability to disrupt Mtb biofilms. The presence of HBHA or pili remains to be analyzed in TRS-induced Mtb biofilms. Interestingly, pili also play a critical role in the architecture of E. coli colonies [137]. The role of pili in the architecture and microanatomy of the Mtb colony remains to be analyzed. Besides containing structural proteins, EPS also has a number of enzymatic activities and quorumsensing molecules to enable communication among the resident cells. A number of reactive oxygen species (ROS)detoxifying enzymes are secreted by biofilm resident cells. These enzymes protect the bacterial cells against ROS generators. However, the enzymes and quorum-sensing molecules of Mtb biofilms have not been characterized and represent an important research prospect.
eDNA
Besides polysaccharides, eDNA is also known to be an important structural component of microbial biofilms [114]. As described earlier in this review, eDNA is found in the biofilms of M. avium [67], M. abscessus, and M. chelonae [69]. In context of Mtb biofilms, the presence of eDNA was reported in leukocyte lysate-induced and TRS-induced biofilms of Mtb [106,108]. Degradation of eDNA using Dnase treatment led to the disruption of leukocyte lysate-induced Mtb biofilms but not of TRS-induced biofilms. These observations suggest different structural roles in the two models of Mtb biofilms. In TRS-induced biofilms, eDNA was observed at the stalk of the microcolonies. These observations suggest that cellulose, along with eDNA, could be involved in attaching the microcolonies to the substratum. In a number of biofilms, the eDNA originates through the altruistic self-killing of a few resident cells [138] or is actively released through membrane vesicles [139]. Currently, we do not know how the eDNA originates in Mtb biofilms and identification of these pathways will improve our understanding of the physiological functions of eDNA. It is important to mention that Mtb aggregates in response to interferon gamma [140]. Mtb is known to produce extracellular vesicles [141] and possesses 88 toxin-antitoxin systems [142], but their role in Mtb biofilms remains unknown. It will be interesting to analyze whether Mtb cells residing in biofilms utilize extracellular vesicles for secreting DNA. The study of the spatiotemporal expression of toxin-antitoxin modules would help us to understand their role in biofilm maintenance. In our view, taking advantage of the models of Mtb biofilms, further research should be focused on understanding the different steps of biofilm formation and maturation. It is plausible that, in response to yet-unidentified signaling molecules, Mtb cells start producing adhesins (or alternatively modulate the cell surface to increase surface hydrophobicity) that facilitate the attachment of bacterial cells to the substratum. After attachment of the Mtb cells to the substratum, more cells start adhering to the surface and start producing polysaccharides along with structural proteins. Other components such as DNA or enzymes are contributed through regulated lysis of a few cells or localized production of extracellular vesicles. The microcolony thus established grows bigger through recruitment of more cells or cell division of resident bacteria. Localized production of EPS-degrading enzymes such as cellulase, protease, and Dnase facilitate the dispersal of Mtb biofilms.
In summary, research on Mtb biofilms has gained momentum with the establishment of three different models of biofilm formation. Although cellulose has been characterized as a key constituent of Mtb biofilms, it is important that the nature of the EPS of Mtb biofilms is further characterized. The demonstration of the presence of Mtb biofilms in animals/humans will further advance research on Mtb biofilms; however, this will require identification of more specific biomarkers for Mtb biofilms. With the deeper understanding of the nature of Mtb biofilms, new interventions in therapy and diagnosis of TB can be facilitated.
IN VIVO PERSPECTIVE OF MTB BIOFILMS
A hallmark of Mtb infection in humans is the presence of granulomas with a caseating necrotic core at the center. Interestingly, several extracellular Mtb cells were detected in the necrotic core and in the acellular rim of necrotic lesions [143]. These bacteria were present either as single cells or as cluster of bacteria encapsulated within a matrix of extracellular polymers. A fraction of bacteria residing in clusters at the necrotic core and acellular rim could survive treatment of guinea pigs with antimycobacterial drugs [143]. Given the critical observations it is tempting to speculate that Mtb may form biofilms in vivo. However, to conclusively demonstrate that these clusters of extracellular Mtb cells are indeed Mtb biofilms, the presence of bacteria-synthesized ECM surrounding these clusters is required. We have recently proposed that cellulose could be used as a marker for the detection of Mtb biofilms in infected animals and humans [108,144]. Additionally, since humans do not produce cellulose, detection of cellulose in human or animal tissues surrounding mycobacterial cells could indicate the presence of Mtb biofilms in vivo. We believe that such a finding would be a major step forward in our understanding of mycobacterial physiology inside the host. It must be noted here that previous studies have also identified short-chain free mycolic acid variants as markers of Mtb pellicle biofilms; thus, the presence of these variants of mycolic acid in human or animal tissue around the Mtb cells could be suggestive of the existence of the Mtb biofilm in vivo. We presume that the demonstration of the presence of Mtb biofilms in animals or humans could be a milestone in the field.
CONCLUDING REMARKS
A number of mycobacterial species such as Mtb and a few NTMs cause chronic infections, and their treatment requires the usage of multiple anti-mycobacterials for a long period of time. Such drug-tolerant chronic infections are often associated with in vivo biofilms. Emerging evidence suggests that few mycobacterial species make in vivo biofilms, thus understanding the bacterial physiology of mycobacteria residing in the biofilms and the nature of ECM is key to our ability to treat such infections. Recent studies have also suggested that free mycolic acids, glycopeptidolipids and other cell wall components could alter the cell-to cell interaction to influence biofilm formation. A few studies have also suggested a structural role of polysaccharides and extracellular DNA in maintaining the structural integrity of the mycobacterial biofilms. Importantly, cellulose has been proposed as the marker of Mtb biofilms. A few studies have also suggested that reductive stress (due to accumulation of reduced counterparts of a redox couple such as NADH or thiols) could initiate biofilm formation. In the view of these findings, further research exploring the genetic pathway used by mycobacterial species to form biofilms in vitro and in vivo and its regulation could be important contributions in the field. | v3-fos-license |
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} | pes2o/s2orc | An extended channel length micro fl ow electrolysis cell for convenient laboratory synthesis
A spiral, extended channel length micro fl ow cell designed for routine and convenient application in an organic synthesis laboratory is described. The performance of the cell is demonstrated using two syntheses and it is shown that high selectivities and high conversions in a single pass can be achieved as well as the formation of products at a rate of up to 25 mmol/h (~3 g/h). The cell is also well suited to carrying out the optimisation of reaction conditions with electrolyses completed on a timescale of minutes.
Introduction
Despite a history going back N 150 years and some examples of industrial applications, organic electrosynthesis has never established a position in routine laboratory synthesis. One reason is the reliance on beaker cells and H-cells when slow conversions and poor/ill-defined conditions hamper yields and reproducibility [1]. There is a clear need for cells for convenient and straightforward use by synthetic organic chemists. In designing and operating such cells, the emphasis is on convenience of use, high yield of product and ease of product isolation through a high conversion of reactant to product, the use of no/low electrolyte concentration and clean counter electrode chemistry. On the scale of laboratory synthesis, current efficiency is only important as far as competing electrode reactions can degrade product purity. Energy consumption only becomes important if and when the laboratory synthesis is to be scaled for manufacture.
One approach to meeting this need employs microflow electrolysis cells with an extended channel length [1][2][3][4][5]. They have been demonstrated to make possible • very high conversions in a single pass • operation with poorly conducting reaction media • rapid electrolysis with residence time of reactant in the cell restricted to minutes • selective chemical change for a number of synthetic reactions The present paper describes a small cell intended for use in a synthetic organic chemistry laboratory. It is an undivided parallel plate reactor where a spacer is employed to achieve a spiral channel with extended length and narrow interelectrode gap. It is designed for studies relating to optimising reaction conditions and for synthesising product on a scale of 100 mg-10 g and is a smaller version of a cell described previously [5]. A variety of electrosynthetic microflow reactors have been reported in the literature [7][8][9][10][11][12][13][14]. The majority of these cells contain a short path lengths (b10 cm), necessitating low flow rates of electrolyte to achieve high conversion in a single pass, which consequently restricts the rate of product formation. The performance of the cell, reported here, is demonstrated using two reactions: the methoxylation of N-formylpyrrolidine (Scheme 1), a reaction used to test performance in earlier microflow cells [2][3][4][5]; and the cleavage of the 4-methoxybenzyl protecting group from 3-phenyl-1-propanol (Scheme 2). This reaction will be reported in more detail in a further publication [15]. In both syntheses, the counter electrode reaction is the reduction of methanol to hydrogen and methoxide, the latter minimising the build-up of protons in the electrolyte along the cell channel.
Electrolysis cell
The cell was manufactured by Cambridge Reactor Design and is now available for purchase as the Ammonite 8 electrolysis cell [6]. The anode was a disc of carbon-filled polyvinylidenefluoride (C/PVDF), type Sigracet BMA5, supplied by Wilheim Eisenhuth GmbH, Germanydiameter 85 mm and thickness 5 mm. The cathode was a circular 316 L
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Electrochemistry Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e l e c o m stainless steel plate (Castle Metals Ltd., UK), diameter 85 mm and thickness 5.0 mm, with a spiral groove, depth 0.5 mm machined into it. The gasket/spacer was cut to fit into the groove and was fabricated from a 1.0 mm thick sheet of KALREZ perfluoroelastomer (James Walker LTD, UK). The C/PDVF composite electrode had a copper disc backing plate to improve the potential distribution along the electrolyte channel. The cell was compressed between an aluminium base plate and a Perspex top via a central bolt and 6 bolts around the perimeter. The cell is rapid to dismantle, clean and reassemble. The solution entered and exited the cell through steel tubing (1/8th inch diameter) to which connection could be made via standard fittings. There were separate reservoirs for reactant and product and solution was pumped with an Ismatec Reglo digital peristaltic pump with flow rates 0.25-3.0 cm 3 min −1 . Electrolyses were controlled with a Rapid Electronics switching mode power supply (85-1903).
Chemicals and analysis
Methanol (Fisher Scientific, HPLC grade) and N-formylpyrrolidine (Sigma-Aldrich, N 98%+) were used without purification. Tetraethylammonium tetrafluoroborate was recrystallized from hot methanol and dried at 363 K in a vacuum oven (~10 mbar) for 48 h. The protected alcohol was prepared by the method of Kern et al. [16].
Conversion and selectivity were determined by gas chromatography using a Shimadzu GC-2014 equipped with an autosampler, FID detector and Agilent Technologies HP5 column. A calibration curve was obtained for starting materials and products of both reactions, by testing a range of solutions of known concentration. Comparison of the unknown reaction solutions to the calibration curve allowed for conversion, yield and selectivity to be calculated for each reaction.
Cell design
The cell is designed to have (a) an extended length electrolysis channel (1 m) in order to achieve a high conversion of reactant to product in a single pass at flow rates compatible with carrying out reactions at a rate of g/h (b) a narrow interelectrode gap to permit use of poorly conducting media including those with low/no electrolyte (c) a short residence time of reactant/product within the cell to minimise the competition of unwanted homogeneous reactions. It is undivided but the absence of a separator is not necessarily a disadvantage. Indeed, it can be turned to advantage since the counter electrode reaction can be used to maintain a constant reaction environment along the channel, e.g. when carrying out oxidations, the cathode can be used to stop the build up of protons along the channel by generating an equivalent quantity of base. The cell is conveniently controlled with a constant cell current. For full conversion, this cell current is calculated using Faraday's laws for the concentration of reactant and flow rate of the solution (provided the desired reaction has a high enough ratepreferably mass transfer controlled [1][2][3][4][5]). It should, however, be recognised that the cell current is, in fact, the integral of the local currents along the channel as they drop from a high value at the channel entry towards zero at the exit (as the reactant concentration drops with conversion) [1][2][3][4][5].
The electrolysis cell is a parallel plate reactor based on two circular plate electrodes, diameter 85 mm, but with a spiral electrolyte channel between them. The spiral electrolyte channel permits an extended channel length within a device with small dimensions and a smooth electrolyte flow regime along the channel. The spiral electrolyte channel is created with a spiral spacer (see Fig. 1) held rigidly in place within a spiral groove machined into one of the electrodes (see Fig. 1). The other electrode is a smooth plate. The interelectrode gap is fixed through the depth of the groove and the thickness of the polymer spacer. In the present cell, the electrodes are 316L stainless steel and C/PDVF composite material and the spacer fabricated from KALREZ perfluoroelastomer; this polymer was selected because of its resistance to oxidation/reduction and its stability in many organic media. The C/ PDVF anode has a copper disc backing plate to improve the uniformity of the potential distribution across its surfaceideally the potential should be constant along the cell channel. The machining of the spacer and the groove leads to an electrolyte channel 1000 mm in length and 2 mm in width while the interelectrode gap is 0.5 mm. The volume of the cell is therefore 1.0 cm 3 and when assembled (see Fig. 2) has the dimensions 110 mm (diameter) × 60 mm (height). Materials and suppliers as well as other details are given in the Experimental section.
Cell performance
The performance of the cell was established using two syntheses, the methoxylation of N-formylpyrrolidine, reaction (scheme 1), and the cleavage of the 4-methoxybenzyl protecting group from 3-phenyl-1propanol, reaction (scheme 2). Table 1 reports the results from a series of electrolyses with Nformylpyrrolidine (Scheme 1). The initial electrolysis were carried out with solutions containing 0.10 M N-formylpyrrolidine and 0.05 M tetrethylammonium tetrafluoroborate and using a cell current~20% above the theoretical optimum value calculated from the equation Scheme 2. The electrochemical cleavage of 4-methoxybenzyl to give 3-phenyl-1-propanol. It can be seen that, indeed, a high conversion in a single pass with a good selectivity can be achieved over a range of conditions. At the slower flow rates, the methoxylation reaction goes close to completion while there is a small decline as the flow rate is increased. Using a higher cell current again leads to a higher conversion. The fractional reaction selectivity is always well above 0.8 and can reach 0.95. At the slowest flow rate the product formation rate is 0.17 g h −1 and this can be increased using a higher flow rate and/or concentration of reactant and several grams per hour is readily achieved. A five-fold increase in reactant concentration necessitates a five-fold increase in cell current but the conversion and selectivity are hardly affected. The increase in reactant concentration allows a large increase in product formation rate. Of course, the amount of product can also be increased using a larger volume of reactant solution and a longer electrolysis time. It should be noted that at the highest flow rate, the residence time of the reactant in the cell is only 20 s. Electrolyses are also possible with lower electrolyte concentration. Table 2 reports data from experiments where the reaction of interest is the removal of a protecting group from an alcohol [15], a reaction where further oxidation of products could lead to loss of selectivity (Scheme 2). Again, the solution was usually 0.10 M reactant and 0.05 M electrolyte and a cell current above the theoretical optimum value used to enhance the conversion. The fractional conversions and fractional selectivities are always high. With increased flow rates, however, there is a slight decay in fractional selectivity and this probably arises from overoxidation with the higher cell currents needed for full conversion. Hence, this synthesis was generally carried out with a lower flow rate.
Both syntheses remain possible with a low electrolyte concentration with no significant loss in performance. For example, the cleavage of the protecting group was repeated using a flow rate of 0.25 cm 3 min −1 and an electrolyte concentration of 5.0 mM; the fractional conversion was 0.99 with a fractional selectivity of 0.83. The drawback is the necessary increase in applied cell voltage,~5.5 V with 5 mM electrolyte compared to~3.5 V with 50 mM electrolyte. This is not a problem for these synthesis and the conditions used. Potentially, however, higher applied voltages can lead to more rapid Joule heating of the cell, changing the reaction dynamics and possibly a loss in selectivity. If necessary, with the cell described, the temperature can be controlled by contact of the base of the reactor with laboratory cooling heating/cooling equipment regulated via a temperature probe fitted into the stainless steel electrode. It should also be noted that in both syntheses the tetrethylammonium tetrafluoroborate is easily recovered by precipitation from the crude reaction mixture, and can be reused many times.
Conclusions
The cell with an extended channel length achieved using a spiral design is shown to be a convenient laboratory tool for electrosynthesis. It allows very high conversion of reactant to product with good selectivity in a single pass of solution through the cell using flow rates that are compatible with the formation of product at a rate of g/h. With complete electrolyses possible in a few minutes, the cell is ideal for exploring the influence of reaction conditions on selectivity and yield. Indeed, several reaction conditions can be investigated with a single filling of the reactant reservoir. Table 2 Performance of the cell for the cleavage of the 4-methoxybenzyl protecting group from 3phenyl-1-propanol (Reaction 2). MeOH/Et 4 NBF 4 (0.05 M). All reactions were performed on 5 cm 3 samples (electrolysis time 10 or 20 min) except the 1.0 cm 3 min −1 experiment when the sample was 10 cm 3 (electrolysis time 10 min). Product analysed by gas chromatography. | v3-fos-license |
2021-05-13T00:04:17.865Z | 2020-12-21T00:00:00.000 | 234408804 | {
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} | pes2o/s2orc | A novel stability indicating RP-HPLC Method for the estimation of ulipristal - A Selective Progesterone Receptor Modulator
The present research article described the novel HPLC technique that was developed for the estimation of ulipristal in an API and tablet formulation. Symmetry, C18, 250mm x 4.6mm .i.d., 5 (cid:22) m Particle size, column with 0.4 ml/min (cid:976)low rate, was used for the chromatographic separation. Phosphate buffer, pH 4.6 and methanol as a mobile phase (20:80) with a (cid:976)low rate of 1 mL/min and eluent at 304 nm was monitored for the mobile phase. In com-pliance with ICH guidelines, the method was validated. Ulipristal was eluted in this technique with a retention time of 1.516 minutes. The limit of detection was 0.075 (cid:22) g/ml and the limit of quanti(cid:976)ication was found 0.25 (cid:22) g/mL. The calibration curve plot was found linear over the concentration ranges 5-150 (cid:22) g/mL, with the regression coef(cid:976)icient (R 2 ) of 0.990. The % assay of the marketed dosage form was found at 97.40 %, which is within the acceptance level. The present method of ulipristal force degradation study was performed as per the guidelines. The results of the force degradation study show the high-est 11.39% degradation in an acidic stressed condition, in comparison to alkaline, peroxide, thermal and photolytic stressed conditions. The experiential evidence of all the study results revealed the suitability of the quanti(cid:976)ication of ulipristal in API and tablet formulation routinely.
INTRODUCTION
Ulipristal, which is used as an emergency contraceptive, is a selective progesterone receptor modulator. As a contraceptive drug, a progestin and a modulator of progesterone receptors, it has a role. It is a steroid of 3-oxo-Delta (4), a steroid ester, an acetate ester, a steroid of 20-oxo, and a tertiary amino compound (Marci et al., 2014) (Figure 1). Ulipristal binds to the progesterone receptor (PR), thus inhibiting gene expression mediated by PR and intervening in the reproductive system with progesterone activity. This agent can also suppress the growth of uterine leiomyomatosis as a result (Puchar et al., 2015). Ulipristal is also used in the treatment of uterine ibroids, also known as myomas, which are non-cancerous womb growths (uterus). Sometimes they can cause heavy or painful cycles, swelling of the tummy and urinary problems (Rabe et al., 2013;Lyseng-Williamson and Croxtall, 2012). Ulipristal is considered an important molecule for the treatment of the abovementioned conditions and not included in any pharmacopoeia. Therefore the determination of ulipristal with a suitable analytical method is important.
The literature review on the available HPLC method for ulipristal reveals that there is an existence of two reported methods. The reported methods have their own limitations as follows, In one reported method (Shi et al., 2014), the reported retention time of 3.04 minute with a narrow linearity range of 20-50µg/mL and used acetonitrile (80%) as a part of the mobile phase, which is generally considered as a costly solvent. In another reported method (Rao et al., 2019) also utilized costly acetonitrile and orthophosphoric acid as a mobile phase. Whereas they also utilized acetonitrile and water as a diluent. Overall this method can be considered as cumbersome. Therefore keeping all facts in mind the efforts were taken to eradicate all the above disadvantages from the reported methods and to develop a reliable, fast method for the estimation of ulipristal using HPLC technique and validate the method as per the requirements of ICH guidelines Harikrishnan et al., 2020).
In this article, the development of a novel method has been represented with several validation parameters and can be utilized for the routine estimation of ulipristal in bulk as well in the marketed dosage form.
Chemicals and Reagents
The present working standards Ulipristal (99.98%) was procured from the Akumentis Healthcare Ltd, Thane, Maharashtra, India. The tablets (Fibristal tablet, manufactured by Akumentis Healthcare Ltd) were purchased from the local market of Hyderabad, India. All required chemicals and reagents were purchased from Finer chemical Ltd, Fisher Scienti ic and Merck.
Standard ulipristal preparation for the analysis
50 mg of ulipristal standard was transferred into 50 ml volumetric lask, dissolved & makeup to volume with the mobile phase. Further dilution was done by transferring 0.2 ml of the above solution into a 10ml volumetric lask and makeup to volume with mobile phase to achieve the inal concentrations of 20 µg/ml.
Preparation of 0.05M Phosphate buffer Solution
About 6.8043 grams of Potassium dihydrogen orthophosphate was weighed and transferred into a 1000 ml beaker, dissolved and diluted to 1000ml with HPLC water. The pH was adjusted to 4.6 with orthophosphoric acid.
Preparation of Mobile Phase
The mobile phase, which is utilised for the analysis of ulipristal, is a mixture of phosphate buffer (pH 4.6 with orthophosphoric acid) and methanol in the volume ratio of 20:80. 800 mL (80%) of Methanol and 200 mL (20%) of the phosphate buffer was well combined and degasi ied for 15 minutes in an ultrasonic water bath. The solution was iltered under vacuum iltration through a 0.45 µm ilter.
Assay of ulipristal marketed formulation
Twenty Fibristal tablets (each tablet 5mg ulipristal) from Akumentis Healthcare Ltd, Thane, Maharashtra, India, were measured correctly and powdered inely in a glass mortar. A 100 ml standard lask was transferred to a weight equal to 100 mg ulipristal. For a duration of 10 min, 10 ml of methanol was added and gently swirled. The clear solution was then iltered and transferred through a Whatmann No 1 ilter paper into the lask, and up to 100 ml of diluent (Methanol: phosphate buffer, 80:20) volume was formed. There was a concentration of 1mg/ml in the resulting solution. To obtain the inal concentration of 100 µg/ml of ulipristal, precisely pipette 1 ml of the above solution into a 100 ml regular lask and make it up to a volume of diluent. Further pipette out 1ml from the 100µg/ ml solution and transferred into 10 ml standard lask and the volume was made up to the mark using a diluent to achieve the concentration of 10µg/ml of ulipristal. The prepared latest solution was injected into the chromatographic system. The chromatogram was recorded.
System suitability
It was conducted (Mondal et al., 2013) to legitimize whether the analytical framework is operating correctly during the method production of ulipristal. The injection of six replicates of the regular solution of ulipristal was performed. The percent RSD of a set of optimized parameters was measured, such as peak area, theoretical plates, retention time and asymmetric factor.
Speci icity
Using 20 mg of a placebo, which is equal to one ulipristal tablet dissolved in 100 ml of the mobile phase. The interference of the placebo test of the ulipristal sample solution was conducted. Then the solution (placebo) was handled as a normal solution. To determine the potential interfering peaks, the solution was injected into the chromatographic device.
Accuracy
Recovery analysis was performed (Mondal et al., 2019) at different levels (80 %, 100 % and 120 %) of pure ulipristal to justify the accuracy of the established method. In order to achieve the different quantities, different amounts of standard ulipristal were incorporated to a set concentration in the ulipristal tablet sample solution. This study was performed three times, and the percentage of recovery and the percentage of mean recovery was calculated. The percent recovery should be between 98.0 per cent to 102.0 per cent for each point.
Precision
This study of the present developed method of ulipristal was studied by determining the ulipristal sample solution. It was evaluated by analyzing the six ulipristal sample solutions in triplicate (n=6). Intraday and inter-day precision were resolute by analysing six times in three different concentrations of ulipristal, i.e 20 µg/ml, 40 µg/ml and 60 µg/ml.
The chromatograms were recorded. The peak area and the retention time of the ulipristal was determined and the relative standard deviation (RSD) was calculated. The percent RSD should not be more than 2 percent for the region of six standard injections performance.
Linearity
An aliquot from this solution was diluted with mobile phase in ive different concentrations to 5-150 µg/ml of ulipristal to execute the standard linearity solution of ulipristal as stated earlier. The calibration curve was plotted for the ulipristal was subjected to regression analysis. In the de ined range, the relationship between the concentration and peak area should be linear and the coef icient of correlation should not be less than 0.99.
Limit of detection (LOD)
Ulipristal solution with a inal concentration of 0.075 µg/ml was prepared from the stock solution for this analysis. The prepared solution was iltered and injected into the chromatographic device. The signal to noise (S/N ratio) value for the LOD solution must be 3. The LOD solution of ulipristal was prepared and injected three times and the area measured for all three HPLC injections was measured. The percent RSD for the six replicate injections region was found to be within limits de ined.
Limit of quantitation (LOQ)
This study involved the preparation of 0.25 µg/ml of ulipristal concentration that was prepared from the series of dilution from the standard ulipristal solution. The prepared sample was transferred into the chromatographic device and injected. The signal to noise (S/N ratio) value for the LOQ solution must be 10. The LOQ solutions were prepared for three injections and calculated the area for all three HPLC injections. The percent RSD for the six replicate injections region was found to be within limits de ined.
Robustness
This feature is used to determine the ability of the developed method to remain unaffected by small, deliberate adjustments in the method's parameters and provides a measure of its reliability during regular use. The low rate, the detection wavelength, and the composition of the organic solvent were deliberately modi ied from the normal chromatographic conditions by injecting the ulipristal working standard solution into the HPLC system.
Force degradation study of ulipristal
Stress testing was carried out in the environmental test chamber (Acamus Technologies, India) at 600C and 75 percent relative humidity (Shobharani et al., 2014), as prescribed by ICH stress conditions such as acidic, oxidative, alkaline, photolytic and thermal stresses.
Acid hydrolysis
Forced degradation in acidic media was achieved by adding 1 ml of 1 M HCl to 1 ml of ulipristal stock solution and the mixture was heated for approximately 2 hours at 80 C and the solution was neutralized by adding 1 M NaOH and held sideways for 24 hours and injected.
Alkaline hydrolysis
Forced degradation of basic media was achieved by adding 1 ml 1 M NaOH to 1 mL of stock solution and the mixture is heated for about 2 hours at 80 • C and the solution is neutralized by including 1 M HCl and held sideways for 24 hours. It is injected with the prepared solution and chromatograms are recorded.
Oxidative Degradation
In a clean and dry 100 ml volumetric lask, 10 mg of pure ulipristal was accurately weighed. To make it soluble, 30 ml of 3% H 2 O 2 and a little methanol were added to it and then held as such for 24 hrs in darkness.
A inal volume of up to 100 ml was produced. Using water to prepare 10 µg/ml of solution and inally dilute it to 10 mL. The above sample was injected against a mobile phase blank in the HPLC method and a chromatogram was obtained.
Photolytic degradation
In a clean and dry Petri dish, about 10 mg of pure ulipristal was taken. It was held without interruption in a UV cabinet at a wavelength of 254 nm for 24 hours. 1 mg of the UV-exposed drug was accurately weighed and transferred to a clean and dry 10 ml volumetric lask. The UV light exposed analyte was irst dissolved in methanol and made up to the mobile phase mark and eventually diluted to 10 mL to prepare a solution of 10 µg/ml. This solution was injected into the HPLC device against a mobile phase blank and obtained a chromatogram.
Thermal degradation
10 mg of pure analyte was correctly measured and transferred to a clean and dry round bottom lask. There was 30 ml of HPLC water applied to it. It was then re luxed uninterruptedly in a water bath at 60 • C for 6 hrs. The mixture of the medication and water was allowed to cool to room temperature after the re lux was over. Up to 100 ml of the inal volume was produced and inally diluted to 10 mL to prepare 10 µg/ml of solution. A blank solution was inserted into the HPLC device against it.
Method development
In the API and tablet formulation of ulipristal, different HPLC chromatographic conditions were hoped to obtain an optimized method for ulipristal estimation. During the original trials, many parameters such as column type, mobile phase composition, mobile phase pH and diluents were ranged. In order to achieve the desired composition of the mobile process for system optimization, different proportions of solvents, the buffer, have been tested. Finally, with the mobile phase phosphate buffer, pH 4.6 and methanol as a mobile phase with a volume ration of 20:80 with 1 mL/min, low rate. Ulipristal was eluted with high-quality peak shape and low retention time. The retention time of 1.516 minutes was observed with the detection at 304 nm for the ulipristal. The developed method was validated in accordance with ICH guidelines. Figure 2 displays an optimized chromatogram.
Method validation
The developed, optimized condition was utilized for the study of validation parameters for ulipristal. The assay result of the ulipristal marketed tablet dosage form of ulipristal shows the percent purity of 97.40%, the obtained chromatogram was depicted in Figure 3 and the result was shown in Table 1. A percentage assay results in the marketed ulipristal The results of the speci icity study, it was observed that no peaks of excipients were detected at the retention time of the ulipristal and supported the method's speci icity. A system suitability study was performed to con irm the satisfactory operation of the equipment used for analytical measurements. The percentage RSD of several criteria have been considered such as peak area, tailing factors, theoretical plates and retention time, and found 0.08, 0.51, 3.80 and 1.33. The results of accuracy as a mean % recovery was found 98.84, and the % RSD was not more than 2 % as shown in Table 2.
The percent recovery was found as a result of accuracy within the appropriate range, i.e. within 95-105 percent as shown in the outcome, supporting the accuracy of the established process. The % RSD of the intra and inter-day precision study was found 0.47 and 0.63. The results of the precision study were also revealed in the table of validation parameters and the results of the precision analysis showed that the new methodology was found to be speci ic.
The linearity study was performed in the range of concentrations 5-150 µg/ml, and the correlation coef icient was obtained is 0.990 for the ulipristal.
The linearity overlay chromatogram, as shown in Figure 4. The obtained linear range indicated the wide determination region of the ulipristal with the required precision in the linearity analysis of the developed method, and the correlation coef icient for the ulipristal was found to be 0.990, indicating its de ined linearity.
Robustness study of the developed ulipristal HPLC method was carried out by changing the parameters from the optimized chromatographic conditions such as changes in mobile phase composition (±2%), detection wavelength (±2 nm), changes in low rate (±0.1ml/min). The % RSD of the tailing factor, which was considered as a parameter was established at 0.43 as shown in the validation parameters table. The % RSD of the tailing factor was found less than 2, which authorize the robustness of the developed method since there was no signi icant changes were observed on the deliberate changes in the optimized parameters.
The sensitivity of the method developed has been demonstrated by the detection limit and the quantitation limit. Concentrations of detection and quantitation found for ulipristal at a very low limit. The detection and quantitation limit of ulipristal were found to 0.075 µg/ml and 0.25 µg/ml, respectively. Degradation studies of ulipristal were performed under the in luence of several stressed conditions such as acid, alkali, oxidation, thermal, photolytic conditions.
Degradation was found in all stressed condition except photolytic in luence. The acidic stressed condition shows the degradation of 11.39%; alkaline stress condition shows 9.48 %, peroxide condition shows 6.22%. The thermal degradation shows 1.28 % of degradation respectively. Table 3 revealed the detailed results and Figure 5 showed the chromatograms.
Results of the degradation analysis of ulipristal showed that stressed conditions of acid, alkaline and peroxide contribute to further degradation compared to other stressed conditions. Thermal degradation results in few degradations, while no degradation has been identi ied in photolytic conditions. The chromatograms of the ulipristal were found very particular in all stressed circumstances.
CONCLUSION
Focused on the experiential evidence of the present established ulipristal method, authors are strongly declaring the method's novelty over the very few methods available. The new HPLC approach is 'rapid' since within 1.516 minutes, it dramatically reduced the total analysis time, which considers the lowest analysis time needed for ulipristal analysis. The present method considered "stability indicating" because under stressed conditions, not as much degradation was observed and excellent separation of ulipristal among the other degraded peaks was also observed. According to the acceptance criteria of ICH Q2B guidelines, the results of the validation parameters were noted and found. Therefore, the present method developed can be used for the routine analytical and quality control assay of ulipristal in bulk as well as tablet dosage form as a novel, accurate, validated method of ulipristal. | v3-fos-license |
2019-04-06T13:04:08.196Z | 2013-01-01T00:00:00.000 | 96929811 | {
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} | pes2o/s2orc | RADIAL VARIATION IN FIBER LENGTH OF SOME LESSER USED WOOD SPECIES IN NIGERIA
Variations in fibre length of ten lesser used wood species were investigated. The mean fibre length varied from 1.07mm in Lannea acida to 2.41mm in Sterculia setigera. Four patterns of within tree radial variations in fibre length were observed in the studied species. In pattern one, fibre length increased from the pith to the bark. This was observed in Butyrospermum paradoxum, where fibre length increased from 1.43 mm in the heartwood to 1.45mm in the transition wood and 1.46mm in the sapwood; and in Parinarium kerstingii, where fibre length increased from 1.33mm in the heartwood to 1.40mm in the transition wood and to 1.42 mm in the sapwood. The same pattern of radial variation was also observed in Detarium senegalense where fibre length increased from 0.95mm in the heartwood zone to 0.96 mm in the transition wood and subsequently to 1.19 mm in the sapwood zone. In pattern two, fibre length decreased from the heartwood (pith) to the bark as observed in Isoberlina doka and Annogeissus leiocarpus. Fibre length decreased from 1.52mm in the heartwood of I. doka to 1.40 mm and 1.31 mm in the transition and sapwood of the plant species respectively. Also fibre length decreased from 1.30 mm in the heartwood of A. leiocarpus to 1.27mm and finally to 1.22 mm in the sapwood of A. leiocarpus. In pattern three, fibre length increased from the heartwood to the transition wood and decreased to the bark. This was observed in Parkia felicoida and Sterculia setigera where fibre length increased from 1.19 mm in the heartwood of P. felicoida to 2.47mm to 1.33mm in the transition wood and decreased to 1.19mm in the sapwood. In Sterculia setigera fibre length increased from 2.47mm in the hearwood to 2.50 mm in the transition wood and decreased to 2.26 mm in the sapwood of the species. In the 4 pattern, fibre length decreased from 1.49 mm in the heartwood to 0.96 mm in the transition wood followed by an increase to 1.19 mm in the sapwood zone of Mitragyna inermis.
INTRODUCTION
The tropical forests in Nigeria have witnessed significant reduction in the availability of economic wood species. The gregarious exploitation of economic wood resources commenced with commercial wood exploitation in Nigeria in 1872 (Aribisala, 1993;RMRDC, 1991). This was complemented by the clearing of forests for housing, infrastructure and agriculture, which proceeded at very fast rates in the 1960's to 1980's. More recently several studies (Aribisala, 1993;Olorunnisola, 2000;RMRDC 2009;Ogunsanwo 2010) have indicated that the overexploitation of the economic wood have had negative impact on capacity utilization in the national forest industries Most of the industries, most especially, the sawmill and furniture industries are now utilizing lesser used wood species, leading to considerable expansion in the number of wood species found in timber markets across the country. Where lesser used wood species are used as replacements of economic wood species, the products are facing acceptance problems in international markets (Jayanelti, 1998;Eastin et al, 2003;Barany et al, 2003). Research studies (Coleman, 1998;Barany, et al 2003) indicated that information on species availability, natural durability, physical properties, working quality and use range which are of utmost importance to manufacturers are lacking. Hence efforts at promoting utilization of lesser used species will not be successful without well documented, readily available concrete information (Arowosoge, 2010).
In view of the above, it is necessary that systematic studies should be carried out on wood quality parameters of lesser used species in the country. As fibres are the principal element responsible for the strength of wood (Panshin and Dezeuw, 1980), it is important to investigate this important parameter in lesser used wood species in Nigerian forests. In diffuse porous hardwoods, an increase in specific gravity is related to fibre characteristics through increase in the proportion of fibres. The amount and quality of fibres has a pronounced effect on wood strength and shrinkage (Panshin and Dezeuw, 1980). Consequently, understanding variation in wood specific gravity, fibre length and related anatomical features provides basis for improved wood utilization (Butterfield et al, 1993). The pattern of radial variation in cell lengths differs across species with radial growth rate (Banan, 1967;Fujiwara and Yang, 2000) which is determined by internal (cambial age) and external factors, such as climatic factors (Nicholls, 1986). In the present study, the patterns of radial variation in the fibre length of ten 15 lesser used wood species growing in the Nigerian tropical forests were studied.
MATERIALS AND METHODS
The hardwood species utilized in the study comprised of Anogeissus leiocarpus (D. C), Guel and Perr; Butyrospermum paradoxum Geartn F. (Aepper); Detarium senegalense J. F. Gmel; Isoberlina doka Graib et. Stapf; Lannea acida, A. Rich; Mitragyna inermis, Parinarium kerstingii Engl; Parkia felicoida keay; Albizia zygia (D. C), J. F. Macbr and Sterculia setigera. The materials for the study were collected from tree species growing in Oke Awon Forest Reserve, near Jebba, Kwara State. The total annual rainfall within the area varied from 1000 to 1250 mm. The tree species were from uneven aged natural stands with ages ranging from 18 to 60 years as deducted from ring counts from samples taken at breast height.
Five trees of each species were felled and disc samples, 7.5cm thick, were taken at breast height.
The sampled discs were immediately wrapped in plastic bags to prevent loss of moisture during transportation The sample discs were debarked at the laboratory using a kitchen knife. Slivers of wood were obtained from the heart wood, transition wood and sapwood zones by cutting out a thin portion of the wood from the pith to the sapwood of the samples.
The samples were macerated in a 1:1 solution of glacial acetic acid to dissolve lignin binding the fibres and in 30% hydrogen peroxide to bleach the fibres as described by Franklin (1946). The mixture was autoclaved at a constant temperature and pressure of 120 o C and 1kgf/cm 2 respectively for 45 minutes. The macerated samples were rinsed three times in distilled water and the samples shaken to separate the fibers. A drop of macerated tissue was placed on the microscope slide and covered with a slide cover. The slides were placed on the stage of a Carl Zeiss single eyepiece microscope with mirror attached for projecting the images on a white paper placed at the same level with the microscope. A total of fifteen whole fibers were measured to the nearest 1.00mm at 33x magnification level.
Statistical Analysis.
The actual length of the fibres was obtained by dividing the length of the measured images by 33. After the calculation of the fibre length per zone, the mean fibre length per species, range and coefficient of variation in fibre length were calculated.
RESULTS AND DISCUSSION
The mean, ranges and coefficient of variation of fibre lengths of the sampled species are shown in Table 1. Fibre length varies from 0.95 mm in the heartwood of D.senegalense to 2.50 mm the transitional of wood of S.setigera. The mean fibre length varies from 0.91.mm in D. senegalense to 2.41 in S. setigera. A comparison of the mean fibre values with those of common hardwood species in Europe and America which varies from 0.95m to 1.88mm (Isenberge, 1951), showed that both values have a very similar distribution. Fibre length is one of the quality parameters for pulpwood. As the hardwoods of similar fibre length have been successfully used for short fibre pulp production, in other countries, these species can be good sources of raw materials for short fibre pulp production locally. The fibre length of S. setigera at 2.41mm is however higher than those reported for hardwoods general. Thus, Osadare (1997) and Ogunwusi (2003) classified S. setigera as a medium/ long fibre wood species and recommended that research and development be directed towards genetic improvement of the plant species for long fibre pulp production in the pulp and paper industry.
Wood is a natural material and therefore is subject to many constantly changing influences (such as moisture, soil conditions, climate and silvicultural treatment). As a result, wood properties vary considerably. The ten wood species used in this study, exhibited various patterns of radial variation in fibre length. In all, four patterns of within tree radial variations in fibre length were observed. These are.
1. Increase in fibre length from the pith to the bark.
This pattern of variation was observed in B. paradoxum, P. kerstingii and D. senegalense (Fig. 1.) 2. Decrease in fibre length from the pith to the bark. This pattern was observed in 1. doka and A.leiocarpus as shown in fig. 2..
3. Increase in fibre length from the pith to the transition wood followed by decrease to the bark. This pattern of variation was observed in P. felicoida, L .acida and S. setigera. as shown in fig. 3. 4. Decrease in fibre length from the pith to the transition zone followed by increase to the bark. This pattern of variation was observed in M. inermis. (Fig. 4). Butterfields et al (1993) in two natural and plantation grown Central American hardwoods; Hyeronma alctorneoides and Vochysia guatamalensis respecively. The radial patterns of fibre length variation observed in 1. doka and A. leiocarpus in which there is a decline in fibre length near in the bark after an initial increase from the pith outwards is the type III pattern described by Panshin and Dezeuw (1980) in timbers such as Fraxinus pennsylvanica, Liriodendron tulipitera, Platanus occidentalism and Salix nigra. Shortening of fibres rear the bark has been attributed to onset of senescence by earlier workers (Dinwoodie, 1965;Panshin and Dezeuw, 1980). The flat curve in the mature wood showing constant fibre length like the Type 1 described by Panshin and Dezeuw (1980) was not exhibited by any of the species studied. However, a more or less constant increase in fibre length from tree centre to periphery (the type II pattern) was displayed by P. kerstingii and B. paradoxum and D. senegalense
A. A. OGUNWUSI
Fibre length is an important property influencing the utilization potentials of wood for a variety of purpose, most especially, for pulp and paper production.The mean fibre values and pattern of radial variation in fibre length in the tested wood species compare favorably with those being used as industrial wood species and for hardwood pulp production in several parts of the globe. This indicated the possible utilization of the wood species in several industries most especially for production of short fibre pulp. However, the fibre length of S. setigera at 2.41mm makes it a possible raw material for long fibre pulp production.
More recently, there has been increasing interest in the development of wood-plastic composites for use as building materials due to abundance, low cost and process ability of wood as filler. In terms of processing, research and development have shown that mechanical properties and fibre length of wood are sensitive to extrusion parameters such as screw configuration and compounding temperature. Research results so far indicated that short fibre wood species resulted in better mechanical properties of wood plastic composites than long fibre species as they are easier to disperse in the High Density Polyethelene Matrix. This may eventually lead to expansion in the frontiers of the utilization potentials of lesser used hardwood species. Consequently, efforts should be made to ensure that the wood quality parameters of the lesser used hardwoods species be classified in order to expand their industrial application and products acceptability in the global market. | v3-fos-license |
2019-03-08T14:16:01.968Z | 2019-02-07T00:00:00.000 | 73437042 | {
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} | pes2o/s2orc | Selection of indigenous starter culture for safety and its effect on reduction of biogenic amine content in Moo som
Objective The aims of this study were to select one strain of Lactobacillus plantarum (L. plantarum) for a potential indigenous safe starter culture with low level antibiotic resistant and low biogenic amine production and evaluate its effect on biogenic amines reduction in Moo som. Methods Three strains of indigenous L. plantarum starter culture (KL101, KL102, and KL103) were selected based on their safety including antibiotic resistance and decarboxylase activity, and fermentation property as compared with a commercial starter culture (L. plantarum TISIR543). Subsequently, the effect of the selected indigenous safe starter culture on biogenic amines formation during Moo som fermentation was studied. Results KL102 and TISIR 543 were susceptible to penicillin G, tetracycline, chloramphenicol, erythromycin, gentamycin, streptomycin, vancomycin, ciprofloxacin and trimethoprim (MIC90 ranging from 0.25 to 4 μg/mL). All strains were negative amino acid-decarboxylase for lysis of biogenic amines in screening medium. For fermentation in Moo som broth, a relatively high maximum growth rate of KL102 and TISIR543 resulted in a generation time than in the other strains (p<0.05). These strain counts were constant during the end of fermentation. Similarly, KL102 or TISIR543 addition supported increases of lactic acid bacterial count and total acidity in Moo som fermentation. For biogenic amine reduction, tyramine, putrescine, histamine and spermine contents in Moo som decreased significantly by the addition KL102 during 1 d of fermentation (p<0.05). In final product, histamine, spermine and tryptamine contents in Moo som inoculated with KL102 were lower amount those with TISIR543 (p<0.05). Conclusion KL102 was a suitable starter culture to reduce the biogenic amine formation in Moo som.
INTRODUCTION
Moo som is a traditional Thai fermented pork which is composed of sliced, cut or diced pork, cooked rice, salt and shopped garlic. The mixture is tightly wrapped in banana leaves or packed in plastic bags and then held at room temperature for 2 to 5 d. This product is consumed both without and with cooking and commonly consumed in the north-eastern region of Thailand [1]. The traditional fermented meat products involve indigenous lactic acid bacteria (LAB) that are generally found in raw materials. LAB convert glucose to lactic acid. This increasing lactic acid was observed with decreasing pH which has a preservative effect against competitive microflora during fermentation. In this case, the fermentation is not controlled which affect the organoleptic quality and safety of the final products. Depending on which species of indigenous microbes were present at the start of fermentation, the appearance of pathogenic species such as Salmonella spp., Escherichia coli and Staphylococcus aureus were especially likely to be found in cuisines with pH value more than 4.6 [2]. Some microflora, Enterobacteriaceae, could convert amino acids to biogenic amines by their decarboxylase activities [3]. A trend has emerged which involves the isolation of indigenous strains from traditional fermented meat to be used as potential starters in meat fermentation [4]. These potential starters possess inherent functional characteristics. They can improve food quality and safety (probiotics) by offering one or more characteristics such as antibiotic susceptibility or negative decarboxylase activity [5,6]. Thus, the use of cautiously selected indigenous strains as starters or co-cultures in fermentation processes can help to achieve in situ expression of the required fermentation, retaining a completely natural product and still function as safety starter cultures where applicable.
Lactobacillus plantarum (L. plantarum) is pervasive lactic acid bacterium and it is detected in environments such as food (fermented meat, dairy products, vegetables, fruits, and beverages), gastrointestinal, respiratory and genital tracts of humans and animals [4]. Various L. plantarum isolates have the ability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. Previous study showed that consumption of L. plantarum reduced contamination of faecal Enterobacteriaceae and decreased certain risk factors for coronary artery disease. It may result in a dose-dependent reduction in the symptoms of Irritable Bowel Syndrome. The functional food products containing probiotic strains of L. plantarum are commercially available [7]. This Lactobacillus is considered as generally regarded as safe in the USA. L. plantarum is shown to confer a health benefit on humans and animals [8]. However, the detection of antibiotic resistant strains of L. plantarum isolated from fermented meat resulted in their recognition as reservoir of antibiotic resistant genes horizontally transmissible to pathogens through the food chain, this being a matter of concern [9,10].
Generally, relatively high levels of biogenic amines have been found in fermented meat products. Biogenic amines are nitrogenous basic compounds which have objectionable physiological effects on human health. They may be responsible for allergic reactions, nausea, hypotension or hypertension, palpitations, intracerebral haemorrhage and death in very acute cases. In fermented meat products, the main biogenic amines are tyramine, putrescine, histamine, cadaverine, tryptamine, spermine and spermidine. These biogenic amines are commonly products of amino acid-decarboxylase activities of microbes. The most frequent foodborne intoxication is caused by biogenic amines, especially tyramine and histamine [11]. The formation of biogenic amines in fermented sausages is dependent upon many variables including the hygiene of the meat ingredients, method of manufacture, qualitative and quantitative composition of the microflora [12]. Furthermore, the choice of starter cultures is based on inhibiting the increase of the potential aminogenic endogenous bacteria together with their own inability to produce biogenic amines. The five strains of L. plantarum demonstrating biogenic amine reduction were apprised. Two strains of L. plantarum presented the highest ability to reduce putrescine and tyramine in culture media [13]. Starter culture L. plantarum was efficient in reducing cadaverine, putrescine, histamine, tryptamine, phenylethylamine, and tyramine accumulation in som-fug [14]. A strains of L. plantarum isolated from fermented sausages had a strong effect on inhibiting the biogenic amines production in sausage fermentation. The selection of suitable inoculating starter culture has been one of the most effective strategies to prevent or minimize the presence of biogenic amines [15].
Our previous studies reported that three strains of L. plantarum, KL101, KL102, and KL103 were isolated from traditional Thai fermented meat for indigenous starter culture strains. These bacteria exhibited antimicrobial activity against Staphylococcus aureus, Escherichia coli, S. Typhimurium and L. monocytogenes [16] and survival in the gastrointestinal environment (acidity, pepsin, bile salt, and pancreatin) [17] and were selected candidate probiotics. In this study, the selection of indigenous safe strain intended as a Moo som starter culture and its influence on biogenic amine accumulation in Moo som was investigated.
Strains and starter culture preparation
Three strains of L. plantarum, L. plantarum KL101 (KL101), L. plantarum KL102 (KL102), and L. plantarum KL103 (KL103) were previously isolated from traditional Thai fermented meat as indigenous starter cultures. Briefly, these strains were selected for their preliminary probiotic function, including antimicrobial activity against foodborne pathogens [16] and survival in the gastrointestinal environment [17]. L. plantarum TISIR543 (TISIR543) as commercial starter culture was purchased from Culture Collection of National Center for Genetic Engineering and Biotechnology (BIOTIC), Thailand. Each strain was stored at -20°C in de Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) containing 20% (v/v) glycerol. Frozen cultures were cross-linked on MRS agar and then incubated anaerobically at 30°C for 24 h prior to use. Cells were harvested by centrifugation at 6,000×g at 4°C for 10 min in Universal 16R refrigerated centrifuge (Hettich Zentrifugen, Tuttlingen, Germany), washed and resuspended in saline solution (0.85% (w/v) NaCl). Finally, the cell concentration of each LAB starter culture was adjusted to 5×10 8 cfu/mL with saline solution and a McFarland standard turbidity of 0.5.
Screening of safety lactic acid bacteria starter cultures
Antimicrobial susceptibility testing: The antimicrobial resistance was estimated using the broth microdilution method which was modified from Dušková and Karpíšková [18]. The following antibiotics were tested in MRS broth: 0.0625 to 256.0000 μg/mL of penicillin G, tetracycline, chloramphenicol, erythromycin, gentamycin, streptomycin, vancomycin, ciprofloxacin and trimethoprim. Each LAB suspension was inoculated in microtiter plate at final concentration of 5×10 5 cfu/mL and then incubated anaerobically at 30°C for 24 h. The minimum inhibitory concentrations (MIC) were determined. The strains were divided as susceptible (MICs <8 μg/ mL), moderately resistant (MICs 8 to 32 μg/mL) or resistant (MICs >32 μg/mL) base on the MIC requirement that the antibiotic inhibit the growth of 90% of bacteria (MIC90) as indicated by Walsh [19].
Testing for the decarboxylase activity of lactic acid bacteria starter cultures: In order to support the enzyme induction before the actual screening test, LAB strains were subcultured 3 times in MRS broth, containing 0.1% (w/v) of each precursor amino acid (Merck, Germany), including L-histidine monohydrochloride, L-ornithine monohydrochloride, L-lysine monohydrochloride, L-arginine monohydrochloride and tyrosine disodium in addition to supplementation with 0.005% (w/v) of pyridoxal-5-phosphate hydrate and 0.01% (w/v) thiamine hydrochloride (Merck, Germany) and then incubated anaerobically at 30°C for 24 h. All strains were streaked on MRS agar, containing 0.1% (w/v) of each precursor amino acid, 0.005% (w/v) of pyridoxal-5-phosphate hydrate and 0.01% (w/v) thiamine hydrochloride and then incubated anaerobically at 30°C for 24 h [20].
All strains of LAB were assayed for decarboxylase activities in screening medium. The decarboxylase activities were tested by inoculating individual LAB colonies from MRS agar, containing 0.1% (w/v) of each precursor amino acid, directly onto plates containing the modified decarboxylase medium, including 0.005% (w/v) of pyridoxal-5-phosphate hydrate, modified from method of Maijala [21]. The medium included the precursor amino acids at 1% (w/v) final concentration. Purple bromocresol was contained as pH indicator. The pH was adjusted to 5.3 and then the medium was autoclaved. The screening medium without amino acid was used as control. The inoculated plates were incubated anaerobically at 30°C for seven days. The conversion of clear zone around colony, color changing from purple to yellow, was determined as amino acid decarboxylase positive. Therefore, another result was determined as amino acid decarboxylase negative.
Microbiological analysis: LAB growth was performed in two replicates during the fermentation. One milliliter of sample was diluted in 9 mL of saline solution. The diluted solution was serially diluted in saline solution and 0.1 mL of each dilution was grown on MRS agar and then incubated anaerobically at 30°C for 24 to 48 h [23]. After that, the maximum growth rate (μ max ) and generation time (λ) were calculated according to Oliveira et al [24] as following equations: Where: X 1 and X 2 are LAB counts; t 1 and t 2 are incubation times. λ = ln 2/μ max (2) Determination of pH and total acidity: Twenty-five milliliters of sample was taken for pH and total acid estimation. Both estimations were estimated triplicates for each supernatant fluid. Direct pH estimation was taken by using pH meter (Mettler Toledo S20, Schwerzenbach, Switzerland). The amount of the total acid as lactic acid produced in the fermentation of Moo som broth was estimated by the standard titration procedure for total titratable acidity according to AOAC [25]. Total acid content estimation was done by titrating the supernatant fluid of the substrates on addition of phenolphthalein as an indicator, 0.1 M NaOH was titrated into the samples. The total acidity was calculated as lactic acid and expressed as g/100 mL.
Effects of commercial starter culture and indigenous strain on the microbial changes and biogenic amine contents during fermentation
Moo som preparation: The Moo som mixture, common recipe, was prepared with lean pork and the ingredients and additives (g/kg): cooked rice, 65; minced garlic, 65; sugar, 4; erythrobate, 4; sodium tripolyphosphate, 3; NaCl, 10; and potassium nitrite, 0.8. Lean pork was stored at 2°C, sliced and mixed with the other ingredients and additives. Three separated batches of Moo som were prepared with different inoculums (non-inoculum [control], TISIR543, and KL102) at initial concentration of 10 5 cfu/g. Each mixture batch was stuffed into plastic casing with a diameter 30 mm (approximately 100 g each) and sealed tightly. All samples were incubated at 30°C for 3 d. They were taken at every 1 d for analysis of microbiology, pH and total acidity and taken at 0, 1, 2, 3, 5, and 7 d for analysis of biogenic amines.
Microbiological analysis: The microbiological analyses were performed in two replicates during the fermentation for LAB, Staphylococci and Enterobacteriaceae. The casings were aseptically removed and 25 g of Moo som sample was diluted in 225 mL of saline solution and homogenised in a stomacher bag mixer (Interscience, Saint Nom la Bretèche, France). The homogenate was serially diluted with saline solution and each dilution was grown in different growth media. The following media and incubated conditions were used: i) MRS agar and then incubated anaerobically at 30°C for 24 to 48 h for LAB count [20], ii) mannitol salt agar (Oxoid, Hampshire, UK) incubated at 30°C for 24 to 48 h for Staphylococci [4], and iii) crystal-violet neutral-red bile dextrose agar (Merck, Germany) incubated at 37°C for 24 h for Enterobacteriaceae count [5].
Determination of pH and total acidity: The sample homogenates for pH and total acidity determination were prepared. Two grams of sample were added with 20 mL of distilled water and then homogenised with Ultra-Turrax IKA (WERKE GMBH & CO.KG, Staufen, Germany) for 60 s. Triplicate determination for each sample was carried out. Direct pH measurement was taken with a pH meter. The homogenate was centrifuged at 3,000×g for 15 min. The supernatant was filtered through Whatman filter paper No. 4 (Merck, Darmstadt, Germany). The filtrate was titrated with 0.1 M NaOH using phenolphthalein as an indicator. The total acidity was calculated as lactic acid and exposed as g/100 g [26].
Determination of biogenic amine
Meat and Moo som samples were prepared for determination of biogenic amines by using a method modified from Tosukhowong et al [5]. Samples were cut into small pieces and blended with a blender (Ronic, Vitry en Charollais, France) for 30 s at 2 times. Five grams of blended sample was taken into plastic bag and extracted with 0.3 M tricholoacetic acid (Sigma Chemical, St. Louis, MO, USA) using the stomacher bag mixer for 8 min. A 500 μL of 77 mM 1, 7-diaminoheptane (Sigma Chemical, USA) was using as an internal standard. The ratio of sample and 0.3 M tricholoacetic acid was 1:5 (w/v). The supernatant was collected by centrifugation at 4,000×g at 4°C for 15 min and then kept at -20°C for high performance liquid chromatography derivatization.
The method for determining biogenic amines was modified from Tosukhowong et al [5]. Freshly prepared dansyl chloride (37 mM in acetone) was used as a derivatizing agent. A 300 μL of supernatant or standard solution was mixed with 60 μL of 2 M NaOH and 90 μL of a saturated solution of sodium hydrogen carbonate. The reaction mixture was leaved at room temperature for 30 min. To this was added 600 μL of fresh dansyl chloride solution and incubated at 40°C for 45 min. The reaction mixture was stopped by 30 μL of 32% ammonia solution (Merck, Germany) and then left at room temperature for 30 min. The sample volume was adjusted with acetonitrile (Sigma Chemical, USA) to 1,500 μL of final volume and gently mixed. Sample was centrifuged at 3,000×g at 4°C for 5 min. The supernatant was filtered through a Minisart RC4 filter (0.45 μm pore size, Sartorius, Goettingen, Germany). After that, 20 μL of filtrate was injected into HPLC for analysis.
Biogenic amine was separated on Luna NH2 column, 4.6× 250 mm, 3 μm (Phenomenex, Torrance, CA, USA) and the analyte quantified on Thermo separation products model ConstaMetric 4100 Bio (Mundelein, IL, USA). The temperature of column was set at 40°C. A 20 μL of derivatized sample or standard was injected. The mobile phase was composed of 0.1 M ammonium acetate (Sigma Chemical, USA) as solvent A and acetonitrile as solvent B. The flow rate was 1.2 mL/min. The isocratic program started at 10% A and 10% B within 10 min and held for 5 min before starting the next run. After that, solvent B was raised to 90% within 25 min. Biogenic amines were detected at wavelength 254 nm by a UV detector (Shimadzu, Kyoto, Japan). The biogenic amine concentration in pork and Moo som was calculated by comparing with the standard concentration.
Statistical analysis
Data are shown as means and standard deviations. Results were based on analysis of the general linear model by SAS 9.0 software (SAS Institute, Cary, NC, USA), except MIC and MIC90 were assessed by the division of antimicrobial agent in three classes [19]. Pearson's correlation coefficients were carried out to determine the relationship among variables using the CORR procedure. A selected indigenous strain of L. plantarum was examined by MICs of all antibiotics as the susceptible class, negative amino acid-decarboxylase in screening medium, the highest maximum growth rate and the lowest generation time in Moo som broth. After that, this selected strain was studied its effect on formation of biogenic amines in Moo som.
Screening of safety lactic acid bacteria starter cultures
The present study determined the lowest antibiotic concen- tration that inhibit (MIC) and 90% (MIC90) of the tested LAB strains. The results showed that TISIR543 and KL102 were observed to be susceptible to all antibiotics while KL101 and KL103 were observed to moderately resistant to tetracycline, gentamycin and streptomycin, ciprofloxacin and trimethoprim (Table 1). Generally, most lactobacilli are intrinsically resistant to aminoglycosides (streptomycin and gentamycin), glycopeptides (vancomycin), inhibitors of nucleic acid synthesis (ciprofloxacin) and inhibitors of folic acid synthesis (trim-ethoprim). However, they are susceptible to penicillins, chloramphenicol, streptomycin, tetracycline and erythromycin [27][28][29]. Additionally, all L. plantarum strains were negative amino acid-decarboxylase for lysis of L-histidine monohydrochloride, L-ornithine monohydrochloride, Llysine monohydrochloride, L-arginine monohydrochloride, and tyrosine disodium in the screening medium. For a safety starter culture, the antibiotic resistant results showed that TISIT543 and KL102 was safer than KL101 and KL103.
Effect of different strain of starter culture during fermentation of Moo som broth L. plantarum count indicated significant differences between Moo som broth inoculated with different strains (p<0.05) during fermentation. At the beginning of fermentation, TI-SIR543 and KL102 counts were higher than KL101 and KL103 after 12 h of fermentation (p<0.05) ( Figure 1A). Due to relatively high maximum growth rate of TISIR543 and KL102 (0.66±0.03 and 0.67±0.02 L/h, respectively) ( Figure 1B), generation times of TISIR543 and KL102 were lower than those of KL101 and KL103 (p<0.05) ( Figure 1C). However, all strains grew up to 24 h of fermentation (<10 10 cfu/g). Thereafter, TISIR543 and KL102 counts were constant throughout until the end of fermentation, while KL101 and KL103 counts decreased moderately over the same fermentation time. The decreased counts of KL101 and KL103 in Moo som broth resulted from low pH values in Moo som broth (pH 3.42 to 3.84) after 36 to 72 h of fermentation (Figure 2A), which was non-optimal pH for L. plantarum growth (pH<4) [30]. Although there were differences in growth rate between strains, the cultures generally exhibited an increase sensitivity at pH values below 4.0. Acid tolerance was accepted as one of the desirable properties used to select potentially probiotic strains [31]. Hence, for growth and count consistence of starter culture, KL102 was a potential indigenous starter culture and selected to be applied in Moo som production as safety starter culture.
The results of pH and total acid content in Moo som broth inoculated with TISIR543, KL101, KL102, and KL103 were observed as the fermentation progressed ( Figure 2). Within the 36 h of fermentation, the pH value was significantly decreased in Moo som broth inoculated with all strains (p<0.05). After that, no differences in pH values were observed between fermentation times until the end of fermentation (after 72 h of fermentation) (p>0.05). Similarly, total acid content in all samples increased rapidly after 48 h of fermentation. Then, total acid contents were constant throughout the remaining fermentation time. Commonly, homo-or heterofermentative LAB produce lactic acid as their major end-product of fermentation via the Embden-Meyerhof pathway (glycolysis) or the phosphoketolase pathway (phosphogluconate pathway), respectively [32]. The sharp decline of pH in early fermenta- tion was accompanied with an increase in L. plantarum counts and total acid (r = -0.819, p<0.01 and r = -0.982, p<0.01, respectively). In addition, a fast growth of LAB leading to rapid pH decline within 36 h is necessary to guard against growth of spoilage and pathogenic bacteria [33].
Microbiological changes in Moo som inoculated with commercial starter culture and indigenous strain during fermentation LAB, Staphylococci and Enterbacteriaceae counts during fermentation of Moo som without and with inoculation of L. plantarum starter culture are summarized in Figure 3. The initial microflora of all samples was obtained from the raw minced pork, except inoculated starter culture samples which TISIR543 and KL102 were inoculated at final concentration of 10 5 cfu/g. For raw minced pork, counts of LAB, Staphylococci and Enterbacteriaceae were 4.63±0.17, 2.78±0.24, and 2.06±0.09 log cfu/g, respectively. During fermentation of all samples, LAB increased rapidly and reached to a maximum 10 8 cfu/g after 3 d of fermentation and then these counts were constant untill the end of fermentation. However, LAB count in inoculated samples was higher than control sample during fermentation (p<0.05) ( Figure 3A). Figure 3B shows that Staphylococci count of inoculated samples decreased slightly at the end of fermentation while this count in the control sample was constant during fermentation. According to previous study, Staphyloccoci counts of Nham without the addition of inoculum remained constant throughout the fermentation period, possibly due to the decrease in pH [5].
On the other hand, Enterobacteriaceae count increased after 1 d of fermentation and then decreased rapidly in all samples, which was lower than 1 log cfu/g after 2 d of fermentation for Moo som inoculated with KL102 and after 3 d of fermentation for Moo som inoculated with TISIR543 and control ( Figure 3C), possibly due to decrease in pH value. The rapid decrease of Enterobacteriaceae count during fermentation was accompanied with an increase in total acid and a decrease in pH (r = -0.862, p<0.01 and r = 0.801, p<0.05, respectively). In Moo som, a rapid growth of LAB caused a total acid increase and pH to decrease to below 4.6 within 3 d, which is essential to inhibit growth of unfavorable microflora [30]. Additionally, various metabolic products of LAB, such as short-chain organic acids, hydrogen peroxide, carbon dioxide, diacetyl and bacteriocin have antimicrobial potential [34].
pH and total acidity measurement The pH and total acid content results are shown in Figure 4. All Moo som samples presented sharp decrease in pH during the first 2 d of fermentation and continuously decreased to a final pH value of lower than 4.6 after 3 d of fermentation (Figure 4A). Similarly, as fermentation time increased, total acid contents of all samples increased quickly during the first 3 d of fermentation and then remained nearly constant throughout 7 d of fermentation ( Figure 4B). The sharp decrease in pH value during fermentation was accompanied with an increase in total acidity (r = -0.897, p<0.01). According to the previous study, the most common amine was spermine, while cadaverine was not found in fresh pork [5]. Biogenic amine accumulation during Moo som fermentation inoculated with commercial starter, TISIR543, or indigenous starter culture, KL102 was observed at 0, 1, 2, 3, 5, and 7 d and compared with that of naturally fermented Moo som, control (Table 2). Overall, tyramine, putrescine, histamine and spermidine were influenced by different starter cultures in Moo som fermentation. Tyramine formation started after 1 and 2 d of fermentation in Moo som for without and with starter culture inoculations, respectively. After 1 and 5 d of fermentation tyramine contents were significantly higher in control sample and sample inoculated with TISIR543, respectively, than in sample inoculated with KL102 at the same fermentation time. Tyramine is the biogenic amine most generally related to various species LAB found in fermented sausage which biosynthesizes tyramine [5,35]. In control Moo som, a rapid increase of tyramine formation during fermentation was accompanied with an increase in LAB loading (r = 0.859, p<0.05). The tyramine toxic level is 100 to 800 mg/kg [36]. In this research, tyramine contents in naturally inoculated Moo som and Moo som inoculated with commercial starter culture after more than 2 and 7 d of fermentation (over fermentation), respectively, were higher than 100 mg/kg. In contrast, in Moo som inoculated with KL102 the tyramine content was lower than 100 mg/kg throughout fermentation, therefore this represents a safe level. The formation of putrescine depended on the inoculum strain. Moo som inoculated with KL102 accumulated constant amounts of putrescine throughout fermentation due to relative decrease in Enterobacteriaceae count during early fermentation. The appearance of putrescine in meat products has been associated with the ornithine-decarboxylase activity of Enterobacteriaceae [3,35]. However, histamine content was lower in Moo som inoculated with KL102 throughout fermentation (p<0.05).
The histamine accumulation in all samples was lower than 100 mg/kg which should be the upper limit for potential risk to healthy individuals [36]. Furthermore, spermine accumulation in Moo som inoculated with KL102 varied slightly during fermentation, while this amine in naturally inoculated Moo som and Moo som inoculated with TISIR543 increased rapidly after 2 and 5 d of fermentation, respectively. A previous study reported that amine oxidase of some indigenous strains of L. plantarum were able to degrade spermine [13]. On the other hand, spermidine content in all samples decreased during early fermentation and was not found after 3 d of fermentation since spermidine can be consumed as a nitrogen source by the microorganisms [37]. Moreover, levels of tryptamine varied slightly during fermentation while its level in all samples was lower than 50 mg/kg. Special concern is devoted to a group of polyamines; putrescine, spermidine and spermine. In humans, polyamines are taken up and used by the fast dividing tissues, consequently polyamines are concentrated in tumors [38]. However, cadaverine was not detectable in all Moo som fermentations. Therefore, biogenic amine formation in Moo som could be prevented by the addition of KL102. Due to the limited knowledge about biogenic amine reduction, the mechanism by which KL102 can degrade some biogenic amines content in Moo som should be further studied.
Results of this study indicate that L. plantarum KL102 is a candidate indigenous starter culture probiotic with low level antibiotic resistance and low decarboxylase activities. Furthermore, this strain showed the fastest fermentation rate as a potential indigenous starter culture. For Moo som fermentation, the reduction of tyramine, putrescine, histamine and spermine content was successfully obtained in Moo som inoculated with KL102. Therefore, the addition of KL102 as indigenous starter culture could minimize the biogenic amine formation in Moo som fermentation.
CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. | v3-fos-license |
2019-10-24T09:16:39.548Z | 2019-10-16T00:00:00.000 | 209445509 | {
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} | pes2o/s2orc | Transforming Endothelium with Platelet‐Rich Plasma in Engineered Microvessels
Abstract Vascularization remains an obstacle when engineering complex tissues for regeneration and disease modeling. Although progress has been made in recreating 3D vascular structures, challenges exist in generating a mature, functional endothelium. It is demonstrated that perfusing engineered microvessels with platelet‐rich plasma, a critical homeostatic component in vivo that is often overlooked in vitro, substantially transforms the endothelium, both maturing endothelial cells and improving functionality in 24 h. Platelets readily adhered to the exposed collagen‐I substrate through small gaps within engineered vessels without forming thrombi. The adherent platelets improve barrier function, enhance endothelial glycolysis, reduce thrombogenicity, and enrich smooth muscle cell growth surrounding the endothelium. These findings demonstrate that platelets are essential to the function of endothelium during vascular maturation and remodeling. This study sheds light on a potential strategy to engineer stable, implantable vascular networks.
Introduction
Engineering complex tissues holds the promise of replacing damaged organs and establishing platforms to model disease and test drug candidates. [1] Efforts have been made to incorporate perfusable vasculature to provide efficient blood supply in support of engineered tissue survival and growth. [2] However, engineered vasculature has thus far not fully mimicked the function of native vasculature which has likely contributed to the limited growth and remodeling of tissues in vitro. One potential strategy for improving the function of engineered vasculature was suggested by studies performed over 50 years ago, when researchers attempted to increase tissue viability and vascular integrity in isolated perfused organs. Perfusion of platelet-rich plasma (PRP) was found to preserve vascular integrity and prevent parenchymal inflammation compared to perfusion of plasma alone. [3] Platelets are known for their critical role in preventing blood loss in response to injury, but their role in safeguarding the endothelium during homeostasis is often overlooked. [4] This function is highlighted by the fact that patients with thrombocytopenia are at risk for spontaneous bleeding even without injury. [5] Over the past 70 years, different roles for platelets have included forming physical plugs to exposed lamina, [3,6] elaborating bioactive vehicles that can improve endothelial barrier function by releasing soluble growth factors, [7] and regulating transendothelial migration of leukocytes and thereby inflammation. [4b] Platelets may also have a role in vascular development, as remodeling of the capillary plexus coincides with the emergence of megakaryocytes [8] and loss of CD41+ cells leads to animal lethality due to vascular defects. [9] However, it is not known how the endothelium changes nor matures when interacting with platelets, nor whether platelets alone are sufficient to enhance vascular function. Better understanding of these questions would provide potential strategies to prevent vascular leakage and improve vascularization in engineered complex tissues.
Studies examining platelet-endothelium interactions have typically relied on either conventional 2D culture or animal models. In 2D transwell culture, transendothelial diffusion of albumin and other fluorescence molecules was reduced following treatment with platelets or platelet releasate, but not with fixed platelets. [7] Removing the platelets reversed this effect, suggesting that growth factors released Vascularization remains an obstacle when engineering complex tissues for regeneration and disease modeling. Although progress has been made in recreating 3D vascular structures, challenges exist in generating a mature, functional endothelium. It is demonstrated that perfusing engineered microvessels with platelet-rich plasma, a critical homeostatic component in vivo that is often overlooked in vitro, substantially transforms the endothelium, both maturing endothelial cells and improving functionality in 24 h. Platelets readily adhered to the exposed collagen-I substrate through small gaps within engineered vessels without forming thrombi. The adherent platelets improve barrier function, enhance endothelial glycolysis, reduce thrombogenicity, and enrich smooth muscle cell growth surrounding the endothelium. These findings demonstrate that platelets are essential to the function of endothelium during vascular maturation and remodeling. This study sheds light on a potential strategy to engineer stable, implantable vascular networks.
from unstimulated platelets were responsible. These early studies presumed that endothelial cells (ECs) did not physically interact with platelets and the presence of platelets only passively changed transendothelial diffusion. To date, few studies have sought to understand how the endothelium is influenced by platelets. Furthermore 2D systems lack a 3D architecture that allows for the formation of a continuous lumen that can be exposed to intra-and transluminal flow and continuous 3D stresses, all of which are integral components of vascular function. Alternatively, animal models have provided confounding observations when investigating the effect of platelets on the endothelium-demonstrating the ability of platelets to both decrease or increase vessel permeability in separate investigations. [10] This is likely due to the complexity of in vivo systems, in which many contributing factors are coupled together. Thus, there is a need to study platelet-endothelium interactions in a system that better mimics the 3D vascular microenvironment in vivo while also allowing for the investigation of individual components in a stepwise approach.
In this study, we exploit an engineered 3D microvessel system to uncover PRP-mediated vascular phenomena that have not been possible using 2D culture. This system comprises vessels within a collagen I matrix that allows for vascular remodeling in response to various types of stimuli: biophysical (hemodynamics), biochemical (angiogenic growth factors), and cellular, and has demonstrated unique utility in studies of angiogenesis and thrombosis. [11] Using this system, we show that perfusing platelets induces substantial changes to vessel diameter, EC ultrastructure, and metabolism, as well as complex physiological processes such as barrier function, antithrombogenicity, and mural cell interactions. These effects were not evident in parallel 2D cultures nor using perfusates such as enriched plasma (EnP) or lyophilized platelets, suggesting endothelial maturation and maintenance requires metabolically active platelets in the context of 3D microvessels. Together, these findings help unveil the role of platelets in diverse vascular functions and provide a promising strategy to enhance the functionality of engineered vessels and complex tissues.
PRP Treatment Alters Vascular Diameter and Barrier Functions in 3D Engineered Microvessels
Engineered microvessels were fabricated in 7.5 mg mL −1 collagen I, seeded with human umbilical vein endothelial cells (HUVECs) and cultured under gravity-driven flow for 3 days, as shown previously ( Figure S1a, Supporting Information). [12] At day 3 after endothelial seeding, each microvessel device was perfused with PRP, plasma that was incubated with platelets for 4 h prior to platelet removal-referred to here as EnP, or control media for 24 h and monitored for changes in vessel morphology and function (Figure 1a,b). The 24 h time frame was chosen for PRP perfusion to eliminate the complications arising from platelet loss or apoptosis after prolonged storage at 37 °C. PRP treatment for 24 h caused vascular remodeling and increased vessel diameter, which reached an average of 120.4 ± 0.9% of the original diameter compared to negligible changes in control media (102.1 ± 2.2%) or EnP (100.9 ± 1.3%) (Figure 1c-f and Figure S1b, Supporting Information). This radial remodeling occurred isotropically (Figure 1b and Figure S1b, Supporting Information), suggesting the establishment of circumferential strain on vessel walls and therefore, increased vessel volume. When imaged in real-time, vessel diameter increased linearly in the initial 2 h before gradually reaching a plateau for the remaining time (Figure 1g). We have noted that consistent increases in diameter occurred even after PRP treatment with platelet concentrations as low as 10% of physiological value (data not shown). We did not observe significant changes in vessel diameter when perfused with washed platelets resuspended in Tyrode's buffer or lyophilized platelets in EnP (101.0 ± 2.8% and 98.6 ± 0.6% of their original diameters, respectively) ( Figure 1f and Figure S1b, Supporting Information). These data suggest that both platelets and plasma contribute to the increased vessel diameters of PRP-treated vessels. ECs displayed similar junctions (i.e., VE-Cad-not shown and CD31) at cell-cell contacts following treatment with PRP, EnP, and washed platelets (Figure 1d,e and Figure S1c, Supporting Information); however, junctions became disrupted when treated with lyophilized platelets in plasma (arrows and arrowheads, Figure S1d, Supporting Information).
We next examined changes to the vessel wall 24 h posttreatment by examining the deposition of basement membrane proteins and barrier function via immunofluorescence analyses. PRP-treated microvessels had a thick and continuous layer of the basement membrane protein, Col-IV, along their vessel walls, whereas control and EnP-treated microvessels had regions of discontinuity (arrowheads, Figure 1h-j). Perfusion of fluorescently conjugated 70 kDa dextran solution in basal media enabled real-time evaluation of fluorescence permeation from vessel lumen into the interstitium. Microvessels treated with EnP resulted in no significant effect compared to control with large and overlapping variations in both conditions, with measured permeability coefficient of (3.5 ± 2.3) × 10 −5 cm s −1 in EnP versus (4.5 ± 3.5) × 10 −5 cm s −1 in control. PRP treatment resulted in decreased fluorescent permeation regardless of the initial vessel conditions, demonstrating improved vessel barrier function compared to control and EnP (Figure 1k-m). The quantification revealed a 45-fold reduction in apparent solute permeability ((9.9 ± 1.4) × 10 −7 cm s −1 ) in PRP-treated vessels with minimal variation compared to control microvessels (Figure 1n,o).
In contrast to engineered 3D microvessels, when PRP was applied to HUVECs cultured in 2D monolayers, we found many platelets adhered at regions between adjacent ECs as seen through phalloidin staining ( Figure S2a, Supporting Information). These platelet-endothelial interactions appeared to have impaired the integrity of VE-cadherin junctions between adjacent cells, resulting in EC retraction ( Figure S2a-ii,iii, Supporting Information). There was a significant decrease in the number of cells per surface area comparing control media Figure 1. PRP perfusion improves 3D engineered vessel integrity and barrier function. a) Schematic of PRP perfusion into microvessels after 3 days of fabrication and b) zoomed diagrams of perfused PRP adhered to exposed collagen substrate and healed the endothelium. c-e) z-Stack projection, cross-sectional and enlarged views of representative confocal images of engineered vessels perfused with c) control media, d) PRP, and e) EnP. Red: CD31, blue: nuclei. f ) Quantification of vessel diameter changes following perfusion with control media, PRP, EnP, washed platelets in Tyrode's buffer (Wash-P), and lyophilized platelets in plasma (Lyo-P). ***: p < 0.001. g) Time course of vessel diameter changes during PRP perfusion. h-j) Representative images of cryo-sectioned microvessels following perfusion with h) control media, i) PRP, and j) EnP with enlarged views of dash boxes in insets. Red: Collagen IV, green: vWF, blue: nuclei. k-m) Fluorescence images after 3 min perfusion of 70 kDa FITC-dextran through microvessels treated with k) control media, l) PRP, and m) EnP. n) Fluorescence intensity comparison across vessel network region (white dash boxes in (k-m)), with left vessel center set as x = 0. o) Quantification and comparison of apparent solute permeability for vessels in three conditions (control, PRP, and EnP). **: p < 0.01. Scale bars: c-e) 100 µm (50 µm in enlargements); h-j) 100 µm (25 µm in enlargements) k-m) 100 µm.
Platelets Can Activate and Form Plugs when a Minimum Endothelium Defect is Exceeded
Platelets are classically believed to become activated following adhesion to exposed lamina then aggregate and recruit other platelets to form a plug during primary hemostasis. When perfused through normal engineered microvessels, some platelets adhered to microvessels but failed to form large aggregates ( Figure S1, Supporting Information). To verify the activation status and aggregation capacity of the platelets in PRP, we created a band injury within individual microvessel branches (Figure 2a). The endothelium in three vessel branches was photoablated to create 150 µm long hyperrectangular defects. The endothelium was removed in the photoablated regions along with small amount of basal collagen ( Figure 2a). Within 30 min following photoablation, vessels were treated with PRP, EnP, or control media for 24 h (Figure 2b-d). In microvessels perfused with control media, ECs grew over the ablation sites, with increased vessel permeability in that region (Figure 2b,e). In the PRP-treated microvessels, a thrombus formed that was confined to the injury site ( Figure 2c). Regions outside the injury site exhibited consistent barrier enhancement and vessel enlargement as seen through fluorescently conjugated dextran perfusion ( Figure 2f). No thrombus was observed with EnP perfusion and the endothelium grew over the ablation sites, with increased dextran permeability, similar to control (Figure 2d,g). When ablated regions were reduced to 10, 25, and 50 µm in diameter and 20 µm depth, cells in the ablated areas were able to heal completely following 24 h PRP treatment with no signs of thrombus formation ( Figure S3, Supporting Information). Together these data suggest that platelets are normally not activated in PRP-treated microvessels but can activate and form plugs when a minimum defect size is exceeded.
Platelets Adhere Abluminally and Cause EC Cytoskeletal Reorganization
We next examined the ultrastructural interactions of platelets and endothelium in 3D microvessels following PRP treatment using transmission electron microscopy (TEM). Pronounced ultrastructural changes occurred to ECs at different time points (i.e., 1 and 24 h) post PRP treatment which were not seen in vessels treated with EnP ( Figure S4, Supporting Information). At 1 h following PRP treatment, the majority of platelets (P) were located on the abluminal side (A) of ECs without significant aggregation (Figure 3a . Ultrastructural analysis of engineered microvessels treated with PRP. a) Following 1 h PRP perfusion, platelet (labeled as P) distribution was only observed i) abluminally (labeled as A) or in some cases ii) internalized within ECs. iii,iv) Increased microtubule activation, mitochondrial (labeled with *) elongation, and fragmentation were visualized. v,vi) Electron dense material, appeared as dark granules, was found evenly distributed along the endothelium and in cell junctions. b) i,ii) After 24 h PRP perfusion, partial activation of platelets (P) could be distinguished through the loss of dense granules and presence of pseudopodia (#). iii,iv) Microtubule and mitochondrial (*) activation were still observed. v) Complex and vi-viii) overlapping cell junctions could be visualized. A: ablumen, L: lumen, P: platelets, C: cells, Col: collagen substrate. of platelets adhered, most of which lost their granules, likely during initial adhesion and partial activation, but maintained intact membranes. Some adherent platelets still contained numerous granules, and occasionally, rounded platelets rich in granules were also found internalized within ECs (Figure 3aii). Increased actin polymerization and microtubules were seen throughout the cytosol of the ECs, with apparent cortical assembly (Figure 3a-iii). Morphological changes were found in mitochondria, including elongation in excess of several micrometers and fragmentation of the membrane (asterisks, Figure 3a-iv). In addition, an electron-dense layer was formed on the luminal surface (L) of the endothelium (Figure 3a-v) and appeared to bridge gaps between adjacent ECs (Figure 3a-vi). At 24 h, intact platelets were again found on the abluminal surface of the endothelium (Figure 3b-i). Cortical actin assembly was appeared to progress into a submembranous web within the endothelium (Figure 3b-iii). Mitochondria showed extended length inside the cells, exceeding several micrometers (asterisks, Figure 3b-iv). Adjacent ECs developed long overlapping junctions with lengths exceeding 30 µm (Figure 3b-vi,vii). Overall, these ultrastructural data suggest that ECs underwent substantial structural reorganization and appeared under tension with partial platelet activation and minimal aggregation.
Molecular Signatures Demonstrate PRP Treatment Leads to Endothelial Maturation
To gain better understanding of molecular characteristics of the endothelial response to PRP, we lysed engineered microvessels and collected RNA for transcription profiling following exposure to three conditions (N = 3, 2, 2 for control, PRP, and EnP conditions, respectively; each replicate was pooled from three microvessels at the same condition). Principle component analysis illustrated distinct clustering of PRP-treated cells versus control, whereas EnP-treated cells were clustered between the two but closer to the control (Figure 4a). Differential expression analysis identified 366 genes differentially expressed with statistical significance in the PRP-treated endothelium compared to the control, with 231 genes upregulated and 135 genes downregulated (fold change >1.5 and false discovery rate (FDR) < 0.05) (Figure 4b). Gene Ontology (GO) terminology analysis showed PRP-treated vessels have significant upregulation of transcripts from genes involved in blood vessel morphogenesis, vascular development, regulation of smooth muscle cell (SMC) proliferation, response to oxygen levels, angiogenesis, and other vascular and circulatory system development (Figure 4c-i). Canonical pathway analysis identified HIF1 signaling as the top upregulated pathway in PRP-treated microvessels. In contrast, EnP-treated vessels showed 56 differentially expressed genes with 28 upregulated compared to the control. Of these 28 genes, 17 were also upregulated in PRP treatment. GO analysis revealed that EnP-treated microvessels had significant upregulation of pathways involved in chemokine and cytokine signaling, regulation of chemotaxis, and inflammatory responses, suggesting EnP-stimulated microvessels with cytokines and chemokines (Figure 4c-ii). This is likely due to the factors released from unstimulated platelets, as suggested in previous work. [7] The top genes that were upregulated in PRP-treated microvessels compared to control include transcription factors such as SMAD7, PPARG, ID1/2/3, CEBPD, ANKRD1, and GATA3, most of which are known to regulate angiogenesis and vascular development; [13] transporters and receptors such as SLC2A1, SLC16A3, SLC2A3, SLC27A3, SLC2A10, SLC12A4, VLDLR; transmembrane proteins such as CLDN3, ITGB4, VCAM1, ENG, LRP1, are known to promote cell adhesion to cells and matrices; growth factors such as
PRP Shifts EC Metabolism from OXPHOS to Glycolysis
Mature endothelium in vivo relies heavily on glycolysis for adenosine triphosphate (ATP) production. We next examined metabolic activities via gene expression and mitochondrial function. Among differentially expressed genes, a subset related to metabolic activity was identified in PRP-treated microvessels that suggested a shift from oxidative phosphorylation (OXPHOS) to glycolysis. In total, 34 genes associated with glycolysis were upregulated including 7 enzymes involved in glucose and lactate transport (PGM1, TPI1, ALDOC, SLC2A3, SLC2A1, ENO2, SLC16A3) that were selected for comparison (Figure 5a). Euclidian distance hierarchical clustering demonstrated that PRP groups were distinct from control and EnP-treated conditions (Figure 5a).
We then used MitoTracker green to directly visualize mitochondrial mass and structure via fluorescence imaging, which showed dense perinuclear mitochondrial networks in control microvessels ( Figure 5b); these became punctate and fragmented at 24 h following PRP treatment (Figure 5c). This is consistent with the ultrastructural analysis, which showed elongation and membrane disruption (asterisks) of mitochondria (M) in endothelium after PRP treatment (Figure 3a-iv,b-iv). In comparison, the mitochondria in EnP-treated microvessels incurred no such changes in mitochondria ultrastructure ( Figure S4a, Supporting Information). These findings suggest a dynamic change to metabolic activity following PRP treatment with general trends in association with the reduction of mitochondrial mass and activity.
To measure OXPHOS activity within ECs after PRP treatment, we perfused tetramethylrhodamine methyl ester (TMRM) and MitoTracker green through microvessels at 1 and 24 h, and quantified mitochondrial membrane potential and total mitochondrial mass, respectively, using confocal fluorescence microscopy ( Figure S5 Fluorescence levels were normalized following the addition of the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to depolarize the mitochondrial membrane. The normalized membrane potential of mitochondria within ECs was diminished following PRP treatment (2.3 ± 0.42 and 2.9 ± 0.48 for 1 and 24 h treatments, respectively) compared to control (3.3 ± 0.97) (Figure 5d-i,ii). The total mitochondrial mass normalized to control was also diminished following exposure to PRP, falling from 1.0 ± 0.17 in control vessels to 0.72 ± 0.26 and 0.54 ± 0.11 after 1 and 24 h PRP exposure, respectively (Figure 5d-iii,iv). However, when normalized to total mitochondrial mass, the activity of remaining mitochondria in PRP-treated microvessels was unaffected after 1 h (3.2 ± 0.58) and elevated after 24 h (5.4 ± 0.89) compared to control (3.3 ± 0.97) (Figure 5d-ii), These results indicate that although total mitochondrial mass and membrane potential decreased, the mitochondria remaining following PRP perfusion were functioning at normal and elevated activity levels at 1 and 24 h. Together, reduced mitochondrial mass and membrane potential support the idea of reduced contribution of OXPHOS to support cellular energetic demand and a shift toward glycolysis.
PRP Enhances SMC Density around Engineered Vessels
Another important component involved in the maturation and stabilization of the developing endothelium is mural cell support. PRP treatment appeared to upregulate the expression of EC genes involved in SMC proliferation (CCL5, EDN1, IGFBP3, NPR3, BMP4, PPARG, TNFAIP3 (PDGFB, RRAS, SMAD6, SMAD9) (Figure 6a). To assess the functional ramifications of these gene changes, we incorporated green fluorescent protein (GFP)-expressing coronary SMCs in the matrix surrounding our microvessels. These co-cultured microvessels were maintained for 3 days prior to the addition of PRP or control medium for 24 h. Vascular patency remained throughout PRP perfusion for 24 h with no thrombus formation (Figure 6b). SMC density significantly increased in the PRP-treated vessels compared to control vessels (1.44 ± 0.17-fold) (Figure 6c). These findings suggest that PRP could potentially promote EC maturation via enhanced mural cell support.
PRP Improves Antithrombotic Potential of Engineered Microvessels
Healthy mature vasculature has antiadhesive and antithrombotic properties. We therefore examined the effect of PRP treatment on blood cell adhesion on engineered vessel walls with or without prior PRP treatment. Whole blood was perfused through the microvessels for 15 min with platelets labeled with CD41a-PE and visualized in brightfield and immunofluorescence microscopy after washing and fixation. There were fewer adherent platelets (arrowheads) and red blood cells (RBCs), and fewer RBC extravasations and petechiae (chevrons) in PRP-treated microvessels compared to control (Figure 6d). PRP-treated microvessels had over a 90% reduction in platelet area coverage, from (8.0 ± 1.8)% in control to (0.8 ± 0.4)% (Figure 6e). CD41a-PE platelets were also seen in larger aggregates in control than that on PRP-treated microvessels. PRPtreated vessels appeared to have abundant residual platelet material (positive for phalloidin and von Willebrand factor (vWF) staining but were negative for CD41a-PE) ( Figure S6, Supporting Information), suggesting these residual platelets were likely in the abluminal side of the endothelium, as seen in ultrastructure analyses.
Discussion
Over 50 years ago platelets were found to be vasculoprotective during ex vivo organ perfusion. [3] Many subsequent studies have sought to understand the mechanism of this phenomenon, which falls outside the normal hemostatic function of platelets. Platelets contain many molecules capable of influencing other cells. With regard to endothelial function, some of these improve barrier function whereas others increase vessel permeability. [15] Although platelets are of obvious importance to endothelial maintenance and functions, the mechanisms by which they carry out these activities have been hard to elucidate. Conventional 2D studies lack the characteristics required for normal physiological interactions between ECs and platelets, e.g., 3D luminal geometry, pressure and flow relationships, and appropriate cell-cell contacts. In this study, we used 3D engineered microvessels to demonstrate that PRP transformed the endothelium by improving vessel barrier function, expanding vessel diameter, thickening the abluminal lining, promoting glycolysis, and enhancing smooth muscle recruitment and antithrombogenicity. These observations are unique in 3D microvessels, whereas in 2D endothelial cultures, bound platelets were found to disrupt EC junctions, leading to cell retraction.
Normal plasma has high protein content, resulting in a high oncotic pressure gradient of approximately 30 cmH 2 O between our engineered microvessel lumen and the surrounding matrix, whereas the hydrostatic pressure gradient across the microvessel lumen is 1 cmH 2 O. Based on the Starling equation, the overall pressure drop would lead to rapid water reabsorption into the vessel, increasing the vessel volume and wall tension. This increase of vessel diameter over time suggests that platelets enhance vessel barrier and maintenance of the oncotic pressure drops across the vessel wall. The increased wall tension is also suggested in our ultrastructural analysis, which identified increased cytoskeleton polymerization in vessels after 1 h PRP perfusion and the formation of a submembranous actin web after 24 h PRP perfusion. The EnP-treated microvessels, though having the same oncotic pressure difference, failed to induce these vessel wall changes. Furthermore, lyophilized platelets perfused in EnP also failed to induce these changes, suggesting physical interactions with metabolically active platelets are critical to induce vessel wall changes in this process. This substantial cytoskeletal remodeling could also be in response to the partial activation of the adhered platelets. Many of the factors released by platelets are associated with inflammatory responses and endothelial reorganization. The long-term effects of these processes on endothelial homeostasis will require further investigation.
Platelets appear to interact with the vessel wall in a biphasic manner. During PRP perfusion in normal vessels, platelets failed to form large aggregates or vascular occlusions. Platelets were found covered and sometimes internalized by the endothelium as soon as 1 h after perfusion and maintained the same morphology after 24 h with no signs of further activation nor aggregation. In contrast, when a large region of the vessel was denuded of endothelium, platelets became locally activated and formed a plug to occlude a vessel branch. When the denuded region was reduced, no platelet plugs formed and the vessel did not become occluded. This suggests healthy endothelium provides negative cues to suppress platelet activation and aggregation.
PRP also promoted vascular maturation and improved functionality. Our transcriptional profiling of 3D engineered microvessels after PRP treatment showed significant upregulation of transcripts involved in vascular development and remodeling. Although genes associated with inflammation were upregulated, GO term and canonical pathway analysis suggest that PRP treatment predominantly affects vascular development and maturation instead of vascular activation. We focused on three aspects of vascular functions supported by corresponding gene regulation: metabolism, mural cell support, and thrombogenicity. Among the >300 differentially expressed genes after PRP treatment, 10% (over 30) were directly associated with glycolysis. The upregulation of transcripts involved in glycolysis coincided with decreased mitochondrial activity in the microvessels, which provide evidence of a shift in EC metabolic activity. In normal blood vessels, glycolysis accounts for over 85% of the total cellular ATP production in ECs, [16] and mitochondrial activity remains low in mature vasculature. It remains unknown how this change in EC metabolism will affect long-term tissue growth and remodeling in vitro or tissue survival after implantation in vivo. It is possible that vascular pre-conditioning toward physiological conditions could promote better survival and integration once implanted.
Our data also showed PRP treatment upregulates transcripts in platelet-derived growth factor signaling and increases mural cell density around microvessels, suggesting platelets could be contributing to vascular development. Though the direct coating of endothelium by mural cells was not obvious, the increase of mural cell density suggests effective paracrine signaling after 24 h PRP treatment. Future long-term studies could facilitate our understanding of the role of platelets in mural cell recruitment and continued vascular development. The decreased platelet binding and blood cell interactions after PRP treatment suggest that PRP could further serve as a pre-conditioning procedure to reduce vascular thrombogenicity prior to anastomosis in vivo.
Overall, our work demonstrates that PRP transforms the endothelium into mature and functional vessels. Although the exact mechanisms of this transformation are yet to be discovered, our work opens up new opportunities to study the role of blood components in the development of a functional vasculature and potentially in engineering complex and functional tissue.
Experimental Section
Unless otherwise stated, all materials were purchased from Sigma Aldrich. Fresh blood was drawn from consenting healthy donors, who were nonsmokers and drug and aspirin free for at least 3 days prior to blood donation, under protocols approved by the Institutional Review Board of the University of Washington.
Perfusate Preparation: PRP were obtained by fractionating 3.2% sodium citrate-treated whole blood at 200 g for 15 min with no brake. The PRP portion was then moved to a separate tube with a transfer pipet and allowed to rest for several hours prior to use. For EnP, PRP was centrifuged at 1200 g for 10 min, the supernatant was collected and centrifuged again at 1200 g for 10 min. The supernatant collected after second spin was gently filtered through a 0.2 µm cellulose membrane to further eliminate platelets from the plasma. EnP was selected as a control to PRP given the presence of soluble growth factors that had been used to decrease EC permeability in 2D monolayers previously, as well as equivalent oncotic pressures resulting from the proteins within plasma. [6] Lyophilized platelet solution was made by adding lyophilized platelets (Bio/Data Corp., Horsham, PA) to the EnP at 2.5 × 10 8 mL −1 .
Despite the inclusion of different blood donors, the same response of PRP, EnP, and control media was repeatedly observed, further supporting the efficacy of PRP as a therapeutic agent.
Cell Culture: HUVECs-a commonly used pooled-population of ECs whose lack of a blood antigen allows for the use of fresh donor blood without the need for donor-matching-were used. HUVECs (Lonza) were cultured according to the manufacturer's guidelines using EBM Basal Medium supplemented with EGM Endothelial Cell Growth Medium SingleQuots Supplements (Lonza). For all experiments, HUVECs were used between passage 3 and 7. For 2D experiments, collagen-coated coverglass was used as a substrate upon which HUVECs were cultured to confluency prior to perfusate treatment. In addition, human coronary artery smooth muscle cells (HCASMCs, Lonza CC-2583) were cultured in SmGM-2 smooth muscle cell growth medium (Lonza, CC-3182). This cell type was transduced with pGIPZ NSC lentivirus overnight in SmGM-2 supplemented with 10 ug mL −1 protamine sulfate (Sigma, P3369) to generate GFP-SMCs. Purified GFP-SMCs were then selected 2 days after transduction in SmGM-2 supplemented with 2 ug mL −1 puromycin (InvivoGen, ant-pr-1) for 1 week. GFP-SMCs were expanded in SmGM-2 and used from P2-4. Microvascular Network Fabrication: Microvascular networks were made from 7.5 mg mL −1 collagen gel as described previously. [11b] Briefly, collagen was injected into a polyethyleneimine/glutaraldehydetreated Plexiglas housing top half that formed negative impression of a microvessel network along with a polydimethylsiloxane (PDMS) stamp. Inlet and outlet ports were formed by inserting stainless steel dowel pins before injecting the collagen. The Plexiglas bottom half was consisted of a flat layer of collagen compressed by a flat PDMS surface that was gelled on top of a standard coverslip. The collagen was allowed to gel for 30 min at 37 °C. After gelation, the PDMS pieces were removed and the two halves were brought together to seal the microvessel network. Culture medium was then added to the reservoirs and incubated for 2 h before cell seeding. To seed the vessels, 10 µL injections of HUVECs at a density of 8 × 10 6 cells mL −1 were delivered to the inlet of an aspirated channel. Flow was allowed by replenishing media in the inlet reservoir twice per day for at least 3 days. Perfusates were then delivered via the inlet reservoir.
Immunofluorescence Analysis: Immunofluorescence images of cells on slides and within intact microvessels in situ were taken using a Nikon A1R Confocal Microscope (Nikon, Tokyo, Japan). Image stacks were acquired with a z-step between successive optical slices of 2-3 µm. Cross-sections, projections, and 3D reconstructions were generated from z-stacks of images using ImageJ software.
In 2D and 3D experiments, vWF expression was quantified by setting a binary threshold on the vWF immunofluorescence channel. The area of vWF was corresponded to the number of pixels counted in the image using ImageJ. This number was divided by the total number of pixels in the region of interest and then, multiplied by 100 to give the percentage of vWF present in a given area of cells. Three images were analyzed for three separate devices. Data for each device were then averaged and used to calculate standard error of the mean (SEM) with n = 3.
Ultrastructural Analysis: Microfluidic vascular networks were examined under TEM as described previously. [17] Briefly, networks were fixed by half-strength Karnovsky's solution (2% paraformaldehyde/2.5% glutaraldehyde in 0.2 m cacodylate buffer) for 20 min followed by full immersion following disassembly in the same fixative solution for several days. Next, samples were post-fixed with 2% OsO 4 in 0.2 m cacodylate buffer, then dehydrated using immersions in graded solutions of ethanol, then propylene oxide (PO), before 1:1 PO/Epon 812 (Ted Pella Inc., Redding, CA) immersion overnight. Epon blocks were cured for 48 h at 60 °C then sectioned into ultrathin sections (70 nm) and placed onto grids. Grids were stained with uranyl acetate for 2 h and lead citrate for 5 min, then imaged using a JEOL JEM-1400 Transmission Electron Microscope (JEOL Ltd., Japan) using a typical acceleration voltage around 100 kV. Images were acquired with a Gatan Ultrascan 1000XP camera (Gatan, Inc., Pleasanton, CA).
For 3D constructs, mitochondrial membrane potential and mass were quantified by confocal microscopy with a Nikon A1R microscope, using a 10x objective. Cells were stained with 30 × 10 −9 m TMRM (Invitrogen) and 50 × 10 −9 m MitoTracker Green (Invitrogen) for 30 min at 37 °C. To achieve baseline TMRM fluorescence, z-stack images of the constructs were acquired every 60 s for 5 min. Mitochondria were depolarized using 20 m CCCP (Sigma-Aldrich) and the resulting fluorescence value was subtracted from the baseline fluorescence to obtain the normalized mitochondrial membrane potential intensity.
Permeability Assessment: Perfusion of 70 kDa FITC-dextran was driven by a hydrostatic pressure drop through the vessel. Using fluorescence microscopy with a 1 s sampling rate for 1 min, the distribution of fluorescence intensity during the transient flow of dextran was used to estimate the averaged apparent permeability of the kidney endothelium using Matlab processing in methods similar to Yuan et al. [18] Fluorescence plot profiles were obtained using ImageJ software.
RNA-seq Data Analysis: Total RNA was extracted from engineered microvessels by perfusing RLT buffer into the network at 250 µL min −1 for 1 min. Using an RNeasy kit (Qiagen), RNA was purified to reach 400 ng at concentration above 50 ng mL −1 for RNA-seq. Base calling was performed using Illumina's Real-Time Analysis software v1.18.66.3. Reads that failed to pass Illumina's base call quality filter were discarded, followed by using Cutadapt v1.11 to trim adapter sequence, discarding paired reads where either read in the pair had a length < 25. RNA-seq samples were aligned to hg38 using Tophat. [19] Gene-level read counts were quantified using htseq-count using Ensembl GRCh37 gene annotations. [20] Genes with total expression above 10 normalized read counts summed across RNA-seq samples were kept for further analysis. The princomp function from R was used for principal component analysis. DESeq was used for differential gene expression analysis with fold changes >1.5 and FDR < 0.1 as criteria to be considered differentially expressed. [21] The topGO R package was used for GO enrichment analysis. [22] Gene expression data set was then submitted to the public database in Gene Expression Omnibus.
Whole-Blood Perfusion: Whole blood was drawn fresh and then fractionated by centrifugation at 200 g for 15 min. PRP was then separated and platelets were labeled by incubating with CD41a-PE for 30 min at room temperature. Whole blood was then reconstituted and perfused at constant pressure for 5 min under live fluorescence imaging with a 1 s sampling rate.
Two-Photon Ablation Injury Model: Vessel injury was performed by multiphoton photoablation using a Mai Tai DeepSee Ti:S laser (maximum power 2.57 W) coupled with an Olympus FV1000 MPE BX61 Microscope fitted with a water-immersion objective lens (25x, NA = 1.05). Vessel regions were first identified for injury by conventional multiphoton imaging of constitutively expressing GFP-expressing HUVECs and by second harmonic generation of collagen microfibers (λ ex = 860 nm, λ detector = 420-460 nm). Areas to be damaged were defined as 3D regions of interest using the Olympus Fluoview software then ablated by laser rastering performed at λ = 800 nm using 100% laser power, a pixel dwell time of 2 µs, and with 25-30 line repeat scans. Laser scanning in the X, Y, and Z dimensions was performed at ≈1 um step sizes. Cellular and collagen damage was confirmed by imaging identical areas after ablation using transmitted light, conventional multiphoton microscopy, or by second harmonic generation of collagen microfibers. Despite the presence of sodium citrate in the donor blood, there was the capability to induce coagulation in microvessels subjected to injury of 150 µm in length, suggesting that the lack of coagulation during PRP perfusion was not an artifact of this system.
Statistical Analysis: For all quantitative measurements, the entire population was used to calculate statistical significance, whereas mean values with n ≥ 3 groups were used to calculate standard error and graphical confidence intervals. Data were then analyzed in RStudio (Boston, MA) using a one-way analysis of variance test, followed by post hoc analysis using Tukey-HSD to determine significance between groups. On each graph, error bars represent ±2 SEM, a 95% confidence interval. A single asterisk was used for p-values < 0.05, two asterisks for p < 0.01, three asterisks for p < 0.001, and four asterisks for p < 0.0001.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. | v3-fos-license |
2018-12-20T14:03:06.059Z | 2018-12-01T00:00:00.000 | 56480025 | {
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} | pes2o/s2orc | Inhibitory Effect of Synthetic Flavone Derivatives on Pan-Aurora Kinases: Induction of G2/M Cell-Cycle Arrest and Apoptosis in HCT116 Human Colon Cancer Cells
Members of the aurora kinase family are Ser/Thr kinases involved in regulating mitosis. Multiple promising clinical trials to target aurora kinases are in development. To discover flavones showing growth inhibitory effects on cancer cells, 36 flavone derivatives were prepared, and their cytotoxicity was measured using a long-term clonogenic survival assay. Their half-maximal growth inhibitory effects against HCT116 human colon cancer cells were observed at the sub-micromolar level. Pharmacophores were derived based on three-dimensional quantitative structure–activity calculations. Because plant-derived flavones inhibit aurora kinase B, we selected 5-methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one (derivative 31), which showed the best half-maximal cell growth inhibitory effect, and tested whether it can inhibit aurora kinases in HCT116 colon cancer cells. We found that derivative 31 inhibited the phosphorylation of aurora kinases A, aurora kinases B and aurora kinases C, suggesting that derivative 31 is a potential pan-aurora kinase inhibitor. The results of our analysis of the binding modes between derivative 31 and aurora A and aurora B kinases using in-silico docking were consistent with the pharmacophores proposed in this study.
Introduction
Flavones are a class of flavonoids having a backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one). They are common in fruits and many plant foods. Some natural flavones show anticancer activity. For example, apigenin (4 ,5,7-trihydroxyflavone), luteolin (3 ,4 ,5,7tetrahydroxyflavone), baicalein (5,6,7-trihydroxyflavone), nobiletin (3 ,4 ,5,6,7,8-hexamethoxyflavone) and tangeretin (4 ,5,6,7,8-pentamethoxyflavone) inhibit the proliferation of breast cancer cells [1][2][3]. Kaempferol (3,4 ,5,7-tetrahydroxyflavone) suppresses the growth of bladder and cervical cancer cells [4]. Chrysin (5,7-dihydroxyflavone) reduces the proliferation of prostate cancer cells [5]. Quercetagetin (3,3 ,4 ,5,6,7-hexahydroxyflavone) induces apoptosis in colon cancer cells [6]. However, the structural features of flavones exhibiting inhibitory effects on cancer cell growth remain unclear. 2 of 13 In order to identify the structural characteristics of flavones exhibiting growth inhibitory effects on cancer cells, we prepared 36 synthetic flavone derivatives containing hydroxy, fluoro, bromo, nitro, methoxy, methyl, styryl or naphthalenyl groups [7]. Colon cancer is the second most diagnosed cancer in women and the third in men [8]. It is also the second most death-causing cancer [8]. Tumor penetration and metastasis can occur in stage II of colon cancer; thus, its survival rate is approximately 70% after stage II. Even when the resection of colon cancer was achieved by the first treatment of colon cancer owing to diagnosis at the early stage, chemotherapy is often required simultaneously with other treatments [9]. Several drugs have been approved as colon cancer chemotherapy. Drugs as adjuvant chemotherapy after surgery are being developed [10]. Here, we used a long-term clonogenic assay to measure the growth inhibitory effects of synthetic flavones in HCT116 human colon cancer cells. The half-maximal cell growth inhibitory concentration (GI 50 ) values of 36 flavone derivatives against HCT116 cells were determined. To derive structural features that exhibit better cell growth inhibitory effects, relationships between the structural properties of the synthetic flavones and their cell growth inhibitory effects were calculated using comparative molecular field analysis and comparative molecular similarity index analysis. Among the 36 synthetic flavone derivatives, we found that 5-methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one (named derivative 31) showed the best GI 50 value (0.49 µM). Because we have shown in our previous study that plant-derived flavones inhibit aurora kinase B (AURKB) [6], we further evaluated the effect of derivative 31 on the inhibition of aurora kinases. The molecular binding modes between the derivative 31 compound and aurora kinases were elucidated using in-silico docking experiments. These results can provide valuable information for designing novel anticancer drugs that target aurora kinases.
Results and Discussion
To investigate the structural features of flavones that influence the growth of cancer cells, we synthesized 36 flavone derivatives and screened candidate compounds by monitoring their growth inhibitory efficacy by a long-term clonogenic survival assay [11], which can efficiently distinguish differences in cell growth rate induced by structurally similar compounds [12,13].
Initially, we tested the inhibitory activity of the derivatives at high (0, 5, 10, 20 and 40 µM) ( Figure 1A) and low concentrations (0, 0.1, 0.5, 1 and 5 µM) ( Figure 1B) on the clonogenicity of cancer cells. Clonogenicity was quantitated by densitometry, GI 50 values were computed using the SigmaPlot software, and the results are shown in Table 1 and Figure 2. Table 1. Names of synthetic flavone derivatives 1-36, and their half-maximal cell growth inhibitory effect (GI 50 ) and the negative logarithmic scales of GI 50 values (pGI 50 ). Table 1. Figure 2. Half-maximal cell growth inhibitory concentration (GI 50 ) values in Table 1.
Their GI 50 values ranged between 0.49 and 41.19 µM. Among the derivatives, derivative 31, 5-methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one, showed the most effective inhibition of clonogenicity (GI 50 value: 0.49 µM). Negative logarithmic scales of the GI 50 values (pGI 50 ) were used as biological data for 3D-QSAR calculation. The 3D structures of the derivatives required for 3D-QSAR calculations were modified using the Sybyl 7.3 program from the X-ray crystallographic structures of derivatives 6 (2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one) and 18 (2-(2,3-dimethoxynaphthalen-1-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one), which were determined by the authors' previous works [14,15]. Three-dimensional quantitative structure-activity relationship (3D-QSAR) was performed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). We divided the 36 derivatives into a training set and a test set. The former was used to generate QSAR models and the latter was used to validate the models generated by the training set. Seven derivatives, namely 7, 10, 14, 17, 22, 29 and 32, were arbitrarily chosen for the test set by one of the data analysis tools, hierarchical clustering analysis [16], as shown in Figure S1. Twenty-nine derivatives in the training set were aligned using the Sybyl DATABASE Alignment module. They were aligned well, which indicated interactions between probe atoms and rest atoms ( Figure S2). Linear correlations between structural properties of the derivatives in the training set and their cancer cell growth inhibitory effects were determined using partial least-squares regression. Among the many CoMFA models generated by iteration, until a good cross-validation correlation coefficient (q 2 ) was found, the model showing 0.772 of q 2 was chosen, where non-cross-validated correlation coefficient (r 2 ), optimal number of components, standard error of estimate, and F value were 0.980, 6, 0.078, and 175.833, respectively. pGI 50 values were predicted based on this model. A comparison of the pGI 50 values obtained from the long-term clonogenic survival assay with the values predicted using the CoMFA model is listed in Table S1, and its graph is shown in Figure S3. The residuals between the experimental data and the predicted values ranged from 0.11% to 8.29%. Likewise, the pGI 50 values of the derivatives contained in the test set were calculated using the same CoMFA model (Table S1). The residuals between the experimental data and the predicted values ranged from 6.79% to 24.96%. These results showed that this CoMFA model was reliable. In this model, the steric and electrostatic field descriptors were 51.7% and 48.3%, respectively. To visualize the field descriptors, we generated contour maps. The steric-bulk-favoring and -disfavoring regions occupied 92% and 8% of the maps, respectively ( Figure S4A). In the steric field map of the CoMFA model, green contours were shown at C2 and C3 positions, meaning that a bulky group was favored in the region. Derivatives containing bulky naphthyl group at the region, such as derivatives 15-18, 22-26, 29-32 and 36, showed good GI 50 values, which were 7.51 µM and less. Derivatives 9-14 have the bulky resveratrol group at C2 and showed good activities, with GI 50 values ranging between 3.63 µM and 2.41 µM. On the contrary, a yellow contour was observed near C4 , meaning that a bulky group was not favored in the region. It is explicable why some of the derivatives containing methoxy group at the C4 position, such as derivatives 4, 8, 33, 34 and 35, showed poor GI 50 values. The electrostatic contour map of the CoMFA model is shown in the Figure S4B. The electrostaticly favoring and disfavoring regions occupied 1% and 99% of the maps, respectively. In the electrostatic field map of the CoMFA model, a small red contour was observed around C2 . This may be the reason derivatives with fluoride or methoxy substitutes at C2 showed low biological activities, as shown by derivatives 1, 2, 5 and 8.
Unlike CoMFA, CoMSIA provides more field descriptors, including hydrogen bond donor and acceptor descriptors and hydrophobic descriptors. To find the best CoMSIA model, many CoMSIA models were generated by iteration. The CoMSIA model showing q 2 of 0.515 was selected, where the r 2 , optimal number of components, standard error of estimate, and F value were 0.952, 6, 0.119, and 73.084, respectively. This model consisted of steric, electrostatic and hydrophobic descriptors, and their contributions were 18.3%, 53.3% and 28.4%, respectively. The pGI 50 values calculated using this model were compared with those obtained from the experimental values (Table S2), and they were graphed as shown in Figure S5. The residuals between the experimental data and predicted values ranged from 0.18% to 10.62%. Likewise, the pGI 50 values of derivatives contained in the test set were calculated using the same CoMFA model (Table S2). The residuals between the experimental data and predicted values ranged from 7.34% to 23.92%. These results showed that this CoMSIA model was reliable. The steric and electrostatic field descriptors were generated to be similar to those obtained from the CoMFA model. A hydrophobic field map ( Figure S6) was additionally obtained from the CoMSIA model. The hydrophobic region occupied 95% of the contour map area, whereas the nonhydrophobic region occupied 5%. The orange-colored contour around C4 suggested that a hydrophobic group was favored at the position. The derivatives 33-35 had a methoxy substituent at the C4 of thier naphthyl moiety and showed lower activities compared to derivatives 22-26, which had naphthyl without a hydrophilic substituent. The pharmacophores obtained based on the CoMFA and CoMSIA models are summarized in Figure 3, which provides insights for rational design of compounds with good inhibitory effects on cell growth. In summary, a bulky group was favored at C2 and C3 but was not favored at C4 . A hydrophobic group was favored at C4 , and an electronegative group was not favored at C2 . One of the criteria for the solubility is logP. The logP values of derivatives containing a 3-hydroxy group ranged between 1.53 and 3.02, those of derivatives with a styryl group, 3.72 and 5.45, and logP values of naphthoflavones ranged from 3.69 to 4.07.
the nonhydrophobic region occupied 5%. The orange-colored contour around C4′ suggested that a hydrophobic group was favored at the position. The derivatives 33-35 had a methoxy substituent at the C4′ of thier naphthyl moiety and showed lower activities compared to derivatives 22-26, which had naphthyl without a hydrophilic substituent. The pharmacophores obtained based on the CoMFA and CoMSIA models are summarized in Figure 3, which provides insights for rational design of compounds with good inhibitory effects on cell growth. In summary, a bulky group was favored at C2′ and C3′ but was not favored at C4′. A hydrophobic group was favored at C4′, and an electronegative group was not favored at C2′. One of the criteria for the solubility is logP. The logP values of derivatives containing a 3-hydroxy group ranged between 1.53 and 3.02, those of derivatives with a styryl group, 3.72 and 5.45, and logP values of naphthoflavones ranged from 3.69 to 4.07. Aurora kinases are Ser/Thr kinases that function as key regulators of chromosome alignment and segregation during mitosis [17]. There are three classes of aurora kinases: aurora kinase A (AURKA), aurora kinase B (AURKB) and aurora kinase C (AURKC). Previously, we showed that plant-derived flavones inhibit AURKB [6]. To investigate whether synthetic flavone derivatives inhibit aurora kinase activity, we selected one of the compounds, derivative 31, which exhibited the best GI50 value, and examined its inhibitory activity against aurora kinases. aurora kinase activity was assessed by its phosphorylation status, as reported previously [18]. Treatment with derivative 31 decreased the phosphorylation of AURKA on Thr-288, AURKB on Thr-232, and AURKC on Thr-198 in a dose-( Figure 4A) and time-dependent ( Figure 4B) manner, suggesting that derivative 31 exhibited pan-aurora kinase inhibitory activity. Aurora kinases are Ser/Thr kinases that function as key regulators of chromosome alignment and segregation during mitosis [17]. There are three classes of aurora kinases: aurora kinase A (AURKA), aurora kinase B (AURKB) and aurora kinase C (AURKC). Previously, we showed that plant-derived flavones inhibit AURKB [6]. To investigate whether synthetic flavone derivatives inhibit aurora kinase activity, we selected one of the compounds, derivative 31, which exhibited the best GI 50 value, and examined its inhibitory activity against aurora kinases. aurora kinase activity was assessed by its phosphorylation status, as reported previously [18]. Treatment with derivative 31 decreased the phosphorylation of AURKA on Thr-288, AURKB on Thr-232, and AURKC on Thr-198 in a dose-( Figure 4A) and time-dependent ( Figure 4B) manner, suggesting that derivative 31 exhibited pan-aurora kinase inhibitory activity. best GI50 value, and examined its inhibitory activity against aurora kinases. aurora kinase activity was assessed by its phosphorylation status, as reported previously [18]. Treatment with derivative 31 decreased the phosphorylation of AURKA on Thr-288, AURKB on Thr-232, and AURKC on Thr-198 in a dose-( Figure 4A) and time-dependent ( Figure 4B) manner, suggesting that derivative 31 exhibited pan-aurora kinase inhibitory activity. AURKA and AURKB are overexpressed in colon cancer [19], and inhibition of aurora kinases triggers mitotic cell-cycle arrest and apoptotic cell death [18]. Therefore, we investigated by flow cytometry whether derivative 31 affects cell cycle progression. After treatment with derivative 31, population of G2/M phase cells increased from 26.2% (0 h) to 43.3% (12 h) and 46.3% (24 h) ( Figure 5A). Notably, the number of sub-G1 phase cells remarkably increased from 3% (0 h) to 31.5% (48 h) AURKA and AURKB are overexpressed in colon cancer [19], and inhibition of aurora kinases triggers mitotic cell-cycle arrest and apoptotic cell death [18]. Therefore, we investigated by flow cytometry whether derivative 31 affects cell cycle progression. After treatment with derivative 31, population of G2/M phase cells increased from 26.2% (0 h) to 43.3% (12 h) and 46.3% (24 h) ( Figure 5A). Notably, the number of sub-G1 phase cells remarkably increased from 3% (0 h) to 31.5% (48 h) as the number of G1 phase cells concomitantly decreased from 59.9% (0 h) to 8.0% (48 h) ( Figure 5B). Because the presence of sub-G1 cell population is indicative of the progression of apoptotic cells, we suggest that derivative 31 induced cell-cycle arrest at the G2/M phase at the early stage but triggered apoptotic cell death in HCT116 colon cancer cells after continued exposure. We thus evaluated the capability of derivative 31 to induce apoptosis. Because the phosphatidylserine localized in the inner surface of the cell membrane translocates to the outer membrane during apoptosis [20], we analyzed the population of apoptotic cells by staining the outer layer of the cell membrane with phosphatidylserine by using annexin V [21]. Propidium iodide was used as a counterstain to label dead cells. Flow cytometry results showed that treatment with derivative 31 at 5 and 10 μM increased the population of annexin V-positive cells from 8% to 25% and 73%, respectively ( Figure 6A). These data suggested that derivative 31 caused apoptotic cell death in HCT116 cells. Caspases regulate the cleavage of many cellular proteins, including the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), to induce apoptosis [22]. Caspases are activated by Because the phosphatidylserine localized in the inner surface of the cell membrane translocates to the outer membrane during apoptosis [20], we analyzed the population of apoptotic cells by staining the outer layer of the cell membrane with phosphatidylserine by using annexin V [21]. Propidium iodide was used as a counterstain to label dead cells. Flow cytometry results showed that treatment with derivative 31 at 5 and 10 µM increased the population of annexin V-positive cells from 8% to 25% and 73%, respectively ( Figure 6A). These data suggested that derivative 31 caused apoptotic cell death in HCT116 cells. Caspases regulate the cleavage of many cellular proteins, including the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), to induce apoptosis [22]. Caspases are activated by proteolytic cleavages [23]. To determine whether derivative 31-induced apoptosis is mediated by caspases, we examined the status of caspase 7 cleavage by Western blotting analysis. We found that the cleavages of caspase 7 and its substrate, PARP, were increased by treatment with derivative 31 in a time-dependent manner ( Figure 6B). Taken together, derivative 31 triggered apoptosis by inhibiting aurora kinases through G2/M cell-cycle arrest and a caspase-dependent mechanism. To elucidate the binding modes between derivative 31 and aurora kinases at the molecular level, in-silico docking experiments were conducted. Among the many X-ray crystallographic structures of AURKA deposited in the protein data bank, 3uod.pdb was selected because its ligand, 4-[(4-[2-(trifluoromethyl)phenyl]amino]pyrimidin-2-yl)amino]benzoic acid (named as TPB) ( Figure S7), is more similar to the synthetic flavones used here than the ligands contained in other crystallographic structures deposited in the protein data bank [24]. Its organism, expression system and resolution To elucidate the binding modes between derivative 31 and aurora kinases at the molecular level, in-silico docking experiments were conducted. Among the many X-ray crystallographic structures of AURKA deposited in the protein data bank, 3uod.pdb was selected because its ligand, 4-[(4-[2-(trifluoromethyl)phenyl]amino]pyrimidin-2-yl)amino]benzoic acid (named as TPB) ( Figure S7), is more similar to the synthetic flavones used here than the ligands contained in other crystallographic structures deposited in the protein data bank [24]. Its organism, expression system and resolution were Homo sapiens, Escherichia coli BL21(DE3) and 2.5 Å, respectively. AURKA consists of 403 residues and 3uod.pdb contains residues between Ser123-Lys401, including a kinase domain. This structure contains its ligand as well as 1,2-ethanediol and di(hydroxyethyl)ether. To prepare the apoprotein of 3uod.pdb, its ligand was extracted using the Sybyl/Biopolymer module (Tripos), but 1,2-ethanediol and di(hydroxyethyl)ether were not deleted. The solution structure of the apoprotein was obtained through energy minimization using the Conjugate Gradient algorithm where Tripos force field and Gasteiger-Hückell charges were used. Because comparing this apoprotein with 3uod.pdb resulted in a root-mean-squared deviation value of 0.7 Å, this apoprotein was used for in-silico docking experiments. As mentioned above, the 3D structure of the title compound was determined based on the X-ray crystallographic structure of derivative 18 (2-(2,3-dimethoxynaphthalen-1-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one) [15]. The results obtained from AutoDock Vina were visualized using PyMol (The PyMOL Molecular Graphics System, Version 1.0r1; Schrödinger, LLC), and analyzed using LigPlot [25]. The binding pocket of AURKA was analyzed using the Ligplot software, and 14 residues were obtained: Arg137, Leu139, Gly140, Val147, Ala160, Leu194, Glu211, Tyr212, Ala213, Thr217, Arg220, Glu260, Leu263 and Ala213 ( Figure S8). The dimensions of the docking box were 16, 8 and 16 for x, y and z, respectively, whereas the centers of x, y and z were 21.494, -21.987 and -10.808, respectively. Because the flexible docking procedure was iterated 30 times, 30 AURKA apoprotein-ligand complexes were generated. Because the original ligand, TPB, was docked into the apoprotein well, in-silico docking of derivative 31 was performed in the same manner as that of the original ligand. Because its binding energies ranged from -9.1 to -7.2 kcal/mol, the thermodynamic stability of the docking process of derivative 31 was considered good for further analysis. The complex with the lowest binding energy was selected. The residues residing in its binding pocket were analyzed using LigPlot: Arg137, Leu139, Val147, Ala160, Leu194, Leu210, Glu211, Tyr212, Ala213, Gly216, Thr217, Arg220, Glu260, Leu263 and Asp274 ( Figure S9). The binding pocket was visualized using the PyMol program as shown in Figure 7. Even the AURKA-derivative 31 complex included one more residue in its binding site than the AURKA-TPB complex, and it does not contain hydrogen bonds, unlike the AURKA-TPB complex where two residues, Arg137 and Ala213, participated in hydrogen bonds. The binding pocket around the naphthalene ring of derivative 31 consisted of mainly hydrophobic residues, Val147, Leu210 and Leu263, and the binding pocket was deep and wide enough to hold a naphthyl or resveratrol group. It is also well explained that a bulky group is not favored at the C4 position because the side chain of Tyr212 could induce steric hindrance with substrates. The results of our analysis of the binding mode of derivative 31 are consistent with the pharmacophores that we proposed.
original ligand. Because its binding energies ranged from -9.1 to -7.2 kcal/mol, the thermodynamic stability of the docking process of derivative 31 was considered good for further analysis. The complex with the lowest binding energy was selected. The residues residing in its binding pocket were analyzed using LigPlot: Arg137, Leu139, Val147, Ala160, Leu194, Leu210, Glu211, Tyr212, Ala213, Gly216, Thr217, Arg220, Glu260, Leu263 and Asp274 ( Figure S9). The binding pocket was visualized using the PyMol program as shown in Figure 7. Even the AURKA-derivative 31 complex included one more residue in its binding site than the AURKA-TPB complex, and it does not contain hydrogen bonds, unlike the AURKA-TPB complex where two residues, Arg137 and Ala213, participated in hydrogen bonds. The binding pocket around the naphthalene ring of derivative 31 consisted of mainly hydrophobic residues, Val147, Leu210 and Leu263, and the binding pocket was deep and wide enough to hold a naphthyl or resveratrol group. It is also well explained that a bulky group is not favored at the C4′ position because the side chain of Tyr212 could induce steric hindrance with substrates. The results of our analysis of the binding mode of derivative 31 are consistent with the pharmacophores that we proposed. Because Western blotting analysis showed that treatment with derivative 31 decreased the phosphorylation of AURKB, the binding mode between derivative 31 and AURKB was elucidated using in-silico docking in the same manner as that of AURKA. Because 4af3.pdb contained the most residues, it was used for in-silico docking [26]. It originated from H. sapiens and was expressed in an E. coli BL21 (DE3) system. Its ligand was cyclopropanecarboxylic acid 4-[4-(4-methyl-piperazin-1-yl)-6-(5-methyl-2h-pyrazol-3-ylamino)-pyrimidin-2-ylsulfanyl]-phenyl]-amide (named as VX6). The binding pocket of AURKB was analyzed using Ligplot: Leu83, Phe88, Val91, Ala104, Lys106, Leu138, Glu155, Tyr156, Ala157, Gly160, Glu161, Leu207, Ala217, Asp218 and Phe219 ( Figure S10). The dimensions and centers of the docking box were the same as those in the AURKA docking condition. Because the original ligand, VX6, was docked into the apoprotein well, in-silico docking of derivative 31 was performed in the same manner as that of the original ligand. The binding energies of 30 AURKBderivative 31 complexes ranged from -9.6 to -7.8 kcal/mol, which showed that the complexes were thermodynamically stable. The complex with the lowest binding energy was selected. The residues residing in the binding pocket of the complex were analyzed using LigPlot: Leu83, Phe88, Val91, Ala104, Lys106, Glu155, Tyr156, Ala157, Glu161, Glu204, Asn205, Leu207, Ala217 and Phe219 ( Figure S11). The binding pocket was visualized using the PyMol program as shown in Figure 8. The AURKB-derivative 31 complex contained fewer residues in its binding pocket than the AURKB-VX6 complex. In addition, the AURK-VX6 complex included two hydrogen bonds at Lys106 and Glu155, whereas the AURKB-derivative 31 complex consisted of only hydrophobic interactions. Like the AURKA-derivative 31 complex, the naphthalenyl group is surrounded by hydrophobic residues, Leu83, Phe88, Ala157 and Leu207, and the side chain of Tyr156 resides in the pocket near the naphthalenyl group. However, the hydrophilic residue Glu161 was near the same pocket; thus, the docking of derivative 31 was not favored compared to that of AURKA. The results of Western blotting analysis showed that even though derivative 31 decreased the phosphorylation of both AURKA and AURKB in a dose-and time-dependent manner, the binding modes of derivative 31 to AURKA and AURKB at the molecular level were different from each other.
In conclusion, 36 synthetic flavone derivatives at micromolar concentrations showed halfmaximal cell growth inhibitory effects against HCT116 human colon cancer cells. The structural conditions that showed good inhibitory effects on the growth of colon cancer cells were derived based on 3D-QSAR calculations, including the CoMFA and CoMSIA methods, where a bulky group was favored at C2′ and C3′ but was not favored at C4′′, a hydrophobic group was favored at C4′, and an electronegative group was not favored at C2′. In our previous study, a flavone derivative inhibited AURKB; thus, Western blotting analysis was performed on derivative 31, which showed the best halfmaximal inhibitory effect on cell growth. Because treatment with derivative 31 decreased the phosphorylation of AURKA, AURKB and AURKC in a dose-and time-dependent manner, this derivative was considered to exhibit pan-aurora kinase inhibitory activity. In addition, flow The AURKB-derivative 31 complex contained fewer residues in its binding pocket than the AURKB-VX6 complex. In addition, the AURK-VX6 complex included two hydrogen bonds at Lys106 and Glu155, whereas the AURKB-derivative 31 complex consisted of only hydrophobic interactions. Like the AURKA-derivative 31 complex, the naphthalenyl group is surrounded by hydrophobic residues, Leu83, Phe88, Ala157 and Leu207, and the side chain of Tyr156 resides in the pocket near the naphthalenyl group. However, the hydrophilic residue Glu161 was near the same pocket; thus, the docking of derivative 31 was not favored compared to that of AURKA. The results of Western blotting analysis showed that even though derivative 31 decreased the phosphorylation of both AURKA and AURKB in a dose-and time-dependent manner, the binding modes of derivative 31 to AURKA and AURKB at the molecular level were different from each other.
In conclusion, 36 synthetic flavone derivatives at micromolar concentrations showed half-maximal cell growth inhibitory effects against HCT116 human colon cancer cells. The structural conditions that showed good inhibitory effects on the growth of colon cancer cells were derived based on 3D-QSAR calculations, including the CoMFA and CoMSIA methods, where a bulky group was favored at C2 and C3 but was not favored at C4", a hydrophobic group was favored at C4 , and an electronegative group was not favored at C2 . In our previous study, a flavone derivative inhibited AURKB; thus, Western blotting analysis was performed on derivative 31, which showed the best half-maximal inhibitory effect on cell growth. Because treatment with derivative 31 decreased the phosphorylation of AURKA, AURKB and AURKC in a dose-and time-dependent manner, this derivative was considered to exhibit pan-aurora kinase inhibitory activity. In addition, flow cytometry results showed that derivative 31 induced apoptosis, and annexin V staining results showed that it triggered apoptosis by inhibiting aurora kinases through G2/M cell-cycle arrest and a caspase-dependent mechanism. The results of binding mode analysis between derivative 31 and AURKA and AURKB at the molecular level using in-silico docking were consistent with the pharmacophores that we proposed. As a result, the synthetic flavone studied here can be developed as a pan-aurora kinase inhibitor and a chemotherapeutic agent.
Preparation of 36 Synthetic Flavone Derivatives
The synthesis and identification of flavone derivatives containing hydroxy, fluoro, bromo, nitro, methoxy, methyl, styryl, and/or naphthalenyl groups were reported previously [7]. The synthetic scheme is provided as Scheme S1 [7]. The names of the derivatives are listed in Table 1. Infrared (IR) spectra were collected using an FT-IR 4200 spectrophotometer (JASCO, Easton, MD, USA) with attenuated total reflection (ATR PR0450-S). IR data as well as the melting points, yields, and purities are provided in the Supplementary Materials. The structures of the derivatives are provided as Table S3. IR spectra are provided in the Supplementary Materials.
Cell Culture
HCT116 human colon cancer cells were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (CellGro/Corning, Manassas, VA, USA) at 37 • C in a 5% CO 2 atmosphere [27].
Clonogenic Long-Term Survival Assay
A long-term clonogenic assay was conducted as described previously [11]. Cells were treated with flavone derivatives (0, 5, 10, 20, and 40 µM) for six days. At seven days after treatment, the cells were stained with 0.1% crystal violet. Among the 36 derivatives, 24 derivatives inhibited the growth of the cancer cells almost completely; thus their clonogenicities were measured at lower concentrations (0, 0.1, 0.5, 1, and 5 µM). The inhibitory activities of flavone derivatives on clonogenicity were measured using densitometry (MultiGuage, Fujifilm, Japan), and GI 50 values were computed using the SigmaPlot software (version 12, SYSTAT, Chicago, IL, USA) [28].
Cell-Cycle Analysis by Flow Cytometry
Cell-cycle status was examined by flow cytometry using propidium iodide [29]. Briefly, HCT116 cells were treated with 5 µM derivative 31 for 0, 12, and 24 h, and fixed in 70% (v/v) ethanol. Next, the cells were stained with 50 µg/mL propidium iodide solution containing 0.1% (v/v) Triton X-100, 0.1 mM EDTA, and 50 µg/mL RNase A. Cellular DNA contents were detected by a NucleoCounter NC-3000 cytometer (ChemoMetec, Allerød, Denmark). Diploid (2N) and tetraploid (4N) cells represented cells at the G1 and G2/M phases, respectively. 2N and 4N cells corresponded to those at the S phase. Cells containing DNA lower than 2N DNA were considered as cells at the sub-G1 phase [30].
Western Blotting Analysis
HCT116 cells were treated with derivative 31 for the indicated times. Cell lysates were prepared and immunoblotted according to standard procedures. Antibody-reactive protein bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Antibodies against phospho-aurora kinase A (T288)/aurora kinase B (T232)/aurora kinase C (T198), cleaved caspase-7 (Asp198), and poly(ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific to GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) [6].
In-Silico Docking
To elucidate the molecular binding modes between the title compound and aurora kinases, in-silico docking was conducted using AutoDock Vina. In addition, preparation of holoproteins and apoproteins, as well as determination of binding site were performed using the Sybyl program (Tripos) [32]. The 3D structures of aurora kinases were obtained from the protein databank. The experiments followed previously reported methods [27].
Statistical Analysis
Statistical significance was analyzed using Student s t-test [6]. A p-value of less than 0.05 was considered statistically significant. All experiments were performed in triplicate.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
2020-01-30T09:15:13.494Z | 2020-01-22T00:00:00.000 | 213915847 | {
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} | pes2o/s2orc | Cordycepin resensitizes T24R2, cisplatin-resistant human bladder cancer cell, to cisplatin by inhibiting the AKT-mediated Ets-1 activation
Gene & Cell Therapy Team, Division of Drug Development & Optimization, New Drug Development Center, Osong Medical Innovation Foundation, Osongsaengmyung-ro 123, Osong-eup, Heungdeok-gu, Cheongju-si 28160, Chungbuk, Republic of Korea Division of Applied Life Science (BK21 Plus), Division of Life Science, PMBBRC, Gyeongsang National University, Jinju, 52828, Republic of Korea Freshwater Bioresources Utilization Bureau, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea
Introduction
Although chemotherapy is the most effective treatment, the patient's clinical response to this treatment varies widely, and survival is often unsatisfactory. The resistance to the anti-cancer drugs often hampers the therapeutic effect of chemotherapy. Conventional anti-cancer drugs actually have different targets and action mechanisms, and most of them kill most cancer cells by inducing apoptosis.
However, due to the imbalanced regulation of the apoptotic machinery, a small subset of cancer cells can acquire resistance to anti-cancer drugs [1].
Resistance to anti-cancer drugs is conferred on a multitude of mechanisms, including the decreased drug influx [2], the increased drug efflux [3], the activation of DNA repair mechanism [4], among others.
The most studied mechanism for drug resistance in tumor cells is an increased efflux of anti-cancer drugs. This mechanism is very effective in preserving intracellular drug concentrations below the apoptosis-triggering threshold by using a variety of ATP-dependent active drug transporters, such as multi-drug resistant protein 1 (MDR1), breast cancer resistant protein (BCRP), and multi-drug resistance-related protein (MRP1) [5,6]. These active transporters in cellular membranes play an important role in not only recognizing anti-cancer drugs but also detoxifying intracellular compartments by pumping these drugs out of intracellular compartments. Novel pharmacologic approaches to overcome the drug resistance in cancer have been established in recent decades. For instance, targeting active drug transporters such as MDR1 can resensitize drug-resistant tumor cells to anti-cancer drugs [7,8].
Cordycepin resensitize T24R2 to cisplatin
The MTT assay was used to confirm the resistance of T24R2 cells to cisplatin. Cell viability was quantified 24 h after the cisplatin treatment of T24 and T24R2 at the concentrations of 1μg/mL and 2μg/mL. As shown in figure 1A, although cell death of T24 cells was increased in a concentrationdependent manner, no significant effect has been observed in T24R2 cells, which displayed clear resistant to cisplatin. To investigate the effect of cordycepin on T24R2, we treated T24R2 cells with various concentration of cordycepin only or mixture of cordycepin and cisplatin, and measured the cell viability using MTT assay ( Figure 1B). Whereas cordycepin-induced cytotoxicity in T24R2 was slightly increased at a high dose of cordycepin (50μg/mL), the combined treatment of cordycepin and cisplatin significantly induced cell death starting at 20μg/mL of cordycepin. Cytotoxicity caused by apoptosis was confirmed by sub-G1/0 ( Figure 1C) and TUNEL assay ( Figure 1D). These data suggest that cordycepin resensitizes T24R2 to cisplatin. cordycepin and 2 g/ml cisplatin was analyzed by TUNEL and DAPI staining. * P < 0.05, ** P < 0.01. *** P < 0.001 by t-test.
Cordycepin-mediated resensitization of T24R2 to cisplatin is induced by apoptosis via the mitochondrial pathway
To investigate the mechanism of cordycepin-mediated resensitization of T24R2 to cisplatin, we assessed the protein expression levels involved in caspase pathway activation. In brief, T24R2 cells were treated with cisplatin (2μg/mL) and cordycepin (20μg/mL) for 24h, and subsequently the levels of caspase-3, -9 and PARP were evaluated by western blotting. Interestingly, the combined treatment of cisplatin and cordycepin induced cleavage of caspase-3, -9, and PARP ( Figure 2A). As shown in Figure 2B, whereas the anti-apoptotic proteins, Mcl-1, Bcl-2, and Bcl-xl were significantly reduced, the pro-apoptotic proteins, Bax and Bak, were considerably increased in T24R2 cotreated with cordycepin and cisplatin. Moreover, to confirm that the combination of cordycepin and cisplatin activated the caspase cascade in T24R2 cells via the mitochondrial pathway, we examined active Bax redistribution. The combined treatment induced active Bax translocation to the mitochondrial membrane as shown in Figure 2C. In addition, the effect of the combined treatment induced a significant loss of the mitochondrial potential, which is considered as a hallmark of apoptosis ( Figure 2D). These data clearly demonstrate that cordycepin combined with cisplatin induces apoptosis in T24R2 cells via the mitochondrial pathway-dependent caspase activation cascade. T24R2 cells were treated with 20 μg/mL cordycepin and/or 2 μg/mL cisplatin for 24h, and then the whole cell extracts were analyzed by western blotting using the each indicated antibody against caspase-3, -9, PARP cleavage (A), anti-apoptotic, and pro-apoptotic proteins (B). α-tubulin was used as an internal control to confirm equal loading of proteins. T24R2 cells treated with cisplatin and/or cordycepin were immunostained with anti-active Bax and analyzed using a confocal microscope. (C) Active Bax was labeled with a FITC-conjugated secondary antibody (green), and the mitochondria and nuclei were stained with Mitotracker CMXRos (red) and DAPI (blue), respectively. (D) The cells were stained with MitoTracker Red CMXRos, and the depolarization of mitochondria was analyzed 8 by flow cytometry.
Reduction of MDR1 expression is involved in cordycepin-mediated resenstization of T24R2
MDR1 expression was more increased in T24R2 compared with T24 ( Figure 3A) and was downregulated by cordycepin at the transcriptional level ( Figure 3B and C). Moreover, to confirm that MDR1 is directly associated with resistance of T24R2 cells to cisplatin, we proceeded to knockdown MDR1 in T24R2. After treatment of 2μg/mL cisplatin for 24h, siMDR1-transfected T24R2 showed very higher cell death rate compared with siGFP-transfected T24R2 ( Figure 3D), reduction of antiapoptotic proteins, Bcl-2 and Mcl-1, and induction of pro-apoptotic proteins, Bax and Bak ( Figure 3E).
To confirm whether cordycepin could inhibit the drug efflux mediated through decrease of MDR1 expression, we assayed the intracellular level of rhodamine in T24, T24R2, cordycepin-treated T24R2, and siMDR1 T24R2. T24R2 cells exhibited very low level of cytosolic rhodamine accumulation, whereas T24 cells contained high levels of rhodamine. As expected, cordycepin-treated T24R2 and siMDR1 T24R2 contained high level of rhodamine ( Figure 3F). Taken together, these suggest that cordycepin-mediated MDR1 reduction is a major mechanism inducing resensitization of T24R2 to cisplatin. Intensity). *** P < 0.001 by t-test.
Cordycepin regulates MDR1 promoter activity
Because the level of MDR1 mRNA was significantly decreased in T24R2 cells treated with cordycepin ( Figure 3B), we investigated whether cordycepin suppressed MDR1 expression by down-regulating MDR1 promoter activity. Using the dual luciferase reporter gene assay system, MDR1 promoter (from -751 to +122 bp) activity was compared in T24 and T24R2 cells. As shown in figure 4A, T24R2 cells showed a higher luciferase activity than T24 cells. Furthermore, cordycepin treatment reduced the promoter activity in T24R2 to the same level as in T24 ( Figure 4B). A previous report suggested that Ets-1 activated the human MDR1 promoter in the human osteosarcoma, Saos-2 [15]. To investigate whether Ets-1 is necessary for MDR1 expression in T24R2 cells, we constructed Ets1 mt-1, Ets-1 mt-2, and Ets-1 mt-1&2 with mutations in the Ets-1 binding sequence of the MDR1 promoter, as shown in Figure 4C. Both Ets-1 binding sequences are necessary for MDR1 promoter activation in T24R2.
To confirm whether Ets-1 binded to MDR1 promoter in T24R2 and whether the binding inhibited by cordycepin treatment, we performed ChiP assay with an anti-Ets-1 antibody. Ets-1 did not bind to the promoter in T24, whereas Ets-1 bound directly to the MDR1 promoter in T24R2. Cordycepin effectively inhibited the binding of Ets-1 on MDR1 promoter ( Figure 4D). Therefore, we suggest that cordycepin downregulates MDR1 expression via inhibition of transcription factor Ets-1 activity, which makes T24R2 sensitize to cisplatin. activity. (D) Using genomic DNA from T24, T24R2, and cordycepin-treated T24R2 cells, we performed chromatin immunoprecipitation (ChIP)-PCR. ** P < 0.01. *** P < 0.001 by t-test.
Cordycepin inhibits AKT activation that phosphorylates Ets-1
To determine whether a correlation exists between the expression of Ets-1 and that of MDR1, the cBioPortal database for cancer genomics (http://www.cbioportal.org) was utilized in January 2018.
When the correlation was analyzed with the mRNA levels of Ets-1 and MDR1 from 413 bladder cancer patients, there was a positive correlation between mRNA expression levels of two genes ( Figure 5A).
Although cordycepin reduced MDR1 expression in transcription level ( Figure 3B and C), transcript level and protein level of Ets-1 were unaffected by cordycepin ( Figure 5B). These data suggested that cordycepin might reduce MDR1 expression through inhibiting Ets-1 activity but not its expression.
Accordingly, we investigated the post-translational modification of Ets-1 affected by cordycepin. The phosphorylation of Ets-1 (T308) was known to induce its activation as a transcription factor [16]. As shown in Figure 5C, the phosphorylation of Ets-1 was attenuated in cordycepin-treated T24R2, which was followed by a reduction of MDR1 expression. In particular, the activation of AKT was significantly inhibited in cordycepin-treated T24R2 ( Figure 5D), and inhibition of AKT using wortmannin, a specific inhibitor of PI3K, also attenuated Ets-1 phosphorylation and reduced MDR1 expression as observed in cordycepin-treated T24R2 ( Figure 5E). To confirm whether AKT inhibition conferred the improved sensitivity to cisplatin in T24R2, these cells were treated with cordycepin or wortmannin in the presence or absence of cisplatin. Wortmannin treatment induced resensitization of T24R2 to cisplatin similar to cordycepin treatment ( Figure 5F). Taken together, inhibition of AKT activation by cordycepin attenuates Ets-1 activity, which leads to resensitization of T24R2 to cisplatin through the reduction of pEts-1. flow cytometry analysis. *** P < 0.001 by t-test.
Discussion
Although chemotherapy is one of the representative and effective methods of tumor therapy, the acquisition of drug resistance in tumor cells is one of the major obstacles for improvement of therapy efficacy. Therefore, the development of strategies to overcome anti-cancer drug resistance should be considered as an important field in chemotherapy. One of these strategies is to combine two or more drugs to the patient, and the establishment of combination therapy strategies and drug selection are considered very important.
Cordyceps militaris is a fungus used as a natural tonic agent in oriental traditional medicine, and is reported to have many biological functions, including anti-inflammation [17], immune modulation [18,19], anti-angiogenesis [11], and having anti-tumor properties [12,13]. Cordycepin, also known as 3'deoxyadenosine, is known to be a primary bioactive component of C. militaris. The tumor-suppressive effect of cordycepin has been reported in various carcinoma cells [20][21][22].
In this study, we found that cordycepin attenuated Ets-1 activity through inhibition of AKT, which binding cassette (ABC) transporter, which is an integral plasma membrane protein [23]. MDR-1 reduces the efficacy of chemotherapy by shortening the retention time of intracellular drugs. In fact, high expression of MDR-1 induces drug resistance in many tumors. Thus the inhibition of MDR-1 is a potential strategy for resensitizing anti-cancer drug resistant cancer cells to the drug [24]. T24R2 cell line showed higher expression of MDR1 compared with T24, and cordycepin inhibited MDR1 expression at the transcriptional level ( Figure 3). The inhibition of MDR1 expression using siMDR1 eliminated the resistance of T24R2 to cisplatin and prolonged the retention of intracellular drug as in the case of cordycepin-treated T24R2 cells. Therefore, cordycepin might resensitize T24R2 cells to cisplatin via reduction of MDR1 expression. MDR1 promoter activity was actually higher in T24R2 compared to T24, and the activity was decreased in cordycepin-treated T24R2. Ets-1 is an essential transcription factor regulating the expression of MDR1 at the transcriptional level and promotes anticancer drug resistance in various tumors [15,25]. The activity of Ets-1 was much higher in T24R2 than in T24, and the was eliminated by mutation of the Ets-1 binding sites on the MDR1 promote activity in T24R2. In addition, the binding of Ets-1 to the MDR1 promoter disappeared in cordycepin-treated T24R2. Therefore, cordycepin inhibits MDR1 expression via inhibition of Ets-1 activity.
Various growth factors, such as hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, increase the mRNA level of Ets-1 through the Ras/Raf/MEK/ERK1/2 pathway. MDR1 expression showed a quantitative linear correlation with Ets-1 expression at transcript level in human bladder cancer patients analysis using cBioPortal for cancer genomics (http://www.cbioportal.org). Although T24 and T24R2 showed similar Ets-1 expression levels of mRNA and protein, MDR1 expression was higher in T24R2 than in T24. Therefore, cordycepinmediated reduction of MDR1 expression should not be dependent on Ets-1 expression. Various posttranslational modifications, such as phosphorylation, ubiquitination, sumoylation, acetylation, and parylation, are known to regulate Ets-1 activity [26]. Among the modifications of Ets-1, Thr-38 phosphorylation of Est-1 by ERK1/2 was well defined [27][28][29]. Strangely, no significant decrease in ERK activation was observed in T24R2 treated with cordycepin, while a significant inhibition in PI3Kmediated AKT activation was observed. PI3K/AKT inhibition using wortmannin significantly inhibited the phosphorylation of Ets-1 and MDR1 expression in T24R2 ( Figure 5E), and also resensitized T24R2 to cisplatin ( Figure 5F). Although it was known that AKT can affect Ets-1 expression levels [30], cordycepin treatment did not affect total Ets-1 expression levels.
These results suggest that cordycepin inhibits MDR1 expression and resensitizes T24R2 to cisplatin through inhibition of PI3K/AKT pathway. Active PI3K/AKT pathway induces transcriptional upregulation of MDR1 through activation of Ets-1. Therefore, cordycepin may be a candidate for combination therapy with cisplatin in the treatment of patients exhibiting cisplatin resistance.
Antibodies and reagents
Cordycepin purified from Cordyceps militaris was purchased from Sigma, and cisplatin was purchased AKT(Thr308), and anti-AKT antibodies were purchased from Cell Signaling Technology (MA, USA).
MTT assay
An MTT assay was performed on the T24 and T24R2 cells to determine cell viability. Cells were plated in 96-well plate (5×10 3 cells/well) and incubated for 24 h in the presence or absence of cisplatin and/or cordycepin. MTT solution (5 mg/mL) was added to the wells and then the cells were incubated for another 3 h. Following incubation, the medium was removed and 200 μl DMSO was added to each well to extract the formazan products produced by viable cells. The absorbance of the solutions was measured on a Bio-Rad 550 microplate reader at 595 nm. The relative cell viability (%) was determined by comparing the absorbance at 595 nm with control, which was treated with DMSO.
PI staining
The
RNA isolation and real-time RT-PCR analysis
Total RNA was extracted from cells using the total RNA isolation solution (RiboEx TM ; GeneAll, Seoul,
Immunoblotting analysis
Total proteins extracted from cells were quantified by using the Bradford method.
Construction of luciferase reporter plasmid and dual luciferase assay
The 873 bp fragment (from -751 to +122) was excised from a pMDR1-1202 cloning recombinant T24, T24R2, and cordycepin-treated T24R2 cells were fixed with 1 % formaldehyde solution.
Chromatin immunoprecipitation (ChIP) assay
Chromatins was prepared from the fixed cells and fragmented by sonication. Immunoprecipitation of cross-linked protein/DNA was performed by using the Ets-1 (C-4) mouse monoclonal antibody. Briefly, 2 μg of the Ets-1 antibody was bound to chromatin mixture at 4 ℃ overnight with rotation, and then 30 μl of protein A magnetic beads (Merck Millipore) was added and additionally incubated at 4 ℃ overnight with rotation. Then real-time PCR analysis using precipitated DNA was performed. The following primers were used for amplification: MDR1 promoter, 5′-CGTTTCTCTACT TGCCCTTTCT-3′ and 5′-CCGGATTGACTGAATGCTGAT-3′.
Statistical analysis
The obtained data were expressed as the means ± SD from three experiments. Differences among means were analyzed using a one-way ANOVA or student's t-test. P < 0.05 was significantly different. | v3-fos-license |
2020-06-25T09:09:23.036Z | 2020-06-01T00:00:00.000 | 220047650 | {
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} | pes2o/s2orc | Computational Insights on the Mechanism of the Chemiluminescence Reaction of New Group of Chemiluminogens—10-Methyl-9-thiophenoxycarbonylacridinium Cations
Immunodiagnostics, in which one of the promising procedures is the chemiluminescent labelling, is essential to facilitate the detection of infections in a human organism. One of the standards commonly used in luminometric assays is luminol, which characterized by low quantum yield in aqueous environments. Acridinium esters have better characteristics in this topic. Therefore, the search for new derivatives, especially those characterized by the higher quantum yield of chemiluminescence, is one of the aims of the research undertaken. Using the proposed mechanism of chemiluminescence, we examined the effect of replacing a single atom within a center of reaction on the efficient transformation of substrates into electronically excited products. The density functional theory (DFT) and time dependent (TD) DFT calculated thermodynamic and kinetic data concerning the chemiluminescence and competitive dark pathways suggests that some of the scrutinized derivatives have better characteristics than the chemiluminogens used so far. Synthesis of these candidates for efficient chemiluminogens, followed by studies of their chemiluminescent properties, and ultimately in chemiluminescent labelling, are further steps to confirm their potential applicability in immunodiagnostics.
Introduction
Chemical demonstrations are very popular and allow the promotion of science, especially among children and youth. Spectacular chemical experiments, especially those that are unexpected, explosive and colorful, attract the full auditoria. One of the most amazing chemical experiments successfully presented at chemical demonstrations is chemiluminescence (CL) [1], in which by mixing several substances a luminous product is formed [2]. In fact, chemiluminescence is a phenomenon of the emission of light as a result of a chemical reaction [3]. In this reaction, a product is formed in an electronically excited state [4,5]. When an electronically excited electron returns to a ground state, an energy is released in a form of light (as a photon) [5]. In addition to cognitive features, chemiluminescence has also found many utilitarian applications in clinical [6,7], pharmaceutical [8], chemical and biochemical analysis [9], and also in environmental [10] and food analysis [11].
The acridinium derivatives are one of the most important groups of compounds used in the chemiluminometric analysis. Their biggest advantage is the relatively high quantum yield of chemiluminescence-up to 7% [34]. It is also important that acridinium esters are easy to synthesize and oxidize in an aqueous alkaline environment [12,43]. However, the search for new derivatives, especially those characterized by the higher quantum yield of chemiluminescence, is one of the aims of the research undertaken. Taking into account previous investigations on the mechanism of chemiluminescence of acridinium derivatives, it is known that the type of the leaving group is affected by the efficiency of the formation of the electronically excited products [12,17,35]. Therefore, the studies on new chemiluminogens, in which a phenoxy group was replaced by a thiophenoxy group, were initiated.
In the current work, we computationally investigate a series of 10-methyl-9thiophenoxycarbonylacridinium derivatives, comparing their susceptibility to oxidation in an alkaline media. We suggest the mechanism of the chemiluminescence reaction of new group of compounds based on acridine moiety, considering both, light-leading to the energy-rich product and dark-leading to competitive products, reaction pathways. Given the possibilities of modern quantum chemistry methods, we focused our study on the ground state reaction profiles and formation of the electronically excited 10-methyl-9-acridinone. By understanding the Scheme 1. Proposed chemiluminescence mechanism of acridinium derivatives.
The acridinium derivatives are one of the most important groups of compounds used in the chemiluminometric analysis. Their biggest advantage is the relatively high quantum yield of chemiluminescence-up to 7% [34]. It is also important that acridinium esters are easy to synthesize and oxidize in an aqueous alkaline environment [12,43]. However, the search for new derivatives, especially those characterized by the higher quantum yield of chemiluminescence, is one of the aims of the research undertaken. Taking into account previous investigations on the mechanism of chemiluminescence of acridinium derivatives, it is known that the type of the leaving group is affected by the efficiency of the formation of the electronically excited products [12,17,35]. Therefore, the studies on new chemiluminogens, in which a phenoxy group was replaced by a thiophenoxy group, were initiated.
In the current work, we computationally investigate a series of 10-methyl-9thiophenoxycarbonylacridinium derivatives, comparing their susceptibility to oxidation in an alkaline media. We suggest the mechanism of the chemiluminescence reaction of new group of compounds based on acridine moiety, considering both, light-leading to the energy-rich product and dark-leading to competitive products, reaction pathways. Given the possibilities of modern quantum chemistry methods, we focused our study on the ground state reaction profiles and formation of the electronically excited 10-methyl-9-acridinone. By understanding the chemiluminescence mechanism, new potential chemiluminogens with specificity and susceptibility could be rationally designed.
Selection of Investigated Models
Our previous investigations [12,13,17] on the mechanism of the chemiluminescence of acridinium derivatives indicate that the type of the leaving group is affected the efficiency of the formation of the electronically excited products. For this reason, we began to analyze the molecules, in which elimination of the leaving group could be much easier than in acridinium esters and thus transformation into cyclic intermediate, leading to the electronically excited product, much efficient. We decided to replace only one atom in the leaving group (Scheme 2) and check how such a small difference in the molecular structure of acridinium esters will affect the efficiency of the chemiluminescence reaction. The selection of the replacing atom was based on the electronegativity of chemical elements (χ in the Pauling scale is equal to 3.44 for oxygen atom and 2.58 for sulphur atom [44]) and structural properties within the leaving group of the investigated compounds. The bond lengths in the thiocarboxyl group are much longer than in the carboxyl group (the C-S bond is equal to 1.8 Å, the C-O bond is ca. 1.3 Å) and the C-C bond between thiocarboxyl group and acridine moiety in acridinium thioester (Scheme 2, ATE) increases slightly compared to the same bond in acridinium ester (Scheme 2, AE). The reduction of electronegativity and increasing the bond lengths within the leaving group suggest easier elimination of the leaving group and thus more efficient transformation to the electronically excited product. Other aspects to consider are feasible for the synthesis of new chemiluminogens after the oxygen-by-sulphur exchange and their stability of using in applications. Based on the literature [1,5,12,13,17], it can be predicted that the synthesis of acridinium thioesters will be similar to those of acridinium esters and will proceed in three steps: the conversion of acridine-9-carboxylic acid into 9-chlorocarbonyl acridine hydrochloride, the esterification of 9-chlorocarbonyl acridine to acridine thioester and the methylation of the endocyclic nitrogen atom to obtain acridinium salt. Considering the physical or/and chemical properties of the sulphur atom, it can be assumed that also acridinium thioesters will be stable in aqueous and organic environments. The chemiluminogens containing sulphur atom are known [13], so we expect the possibility of application of acridinium thioesters as a chemiluminogenic fragment in chemiluminescent labels. chemiluminescence mechanism, new potential chemiluminogens with specificity and susceptibility could be rationally designed.
Selection of Investigated Models
Our previous investigations [12,13,17] on the mechanism of the chemiluminescence of acridinium derivatives indicate that the type of the leaving group is affected the efficiency of the formation of the electronically excited products. For this reason, we began to analyze the molecules, in which elimination of the leaving group could be much easier than in acridinium esters and thus transformation into cyclic intermediate, leading to the electronically excited product, much efficient. We decided to replace only one atom in the leaving group (Scheme 2) and check how such a small difference in the molecular structure of acridinium esters will affect the efficiency of the chemiluminescence reaction. The selection of the replacing atom was based on the electronegativity of chemical elements (χ in the Pauling scale is equal to 3.44 for oxygen atom and 2.58 for sulphur atom [44]) and structural properties within the leaving group of the investigated compounds. The bond lengths in the thiocarboxyl group are much longer than in the carboxyl group (the C-S bond is equal to 1.8 Å, the C-O bond is ca. 1.3 Å) and the C-C bond between thiocarboxyl group and acridine moiety in acridinium thioester (Scheme 2, ATE) increases slightly compared to the same bond in acridinium ester (Scheme 2, AE). The reduction of electronegativity and increasing the bond lengths within the leaving group suggest easier elimination of the leaving group and thus more efficient transformation to the electronically excited product. Other aspects to consider are feasible for the synthesis of new chemiluminogens after the oxygen-by-sulphur exchange and their stability of using in applications. Based on the literature [1,5,12,13,17], it can be predicted that the synthesis of acridinium thioesters will be similar to those of acridinium esters and will proceed in three steps: the conversion of acridine-9-carboxylic acid into 9-chlorocarbonyl acridine hydrochloride, the esterification of 9-chlorocarbonyl acridine to acridine thioester and the methylation of the endocyclic nitrogen atom to obtain acridinium salt. Considering the physical or/and chemical properties of the sulphur atom, it can be assumed that also acridinium thioesters will be stable in aqueous and organic environments. The chemiluminogens containing sulphur atom are known [13], so we expect the possibility of application of acridinium thioesters as a chemiluminogenic fragment in chemiluminescent labels. The models selected to investigation are 10-methyl-9-(thiophenoxycarbonyl)acridinium cations substituted with various alkyl groups or an electronegative methoxy group, nitro group or halogen in the thiophenyl fragment (Scheme 3, a-h) and also in the acridine moiety (Scheme 3, i-l). These compounds were selected for investigations since the electron-attracting features of the substituents in the thiophenyl fragment may substantially affect their affinity for nucleophiles and susceptibility to oxidation (chemiluminescence efficiency), and consequently their applicability. The models selected to investigation are 10-methyl-9-(thiophenoxycarbonyl)acridinium cations substituted with various alkyl groups or an electronegative methoxy group, nitro group or halogen in the thiophenyl fragment (Scheme 3, a-h) and also in the acridine moiety (Scheme 3, i-l). These compounds were selected for investigations since the electron-attracting features of the substituents in the thiophenyl fragment may substantially affect their affinity for nucleophiles and susceptibility to oxidation (chemiluminescence efficiency), and consequently their applicability.
Orbitals
As is known from previous studies [12,13,17], acridinium derivatives may react with oxidants in alkaline media relatively easily, yielding electronically excited products that are able to emit light. In the environment of the reaction (alkaline media), an oxidant dissociates to the anionic form and then reacts with acridinium cation, which initiates the chemiluminescence reaction. The anion of the oxidant is the nucleophile and it will attack the electrophilic center in the acridinium cation. To better understand where the electrophilic center of the molecule is located, it can refer to the frontier molecular orbital theory [45]. According to this theory, which describes the electronic structure of molecules, the bonding between the molecules approximates the molecular orbitals as linear combinations of atomic orbitals (LCAO). Therefore, the Lowest Unoccupied Molecular Orbital (LUMO) distribution of an electrophile determines the molecular center sensitive to nucleophilic attack. The computationally predicted LUMO orbitals of acridinium thioester with indicating potential electrophilic centers: the endocyclic C9 and the carbonyl C15 atom, and the values of the LCAO coefficient of the pz atomic orbital in LUMO orbitals at these centers are shown in Figure 1. The average value of the calculated LCAO coefficient of the pz LUMO orbital of the investigated ATEs is equal to 0.317 at the endocyclic C9 atom and 0.014 at the carbonyl C15 atom (for more details see Table S1 in the Supplementary Materials). The ca. 20 times higher LCAO coefficient at C9 than at C15 atom implies that C9 rather than C15 makes the site of the primary nucleophilic attack of the anionic form of the oxidants. The Mulliken partial charges at the same potential electrophilic center are also shown in Table S1 in the Supplementary Materials. The computational result of Mulliken charge at the C9 is lower than at the C15 (ca. 2 times). These results suggest that the population analysis shows which atom has the largest deficit of the negative charge and does not indicate well the location of the electrophilic center. In this subject, the more appropriate approach is to use the frontier molecular orbital theory.
Orbitals
As is known from previous studies [12,13,17], acridinium derivatives may react with oxidants in alkaline media relatively easily, yielding electronically excited products that are able to emit light. In the environment of the reaction (alkaline media), an oxidant dissociates to the anionic form and then reacts with acridinium cation, which initiates the chemiluminescence reaction. The anion of the oxidant is the nucleophile and it will attack the electrophilic center in the acridinium cation. To better understand where the electrophilic center of the molecule is located, it can refer to the frontier molecular orbital theory [45]. According to this theory, which describes the electronic structure of molecules, the bonding between the molecules approximates the molecular orbitals as linear combinations of atomic orbitals (LCAO). Therefore, the Lowest Unoccupied Molecular Orbital (LUMO) distribution of an electrophile determines the molecular center sensitive to nucleophilic attack. The computationally predicted LUMO orbitals of acridinium thioester with indicating potential electrophilic centers: the endocyclic C9 and the carbonyl C15 atom, and the values of the LCAO coefficient of the p z atomic orbital in LUMO orbitals at these centers are shown in Figure 1. The average value of the calculated LCAO coefficient of the p z LUMO orbital of the investigated ATEs is equal to 0.317 at the endocyclic C9 atom and 0.014 at the carbonyl C15 atom (for more details see Table S1 in the Supplementary Materials). The ca. 20 times higher LCAO coefficient at C9 than at C15 atom implies that C9 rather than C15 makes the site of the primary nucleophilic attack of the anionic form of the oxidants. The Mulliken partial charges at the same potential electrophilic center are also shown in Table S1 in the Supplementary Materials. The computational result of Mulliken charge at the C9 is lower than at the C15 (ca. 2 times). These results suggest that the population analysis shows which atom has the largest deficit of the negative charge and does not indicate well the location of the electrophilic center. In this subject, the more appropriate approach is to use the frontier molecular orbital theory.
Orbitals
As is known from previous studies [12,13,17], acridinium derivatives may react with oxidants in alkaline media relatively easily, yielding electronically excited products that are able to emit light. In the environment of the reaction (alkaline media), an oxidant dissociates to the anionic form and then reacts with acridinium cation, which initiates the chemiluminescence reaction. The anion of the oxidant is the nucleophile and it will attack the electrophilic center in the acridinium cation. To better understand where the electrophilic center of the molecule is located, it can refer to the frontier molecular orbital theory [45]. According to this theory, which describes the electronic structure of molecules, the bonding between the molecules approximates the molecular orbitals as linear combinations of atomic orbitals (LCAO). Therefore, the Lowest Unoccupied Molecular Orbital (LUMO) distribution of an electrophile determines the molecular center sensitive to nucleophilic attack. The computationally predicted LUMO orbitals of acridinium thioester with indicating potential electrophilic centers: the endocyclic C9 and the carbonyl C15 atom, and the values of the LCAO coefficient of the pz atomic orbital in LUMO orbitals at these centers are shown in Figure 1. The average value of the calculated LCAO coefficient of the pz LUMO orbital of the investigated ATEs is equal to 0.317 at the endocyclic C9 atom and 0.014 at the carbonyl C15 atom (for more details see Table S1 in the Supplementary Materials). The ca. 20 times higher LCAO coefficient at C9 than at C15 atom implies that C9 rather than C15 makes the site of the primary nucleophilic attack of the anionic form of the oxidants. The Mulliken partial charges at the same potential electrophilic center are also shown in Table S1 in the Supplementary Materials. The computational result of Mulliken charge at the C9 is lower than at the C15 (ca. 2 times). These results suggest that the population analysis shows which atom has the largest deficit of the negative charge and does not indicate well the location of the electrophilic center. In this subject, the more appropriate approach is to use the frontier molecular orbital theory.
Mechanism of Chemiluminescence
Considering the reaction of acridinium derivatives with oxidants (e.g., OOH − ) in alkaline environment (e.g., in presence of OH − ), two processes should be considered: the reaction leading to the light emission (chemiluminescence) and other competitive processes (so-called dark processes or non-CL processes). Schemes 4 and 5 present the processes that account for CL and non-CL processes, respectively (the summary of all investigated processes is shown in Scheme S1 in the Supplementary Materials). The theoretically predicted thermodynamic data for all reaction steps are given in Table 1, while Table 2 contains kinetic characteristics of the selected steps. Additionally, the density functional theory (DFT)-optimized geometries originating from the acridine molecules produced by the reaction of ATEs with OOH − or OH − are shown in results of computational study on excited states of 1,2-dioxetane suggest that in the first step the O−O bond is broken and the molecule enters in a region of biradical character, and next the chemiexcitation occurs concomitantly with C-C bond breaking [52,53].
Non-CL Processes
The values of quantum yield of chemiluminescence of acridinium derivatives (2-7% in aqueous environments [54,55]) indicate such a small portion of chemiluminogen is transformed into electronically excited product, which leads to the light emission. Unfortunately, most of the molecules undergo competitive reactions (the dark processes), that do not lead to the formation of electronically excited 10-methyl-9-acridinone, but 10-methyl-9-acridinone in the ground state. The first group of transformation of acridinium derivatives, which not leading to the electronically excited products, is all reactions, which transform of the acridinium cation to the non-emitting product (Scheme 5, pathway starting with step VI), and the second group is the hydrolysis of the acridinium molecule (Scheme 5, pathway XI). Both above-mentioned groups significantly reduce the chemiluminescence quantum yield of acridinium derivatives. The first group of the dark processes starts from the nucleophilic attack of OHat the endocyclic carbon atom (C9) of acridine moiety, creating of so-called 'pseudobase'. The computationally predicted thermodynamic data (Table 1) indicated that the two steps of competitive processes-leading to the CL (step I, addition of the OOH -) and the formation of the 'pseudobase' (step VI, addition of the OH -) are possible for all of investigated ATEs. Considering the values of Gibbs' free energies in aqueous environment, one can be noted that the addition of OHinto position 9 of the acridine moiety is thermodynamically more preferred than the OOH -. In this case, the kinetic factors will be determined the preference for the addition of ions. All proposed non-CL steps (VI-X) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table 2) predicted for step VIII are relatively small (in the range of 0.3-2.8 kcal mol -1 ). On the other hand, the thermodynamic data show that the energy released in this step exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Therefore, steps VI-VIII will not lead to the formation of electronically excited 10-methyl-9-acridinone. The thermodynamic data suggest that step X can lead to the formation of product in electronically excited state (Table 1), but the activation barriers (Table 2) predicted for this step are very high (43.3 and 36.1 kcal mol -1 for unsubstituted (compounds a-h) and substituted (compounds i-l), respectively), so the step X will not occur. In this situation, both the above pathways may account for the dark transformation of ATEs. Comparing the thermodynamic and kinetic results of non-CL pathway, starting with the formation of 'pseudobase', of unsubstituted (compounds a-h) and substituted (compounds i-l) in the acridine moiety derivatives (Tables 1 and 2, steps VI-X), no significant differences in the values of enthalpies and Gibbs free energies can be 1 ∆ a,298 H 0 and ∆ a,298 G 0 (both in kcal mol −1 ), respectively, represent the enthalpy and Gibbs' free energy (gaseous phase) or free energy (aqueous phase) of activation at standard temperature and pressure; 298 k 0 (in s −1 ) and 298 τ 99 , respectively, denote the rate constant and the time after which the reaction is 99% complete.
Chemiluminescence Pathway
The chemiluminescence reaction begins from nucleophilic attack of anionic form of oxidant (e.g., OOH − ) at the C9 atom on acridinium moiety (step I). In the next steps, the addition product 2 reacts with an OH − ion and after elimination of the thiophenyl anion, forms cyclic intermediate 3 (step II), which after the elimination of carbon dioxide leads to the energy-rich 10-methyl-9-acridinone (5 *, step III), and then followed by the relaxation of electronically excited 10-methyl-9-acridinone to the ground state with the emission of light (step IV).
The computationally predicted thermodynamic data (Table 1) indicated that all proposed CL steps (I-V) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table 2) predicted for step III (formation of electronically excited product) are similar to the activation barriers of the same step predicted for acridinium esters (ca. 15.4 kcal mol −1 ) [12]. Boużyk et al. have shown that the necessary value of energy to cause the electronic excitation of 10-methyl-9-acridinone is 88.2 kcal mol −1 [46]. Our calculation reveal that the energy released in step V (Table 1) exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Comparing the thermodynamic and kinetic results of CL pathway (Tables 1 and 2, steps I-V) of unsubstituted (compounds a-h) and substituted (compounds i-l) in the acridine moiety derivatives, no significant differences in the values of enthalpies and Gibbs free energies can be seen. However, after analyzing the Gibbs free energy profiles of the full reaction leading to the generation of light ( Figure 2) for acridinium thioesters without any substituent in the acridine moiety (derivative a) and with the methoxy group in the 2 position in the acridine moiety (derivative i), one can conclude that the addition of an electron-donor substituent, such as -OCH 3 , into the acridine moiety reduces the activation barrier with similar or slightly lower values of thermodynamic characteristics of individual steps of CL pathway. Comparing the chemiluminescent properties of acridinium thioesters, the nature of the decomposition of cyclic intermediates (structure 3, Scheme 4) should be considered. This entity has two important bonds (O-O and C-C), which have an effect on the transformation in the electronically excited product-10-methyl-9-acridinone. In geometrical terms, the bond lengths of C-C and O-O are 1.532 Å and 1.491 Å, respectively and both appear to be covalent. The VDD population analysis results also present a typical distribution of charges (see Table S2 in the Supplementary Materials)oxygen atoms have a negative charges and carbon atoms have positive charges. It can be concluded that the combination of negative charges of both oxygen atoms and the small difference between charge indicates a repulsive electrostatic interaction between oxygen atoms. In the case of carbon atoms, the charge differences are significantly higher, which also indicates a repulsive effect of interaction, but attenuated by the polarization effect. To better understand the nature of intramolecular interactions in a dioxetanone entity, the calculations were performed using the quantum theory of atoms in molecules (QTAIM) [47]. The topology of the electron localization function using a bond critical point (BCP) (see Table S2 in the Supplementary Materials) indicates that the C-C bond is a typical covalent bond (combination of negative value of Laplacian (∇ 2 ρ(r)) with high value of electron density distribution (ρ(r)) and the O-O bond is charge-shifted (combination of positive value of Laplacian (∇ 2 ρ(r)) with high value of electron density distribution (ρ(r)) [48,49]. To determine σ or π bond character, the values of the ellipticity (ε) were analysed (see Table S2 in the Supplementary Materials). The analysis of these parameters demonstrates that both O-O and C-C bonds represent a sigma bond character (larger values (>0.1) indicate π bond character and lower values indicate σ bond character [50]). However, the value of ellipticity for O-O bond is very close to the value that indicates π bond character. Finally, the ratio of -(G(rb)/V(rb)) was used as a criterion of the nature of bonds (Table S2, Comparing the chemiluminescent properties of acridinium thioesters, the nature of the decomposition of cyclic intermediates (structure 3, Scheme 4) should be considered. This entity has two important bonds (O-O and C-C), which have an effect on the transformation in the electronically excited product-10-methyl-9-acridinone. In geometrical terms, the bond lengths of C-C and O-O are 1.532 Å and 1.491 Å, respectively and both appear to be covalent. The VDD population analysis results also present a typical distribution of charges (see Table S2 in the Supplementary Materials)-oxygen atoms have a negative charges and carbon atoms have positive charges. It can be concluded that the combination of negative charges of both oxygen atoms and the small difference between charge indicates a repulsive electrostatic interaction between oxygen atoms. In the case of carbon atoms, the charge differences are significantly higher, which also indicates a repulsive effect of interaction, but attenuated by the polarization effect. To better understand the nature of intramolecular interactions in a dioxetanone entity, the calculations were performed using the quantum theory of atoms in molecules (QTAIM) [47]. The topology of the electron localization function using a bond critical point (BCP) (see Table S2 in the Supplementary Materials) indicates that the C-C bond is a typical covalent bond (combination of negative value of Laplacian (∇ 2 ρ(r)) with high value of electron density distribution (ρ(r)) and the O-O bond is charge-shifted (combination of positive value of Laplacian (∇ 2 ρ(r)) with high value of electron density distribution (ρ(r)) [48,49]. To determine σ or π bond character, the values of the ellipticity (ε) were analysed (see Table S2 in the Supplementary Materials). The analysis of these parameters demonstrates that both O-O and C-C bonds represent a sigma bond character (larger values (>0.1) indicate π bond character and lower values indicate σ bond character [50]). However, the value of ellipticity for O-O bond is very close to the value that indicates π bond character. Finally, the ratio of -(G(r b )/V(r b )) was used as a criterion of the nature of bonds (Table S2, Supplementary Material). For O-O bond, value of these parameters demonstrate covalent bond, and for C-C bond, the -(G(r b )/V(r b )) ratio is lower than the range indicating partially covalent bond (larger values (-(G(r b )/V(r b )) > 1.0) indicate noncovalent bond character and values in the range (0.5 < -(G(r b )/V(r b )) < 1.0) indicate partially covalent bond character [51]). The results of calculation using quantum theory of atoms in molecules does not clearly indicate the covalent nature of the O-O and C-C bonds in the cyclic intermediates. On the other hand, the results of computational study on excited states of 1,2-dioxetane suggest that in the first step the O−O bond is broken and the molecule enters in a region of biradical character, and next the chemiexcitation occurs concomitantly with C-C bond breaking [52,53].
Non-CL Processes
The values of quantum yield of chemiluminescence of acridinium derivatives (2-7% in aqueous environments [54,55]) indicate such a small portion of chemiluminogen is transformed into electronically excited product, which leads to the light emission. Unfortunately, most of the molecules undergo competitive reactions (the dark processes), that do not lead to the formation of electronically excited 10-methyl-9-acridinone, but 10-methyl-9-acridinone in the ground state. The first group of transformation of acridinium derivatives, which not leading to the electronically excited products, is all reactions, which transform of the acridinium cation to the non-emitting product (Scheme 5, pathway starting with step VI), and the second group is the hydrolysis of the acridinium molecule (Scheme 5, pathway XI). Both above-mentioned groups significantly reduce the chemiluminescence quantum yield of acridinium derivatives. The first group of the dark processes starts from the nucleophilic attack of OH − at the endocyclic carbon atom (C9) of acridine moiety, creating of so-called 'pseudobase'. The computationally predicted thermodynamic data (Table 1) indicated that the two steps of competitive processes-leading to the CL (step I, addition of the OOH − ) and the formation of the 'pseudobase' (step VI, addition of the OH − ) are possible for all of investigated ATEs. Considering the values of Gibbs' free energies in aqueous environment, one can be noted that the addition of OH − into position 9 of the acridine moiety is thermodynamically more preferred than the OOH − . In this case, the kinetic factors will be determined the preference for the addition of ions. All proposed non-CL steps (VI-X) are possible for all investigated compounds in gaseous and aqueous phases. Our calculations reveal that the activation barriers (Table 2) predicted for step VIII are relatively small (in the range of 0.3-2.8 kcal mol −1 ). On the other hand, the thermodynamic data show that the energy released in this step exceeds that which is necessary to cause the electronic excitation of product of CL reaction. Therefore, steps VI-VIII will not lead to the formation of electronically excited 10-methyl-9-acridinone. The thermodynamic data suggest that step X can lead to the formation of product in electronically excited state (Table 1), but the activation barriers (Table 2) predicted for this step are very high (43.3 and 36.1 kcal mol −1 for unsubstituted (compounds a-h) and substituted (compounds i-l), respectively), so the step X will not occur. In this situation, both the above pathways may account for the dark transformation of ATEs. Comparing the thermodynamic and kinetic results of non-CL pathway, starting with the formation of 'pseudobase', of unsubstituted (compounds a-h) and substituted (compounds i-l) in the acridine moiety derivatives (Tables 1 and 2, steps VI-X), no significant differences in the values of enthalpies and Gibbs free energies can be seen. The Gibbs free energy profiles of the full non-CL pathway ( Figure 3) for acridinium thioesters without any substituent in the acridine moiety (derivative a) and with the methoxy group in the 2 position in the acridine moiety (derivative i), show that the addition of an electron-donor substituent, such as -OCH 3 , into the acridine moiety reduces the activation barrier with similar or slightly lower values of thermodynamic characteristics of individual steps of CL pathway. Scheme 5. Non-CL pathways of ATEs: (a, blue) the transformation of the acridinium cation to the non-emitting product, and (b, orange) the hydrolysis of the acridinium molecule. and IX (hydrolysis of acridinium cation) steps. It can be assumed, that the addition of an electron-acceptor substituents in 2 and 6 positions or an electron-donor substituents in 2 and 6 position while an electron-acceptor substituent in 4 position, will affect the more efficient transformation into electronically excited product, but also sensitizes to the hydrolysis of ATEs.
Considering the utilitarian perspectives of the studied compounds, we compared the energetical values of the main steps (the chemiluminescence pathway and the formation of the pseudobase as affecting the whole process) between acridinium thioesters and currently used in the analysisacridinium esters. Figure S2 in the Supplementary Materials presents the Gibbs free energy profiles of the CL pathway and the first step of the non-CL pathway (formation of the pseudobase) of acridinium thioesters and acridinium esters. The results show slight differences of the values of Gibbs free energies for step I (addition of OOHat the C9 atom on acridinium moiety; -44.8 and -43.1 kcal mol -1 for AE and ATE, respectively) and VI (formation of the pseudobase; -62.8 and -61.9 kcal mol -1 It can be assumed, that the addition of an electron-acceptor substituents in 2 and 6 positions or an electron-donor substituents in 2 and 6 position while an electron-acceptor substituent in 4 position, will affect the more efficient transformation into electronically excited product, but also sensitizes to the hydrolysis of ATEs. Considering the utilitarian perspectives of the studied compounds, we compared the energetical values of the main steps (the chemiluminescence pathway and the formation of the pseudobase as affecting the whole process) between acridinium thioesters and currently used in the analysis-acridinium esters. Figure S2 in the Supplementary Materials presents the Gibbs free energy profiles of the CL pathway and the first step of the non-CL pathway (formation of the pseudobase) of acridinium thioesters and acridinium esters. The results show slight differences of the values of Gibbs free energies for step I (addition of OOH − at the C9 atom on acridinium moiety; −44.8 and −43.1 kcal mol −1 for AE and ATE, respectively) and VI (formation of the pseudobase; −62.8 and −61.9 kcal mol −1 for AE and ATE, respectively) and significant differences of the values of Gibbs free energies for step II (formation of the cyclic intermediate; −47.6 and −69.0 kcal mol −1 for AE and ATE, respectively) between acridinium esters and acridinium thioesters. A comparison of these values suggests the more thermodynamically preferred formation of the cyclic intermediate for acridinium thioesters, and thus its decomposition to the electronically excited product. It can be concluded that acridinium thioesters will be more efficient chemiluminogens than acridinium esters.
Electronically Excitation
The mechanism of chemiluminescence reaction of ATEs indicates that CL pathway leads to energy-rich 10-methyl-9-acridinone, which returning to the ground state emits light. In our previous studies on acridinium esters [12,17], we reported formation of electronically excited product after elimination of phenyl carbonate anion [17] or firstly, phenoxy anion and secondly, carbon dioxide from dioxetane intermediate [12]. As mentioned in Section 2.3.1., the formation of energy-rich 10-methyl-9-acridinone in the CL reaction of acridinium thioesters follows by the transformation to the dioxetane intermediate after the elimination of the thiophenyl anion, and then the elimination of the carbon dioxide (Scheme 4, step III and IV). The formation of cyclic intermediate 3 (Scheme 4) and then its decomposition is a rate determining step of the chemiluminescence.
To better understand the nature of an electronically excited product, an investigation of excited states was carried out using the time dependent (TD) DFT calculation. As is known, the intersystem crossing (ISC) mechanism, which is the nonradiative transition between two electronic states of different multiplicity, plays an important role in photochemistry and explains well the properties of organic chromophores [56,57]. Taking into account the previous reports about the decomposition mechanism of 1,2-dioxetane [52], 1,2-dioxetanedione [58] and acridinium esters [5], the spin-orbit coupling between the S 0 and the T 1 states occurs along the chemiluminescence reaction pathway. The computed energy profiles on the singlet (S 1 ), triplet (T 1 ) and ground (S 0 ) states started from the transition state of decomposition of cyclic intermediate 3 (TS-III) show that the potential energy curve of the triplet state (T 1 ) lays closer than the potential energy curve of the singlet state (S 1 ) to the potential energy curve of the ground state (S 0 ). Figure 4 and Figure S3 in the Supplementary Materials show the energy diagram of CL pathway in aqueous and gaseous phases, respectively, including thermal decomposition (S 0 ) and formation of excited states (S 1 and T 1 ). The vertical excitation energies in aqueous phase of 10-methyl-9-acridinone in equilibrium geometry are 2.51 and 2.26 eV for T 1 state and 3.29 and 3.33 eV for S 1 state for unsubstituted and substituted in the acridine moiety, respectively. Therefore, the emission energy from T 1 corresponds well to experimental results that show the maximum emission speak at 451 nm (2.75 eV) for 10-methyl-9-acridinone [46]. It should be noted that the emission energy from S 1 state in gaseous phase for unsubstituted in the acridine moiety product of the chemiluminescence reaction are higher than that for the product substituted in position 2 in acridine moiety (4.82 and 3.23 eV, respectively). This difference decreases in the aqueous phase as indicated above. According to this, the photoemission of the mixed state (from T 1 and S 1 states) of 10-methyl-9-acridinone in water solution is expected. On the other hand, the phosphorescence will be quenched in solution, and we expect that it may not play a significant role in the chemiluminescence of acridinium derivatives.
Materials and Methods
Unconstrained geometry optimizations of isolated molecules were carried out at the DFT [59] and TD DFT [60] level of theory to determine ground and excited states, respectively. All the calculations were conducted using the popular Becke's three-parameter hybrid functional B3LYP [61,62] and the 6-31G** basis set [63,64] with the Gaussian09 package [65]. B3LYP functional is known to produce reliable results [5,12,17] for the thermodynamic data and have been proven to provide accurate qualitative results for the mechanism of chemiluminescence reaction of investigated molecules. After completion of each optimization, the Hessian (second derivatives of the energy as a
Materials and Methods
Unconstrained geometry optimizations of isolated molecules were carried out at the DFT [59] and TD DFT [60] level of theory to determine ground and excited states, respectively. All the calculations were conducted using the popular Becke's three-parameter hybrid functional B3LYP [61,62] and the 6-31G** basis set [63,64] with the Gaussian09 package [65]. B3LYP functional is known to produce reliable results [5,12,17] for the thermodynamic data and have been proven to provide accurate qualitative results for the mechanism of chemiluminescence reaction of investigated molecules. After completion of each optimization, the Hessian (second derivatives of the energy as a function of the nuclear coordinates) was calculated and checked for positive definiteness to assess whether the structures were true minima. The solvent (water) effect included in the DFT calculations at the level of the polarizable continuum model (PCM) was used to mimic water environment (UAHF radii were used to obtain the molecular cavity) [66,67]. The transition energies for the electronically excited product of chemiluminescence reaction of acridinium thioesters-10-methyl-9-acridinone-were calculated from an excited state optimized structure using the state-specific approach. The Voronoi deformation density (VDD) population analysis was performed by using the Multiwfn code [68] with the same level of theory and basis set as geometry optimization. The electron density at the bond critical point (BCP) were analysed using the quantum theory of atoms in molecules (QTAIM) methodology [47]. Visualization of the results of the calculations of equilibrium structures of all molecules was made using the ChemCraft software [69].
The enthalpies (∆ r,298 H 0 ) and Gibbs free energies (free energies in the case of DFT(PCM)) (∆ r,298 G 0 ) of the reactions (r), as well as the enthalpies (∆ a,298 H 0 ) and Gibbs free energies (free energies in the case of DFT(PCM)) (∆ a,298 G 0 ) of activation (a) were calculated by following the basic rules of thermodynamics [44]. The rate constants ( 298 k 0 ) for the gaseous phase reactions were obtained by applying the equation: Resulting from the transition state theory, and reaction completion time ( 298 τ 99 ) from the formula: where R, T, N and h denote the gas constant, temperature (298.15 K), Avogadro number and Planck's constant, respectively [44].
Conclusions
Due to the rapid growth of population and factors which negatively affect health, the increasing number of diseases motivates the intensive search for new effective diagnostic methods. One of the promising immunological diagnostic procedures is chemiluminescent labelling, in which efficient chemiluminogens are used. Searching for new chemiluminogens with better features, especially higher quantum yields, is the main aim of this work. As acridinium esters are an efficient chemiluminogens, we modelled new group of compounds based on acridinium moiety-10-methyl-9-thiophenoxycarbonylacridinium derivatives. The proposed chemiluminescence reaction mechanism of these compounds was based on our previous computational and experimental studies [12,13,17]. The CL reaction begins with the nucleophilic attack of hydroperoxide anion, then forms highly strained dioxetanone ring after addition of hydroxyl anion, and finally energy-rich 10-methyl-9-acridinone as a result of decomposition of cyclic intermediate. The step, in which the decomposition of the cyclic dioxetanone ring is observed, is the rate-determining step in the whole process. All the considered compounds were computationally proven to easily transform into electronically excited products as a result of the chemiluminescence reaction. However, all investigated derivatives could be divided into two groups: unsubstituted and substituted in the acridine moiety. The addition of an electron-donor substituent, e.g., methoxy group, into the acridine moiety reduces the activation barrier with almost similar values of thermodynamic characteristics of individual steps of CL pathway. Additionally, the studied acridinium derivatives are susceptible to two reactions competing with chemiluminescence: formation of 'pseudobase' and hydrolysis. The computational results show that the possible pathway of the reaction, CL or one of the competitive pathways, seems to be exclusively dependent on kinetic factors. The most promising are the addition of an electron-acceptor substituents in the 2 and 6 positions or an electron-donor substituent in the 2 and 6 position while an electron-acceptor substituent in the 4 position moves into the thiophenoxy group. These substitutions will affect the more efficient transformation into an electronically excited product, but also sensitize it to the hydrolysis of investigated compounds. The computationally predicted emission energy from T 1 for electronically excited products of CL reaction-10-methyl-9-acridinone corresponds well to experimental results. The theoretical studies of the excited state indicate that the photoemission of the mixed state (from T 1 and S 1 states) of 10-methyl-9-acridinone in water solution is expected.
To sum up, with the use of a simple computational approach, we were able to identify potential chemiluminogens for further experimental studies. Synthesis, followed by studies of their chemiluminescent properties, and ultimately in chemiluminescent labelling, are further steps to confirm their potential applicability in immunodiagnostics.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/12/4417/s1. Figure S1. The VDD population analysis results of the investigated dioxetanone molecules (a) unsubstituted and (b) substituted in the acridine moiety. Figure S2. Relative Gibbs free energies of the CL pathway (reaction steps: I-IV) and non-CL step VI (formation of pseudobase) of acridinium thioesters: (a, black) and acridinium esters (c, violet). Figure S3. Energy diagram of chemiluminescence reaction in the gaseous phase with energy of the lowest triplet (T1) and singlet (S1) states. Scheme S1. Pathways of the reactions of ATE with OOH− and OH− (CL pathway (green arrows), non-CL pathways: transformation of ATE to the non-emitting product (blue arrows) and hydrolysis of ATE (orange arrows)). Table S1. The calculated Mulliken atomic charges and LCAO coefficient of the pZ LUMO orbital at the endocyclic C9 and the carbonyl C15 atoms. Table S2. The VDD population analysis results of the investigated dioxetanone molecules (a) unsubstituted and (b) substituted in the acridine moiety. Table S3. Cartesian coordinates and structures of the lowest energy and transition state structures of investigated molecules.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
2019-08-23T13:03:47.398Z | 2019-09-24T00:00:00.000 | 201274803 | {
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} | pes2o/s2orc | Enhancing the Antioxidant Activity of Technical Lignins by Combining Solvent Fractionation and Ionic‐Liquid Treatment
Abstract A grass soda technical lignin (PB1000) underwent a process combining solvent fractionation and treatment with an ionic liquid (IL), and a comprehensive investigation of the structural modifications was performed by using high‐performance size‐exclusion chromatography, 31P NMR spectroscopy, thioacidolysis, and GC–MS. Three fractions with distinct reactivity were recovered from successive ethyl acetate (EA), butanone, and methanol extractions. In parallel, a fraction deprived of EA extractives was obtained. The samples were treated with methyl imidazolium bromide ([HMIM]Br) by using either conventional heating or microwave irradiation. The treatment allowed us to solubilize 28 % of the EA‐insoluble fraction and yielded additional free phenols in all the fractions, as a consequence of depolymerization and demethylation. The gain of the combined process in terms of antioxidant properties was demonstrated through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH.) radical‐scavenging tests. Integrating further IL safety‐related data and environmental considerations, this study paves the way for the sustainable production of phenolic oligomers competing with commercial antioxidants.
Introduction
Industryi si ncreasingly demanding of biobasedp henolic compounds, to be used as building blocks for polymer synthesis or valued for their antiradicala ctivity,e specially in the field of polymers, materials, and cosmetics. Phenolics derived from plant biomass are in particular potentiala lternatives to synthetic commercial antioxidants such as Bisphenol A [1] and tert-butylated hydroxytoluene( BHT). [2] Amongt hem, ferulic acid is already knownf or its potential for various applications, [3] but its low availability from plant sourcesm ight hinder its industrial development. As polymers of phenyl propanoids representing up to 30 %o fthe plant biomass, ligninsr epresent one of the major potential sources of biobased phenolics. [4] Moreover, the valorization of technical lignins generated as industrialb yproducts would increase the sustainability of lignocellulosebased biorefineries. [5] Various strategies driven by green chemistry principles have been designed to convert technical lignins into functional moleculeso ra ssemblies while mitigating their variability processing from botanical origin and transformationsd uring industrial treatments. [6] These strategies include fractionation and depolymerization. Fractionation can be performed by ultrafiltration, potentially directly integrated into the pulping process, [7] or by solvente xtraction, whichi sa dvantageously performed at ambient temperature andp ressure and does not requires pecific equipment. [8] In contrast, among the depolymerization strategies (thermochemical, biological, or chemocatalytic), [9] the recently reported implementationo fm ethyl imidazolium bromide ([HMIM]Br) ionic liquid (IL) providesaw ay to produce lignin oligomers with increasedf ree phenol content (PhOH) and decreased polymerization degree, as ac onsequence of the selectivecleavage of aryl-alkyl ether bonds (Figure 1). [10] In particular,t his was shown to generate new phenol groups from the methoxy groups of lignin syringyl and guaiacyl units.S uch Ag rass soda technical lignin (PB1000) underwent ap rocess combining solvent fractionation and treatment with an ionic liquid (IL), and ac omprehensive investigation of the structural modifications was performed by using high-performance sizeexclusion chromatography, 31 PNMR spectroscopy,t hioacidolysis, and GC-MS. Three fractionsw ith distinct reactivity were recoveredf rom successive ethyl acetate (EA), butanone, and methanol extractions.I np arallel, af raction deprived of EA extractives was obtained. The samples were treated with methyl imidazolium bromide ([HMIM]Br) by using either conventional heatingo rm icrowavei rradiation. The treatment allowed us to solubilize2 8% of the EA-insoluble fraction and yieldeda dditional free phenols in all the fractions,a saconsequence of depolymerization andd emethylation. The gain of the combined processi nt erms of antioxidant properties was demonstrated through2 ,2-diphenyl-1-picrylhydrazyl (DPPHC)r adical-scavenging tests. Integratingf urtherI Ls afety-related data and environmentalc onsiderations,t his study paves the way for the sustainable production of phenolic oligomers competing with commercial antioxidants. phenolic oligomers have great potentiala sa ntioxidant additives for the formulation of materials because they are likely to combine miscibility in the polymer matrix and limited migration towards the environment owing to their oligomeric structure. [11,12] Despite this potential, the process has, until now, only been applied to lignin models, and the antioxidantp roperties of the oligomers have not been assessed yet. The advantages of using [HMIM]Br for the transformationo fl ignins are foreseen to potentially ensure the homogeneity of the reaction medium, to avoid the use of additional chemical reagents, and to perform the reactioni nm ild conditions (T < 110 8C, t < 40 min) compared with most lignin catalytic conversion processes. Moreover,i nt his context of use, the IL can be recycled. All these advantages also render the process attractive from an environmental point of view.H owever,t he sustainability assessment of the process requires further health and safety information on [HMIM]Br,w hich has not been provided so far.T he present paper aims at assessing the possibility of transferring the newly developedI Lp rocess [10] to technical lignin fractions. The objective was to recover antioxidant extracts soluble in ethyl acetate (EA), one of the conventional solvents recommended for their relativelyl imited health hazard and environmental footprint as assessed from the Health and Safety Executive (HSE) criteria compiled from variouss ources. [13] EA was selected here to separate extractives from the IL/waterl ayer.B ecausea lkali grass lignin Protobind 1000 (PB1000)f rom GreenValue LLC is available at an industrial scale and is already knownf or its antioxidant properties, [14] it was selected as the starting material for this study.T hisl ignin was subjected to at hree-step semi-continuouss olvent fractionation process to recover three structurally distinct soluble fractions (F1-F3). In parallel, the same lignin sample was submitted to an extensive EA washing to recover an EA-insoluble residue (F4) and validate the possibility of recovering soluble compounds through chemical treatment of this fraction. PB1000 and the four fractions were submitted to the previously optimized IL treatment by using microwavei rradiation (MW) or conventional heating (CH) as heating processes. The advantage of MW is the short reactiont ime (10 s), whereas CH was used to generate more severe modifications relevant to investigation of the structure-properties relationships. The phenolic monomers and oligomers were extracted from the reaction media with EA, and the ethyl acetate extracts( EAE) were analyzed by chromatographic and spectroscopic methods to assess the efficiency of the conversion, elucidate mechanisms, and identify molecules of interestf or furtherd evelopments. The antioxidant properties of PB1000 and the EAE as well as those of the insoluble residues recoveredf rom the most drastic process (CH, 40 min) were assessed through 2,2-diphenyl-1picrylhydrazyl (DPPHC)r adical-scavenging tests. The half-maxeffective concentrationE C 50 was used as the criterion to compare the performance of the different samples.T he study demonstrates the technical advantage of implementing the [HMIM]Br-based treatment in ac ascadinga pproachc ombining fractionation and depolymerization. In addition, sustainability considerations of the process are further discussed according to safety data obtainedf or the IL.
Results and Discussion
Recovery of different PB1000 fractions and their contrasted reactivitytowards [HMIM]Br treatments
PB1000f ractionation
PB1000w as fractionated at as emi-pilot scale (1 kg dry powder) through as emi-continuous process intended for future industrial development. [8] This process consisted of a three-step sequential extraction with solvents of increasing polarity ( Figure 2) and yielded 31 wt %f or the EA-soluble fraction F1, 19 wt %f or the butanone-(MEK)-solublef raction F2, and 23 %f or the MeOH-soluble fraction F3. Because F2 and F3 contained residual EA-soluble compounds (37 and 7%,r espectively), PB1000 was also submitted, at lab scale [g],t oadrastice xtensivew ashing with EA yieldinga nE A-insoluble fraction F4 (50 %o ft he weighto ft he startingP B1000) subsequently used only to validate the depolymerization process.
One main characteristic of this set of four fractionsw as the increasing proportion of lignin inter-unit aryl-alkyl b-O-4 linkages from F1 to F4, as reflected by the increasing thioacidolysis yields (Table 1). [15] Because our previouss tudy performed on dioxan-isolated model lignins showedt he b-O-4 linkages to be the mostp rivileged target of the IL treatment allowing partial depolymerization, [10] these fractions were thus expected to show increasing reactivity towards the IL treatment.
To determine the influence of structural variations on the efficiency of the depolymerization process, PB1000 and its four derived fractions were subjected to heating treatments in [HMIM]Br,e ither under MW (10 s, 110 8C) or under CH [110 8C, 20 (CH20) or 40 min (CH40)].I nl ine with the objective of the treatment to recover EA-soluble functional extracts, the efficiency of the treatments was assessed on the basis of the recovery yields of the EAE after treatment and the characteristics of the extracts, selecting average molar masses and PhOH as the major criteria for furthera pplications (Table 2). Moreover, to estimate the solubility gain, the proportion of EA-soluble compounds with respect to the total products recovered was calculated and compared with the EA solubility of the initial sample ( Figure 3).
Effect of the [HMIM]Br treatment on the recovery of EAE
As ac onsequence of the fractionation process, the four fractions exhibited initial contrasted solubility in EA:F 1w as 100 % EA-soluble, whereas F4 was poorly soluble, and F2 and F3 only partially (37 and 7%,r espectively). The[HMIM]Br treatments induced ad ecrease in the proportion of EA-soluble compounds of all samples, except for F3 and F4,w hich conversely exhibited an increase. The highest effect of the [HMIM]Br treatment was observedf or F4 under the MW conditions. Indeed, 15 %o f this insoluble fraction could be solubilized, and 28 %o ft he products formed was soluble. The EAE represented 8% of PB1000.T hree-fold andt wo-fold lower solubilities wereo bserved with CH20 and CH40 conditions, respectively.
In contrast, the treatment of the totally soluble fraction F1 led to the decrease of the solubilityw ith production of insoluble material (16-40 %d epending on the treatment). The maximum solubility decrease was observed with the CH conditions, indicating that these conditions potentially favored recondensation reactions of the EA-soluble compounds owing to the longer reaction time. In the case of the only partially EA-soluble fractionsF 2a nd F3, two contrasting behaviors were observed:t he F2 behavior was similart oF 1w ith as olubility decrease enhanced in CH conditions, and the F3 behavior was similar to F4 with solubility increase (up to 19 %s olubility increase)u nder MW conditions. The EAE recovered from F3 accounted for 21 %o ft he fractiona nd 5% of PB1000.T he treatment of PB1000w ithout any previousf ractionation led to an intermediate behavior with as olubility decrease observed only in the most severe conditions, CH40. In conclusion, the [HMIM]Brt reatment could produce EA-soluble compounds from fractions showingp oor initial EA solubility,a nd the presence of EA-soluble compounds in the initial sample led to an apparent decrease in EA solubility,w hich was most probably owing to recondensation reactions favored by CH conditions. Besides initial EA solubility,t he fraction-dependent effect of [HMIM]Brt reatment on the amount of EAE recovered could be related to the structurald ifferences of the fractionsi nt erms of b-O-4 bond content. Indeed, in the case of F1 and F2, which exhibit the lowest amount of b-O-4 bonds according to thioacidolysis yields, the proportion of EA-soluble compoundsi n the reaction mixturesa fter treatment decreased, whereas in the case of F3 and mainly F4, this proportion significantly increased. The resultsa re in agreement with our previously reported statements [10] and suggestthat b-O-4 linkages are effectively the most privileged targets of the IL treatment. This also demonstrates that the efficiency of the depolymerization process is able to counterbalance the effect of recondensation reactions occurring during the treatment, and that may be particularly important in the case of long reaction times (CH compared with MW).
In all cases, and whatever the treatment, the yield of the reaction was good but never reached 100 %o wing to losses of compounds, mainly volatiles (not identified nor quantified), during the reactiona nd soluble andi nsoluble compounds during the separation process (precipitation of the products and removal of the water/ILl ayer).
Molar mass distributiono fthe EAE
Whatever the sample, the weight-average molar mass (M w )o f the EAE, before or after treatment, did not exceed 2000 gmol À1 ,i ndicating that the EAE were essentially composed of oligomers (less than ten phenylpropaneu nits). Nevertheless, some variations in molar distribution appeared between the samples upon treatments. Increasing EAE average molar masses was observed from F1 to F3, in agreement with the use of solventw ith increasing polarities for the PB1000 fractionation process. [16,17] The effect of the [HMIM]Br treatment on the molar masses depended on the fraction and conditions used. In the case of F3, all treatments induced ad ecrease in average molarm asses with an increasing effect from MW to CH20 and CH40. For F1 and F2, ad ecrease in the molar mass was only observed in CH conditions,a nd the use of MW by contrastl ed to as light increase. In thec ase of F4, the EAE produced by the treatments exhibitedm olar masses 1.4-to 1.6fold lower than that of the initial F4 insoluble fraction,a nd of the same range as the non-modified EAE of the other fractions. As light increasew as observed in the CH conditions. This result supported the hypothesist hat recondensation reactions were favored by the CH conditions.
Evidence of demethylation and depolymerization upon [HMIM]Br treatment
GC-MS analysis was performed to investigate the phenolic monomers present in the EAE (Supporting Information, S1). It revealed the presence of am ixture of compounds (phenolic acids, ketones, and aldehydes accounting in total for 1.2 %o f PB1000)i nF 1, the absence of monomeric compounds in F2 and F3 before treatment, and the formation of new compoundsf or all fractionsa fter treatment. The chromatograms of the EAE recovered after treatment revealed the presenceo f phenolicm onomers diagnostic of demethylation (all fractions) and/or depolymerization (F2 and F3). In F1, the treatment led to the total conversion of acetosyringone, the major EAE phenolic monomer before treatment, into its once-and twice-demethylated counterparts [1-(3,4-dihydroxy-5-methoxyphenyl)ethan-1-one C and 1-(3,4,5-trihydroxyphenyl)-ethan-1-one D] and to the disappearance of all other phenolic extractives, most probablyi nvolved in recondensation reactions. In F2 and F3, the treatmentl ed to the production of two ketones A and B previously identifieda sa cidolysis ketones from experiments on lignin models. [10] In F2, only the demethylated form B was detected whereas in F3 both were formed. The analysiso ft he thioacidolysis products also indicated that demethylation took place within lignin units linked through b-O-4 bonds (Supporting Information, S2).I ndeed,c atechol, 5-hydroxyguaiacyl, and 3,4,5-trihydroxyphenyl thioethylated derivatives were detected after thioacidolysis of all treated samples. The proportion of these demethylated units was higheri nt he CH-treated samples, with an increased effect after 40 min of CH. An important feature arising from theseresults is that demethylation and depolymerization throught he cleavage of b-O-4 bondss eem to be concomitantb ut independentp rocesses. Both the demethylation and depolymerization reactions diagnosed herein allow the appearance of new phenolicg roups initially involved in ether bondsa nd could thus account for the increase of PhOH.
PhOH functionality gained by IL treatment combinedwith EA extraction
To assess the functionality gained throughI Lt reatment combined with subsequent extraction, the PhOH content of the EAE after treatment were compared with the initial PhOH content of the samples. As shown by the higherP hOH content of F1 (the PB1000 EA-soluble fraction) compared with PB1000,E A extraction alone provided aw ay to recover compounds with higher functionality.H owever,t his functionality was even higher when the IL treatment was applied beforer ecovery of the EAE (3.95-11.94 versus 3.88 mmolg À1 ), except for F3-MW (2.78 mmolg À1 ). Moreover,w hatever the sample and the conditions, the [HMIM]Br treatment led to an increase of PhOH compared with the startings ample. This effect increased from F1 to F3, for which am aximum ten-fold increase was obtained ( Table 2). In the case of F3, the increase in PhOH was concomitant to the decrease in average molar masses, suggesting that the treatment induced the cleavageo fe ther bonds with subsequent release of phenol groups, as previously demonstrated on lignin models. [10] The PhOH enrichment (DPhOH, mmol g À1 )t hrough IL treatment and subsequentE Ae xtractioni ncreased with the thioacidolysis yield of the initial fraction, whatever the treatment Figure 4). This was consistent with the major contribution of depolymerization reactions through b-O-4 bond cleavage to form new phenol groups. However,P B1000 and its residual F4 fraction did not followed the same tendency as the other three fractions. In particular, the DPhOH upon CH treatments was lower than expected from the behavior of these fractions. This suggested that ah igherp roportion of phenolsf ormed by depolymerization was present in the EA-insoluble residues compared with the other fractions.M oreover,t he weak differences in terms of mass distribution, and in fact the unclear correlationb etween thioacidolysis yield of the starting fraction and the M w of the obtained EAE,o nce more indicated that in the case of fractions for which the depolymerization process could be highly efficient (F3 or F4), recondensation reactions also occurred. Interestingly,t hese recondensations reactions seemed not to impact the global PhOH content. Thisc ould be also proofo fa synergistic effect between both demethylation and depolymerization processes. In all cases, the efficiency of the treatment conditions in terms of phenols production was increased from MW to CH20 and CH40. As am ajor feature, the [HMIM]Br treatment was shown to induce an increaseo fP hOH content, which may be the consequence of both depolymerization and transformation of methoxy groups into phenol groups.
To assess the effect of the PhOH increase on the antioxidant properties, the EAE showingt he highest PhOH content (CH40s amples) were selected fort he furtherr adical-scavengingt ests. The corresponding EA-insoluble residues werea lso tested in view of the potentialuse of all fractions of PB1000.
Interesti nt he products of fractionation and [HMIM]Brtreatment as potential antioxidants
Antioxidant properties (AOP) of the phenolic monomers compared withreference antioxidants
The main phenolic monomers detectedi nt he EAE after treatment, namely acidolysis ketones (compounds A and B, Figure 5) and acetosyringone demethylated once or twice (compounds C and D,F igure 5), were tested for their DPPHC radical-scavengingc apacity according to at est previously used to compare the AOP of lignin modelsa nd technical lignins. [14] EC 50 (concentrationo ft ested sample necessary to reduce 50 % of the radicals) was used for this comparison.
A, B, C,a nd D showed higher AOP (EC 50 < 0.2 gL À1 )t han the other lignin model compounds tested (ferulic acid, coniferyl alcohol, and p-coumaryl alcohol;F igure 6). The lowest EC 50 was reachedw ith the twice-demethylated acetosyringone (compound D)-four times lower than that of ferulica cid (0.21 gL À1 ), which is ar eference for natural antioxidants. The radical-scavenging capacity of lignins,and phenolics in general, relies on the ability of phenols to trap free radicals in the mediuma fter the loss of ap roton [18] followed by the stabilization of this radicalb ym esomerism.T he best performance of compounds C and D is consistent with the presence of highly electron-withdrawing groups conjugated with the aromatic ring and with the presence of several phenols carriedb ya djacent carbons. [19] Interestingly,a ll tested compounds were competitive with a commercial antioxidant such as BHT (EC 50 = 0.19 gL À1 ). Thus, these compounds might be advantageously purified or synthesized and used as antioxidants for commercial applications. However,t oa void costly purification steps and to profit from the possible synergies between phenolic compounds, the AOP of the mixtures of products recovered from the treatments [EAE and EA extractionr esidues (EAR)] was considered.
AOP of EAE andEAR compared with PB1000 and its fractions
All fractions (F1-F3) and the products recovered from the CH40 IL treatment of these fractionse xhibited DPPHC radicalscavenging capacities similar to or higher than that of the PB1000 reference sample, according to the EC 50 data (Table 3).
Concerning the untreated fractions, the resultsa re in accordance with previous studies on the fractionation of other technical lignins (organosolv lignin BIOLIGNIN and LignoBoost Kraft lignins), showingthe generally higherradical-scavenging activity of the EA-soluble fraction compared with the other ones. [20] The IL treatment of the fractions led to the production of EAE with enhanced radical-scavenging activity compared with PB1000 and the fraction considered. This enhancement could be explained by the increased PhOH contenta fter treatment and the presence of the highly antioxidant phenolicmonomers in EAE. In addition, EAR also exhibited interestingly higher activity than their corresponding startingf ractions, except for F1. In the case of F3, the AOP of the insoluble residue was even twice that of the EAE, which confirmed that some new-formed phenol groups were carriedb ys ome compounds of higher molar mass insoluble in EA after treatment. According to the EC 50 ,F 2a ppeared as the most interesting fraction with respect to the production of antioxidantst hroughI Lt reatment because the radical-scavenginga ctivity of both the EA-soluble and -insolublef ractions after treatment were the best.
Benefit of combining fractionation and IL treatment
Owing to PB1000 fractionation and the subsequent IL treatment of the fractions, as et of functional antioxidant products with distinct characteristics wasg enerated.T he mapping of the different samples according to their antioxidant property (EC 50 ), molar mass distribution (M n , M w )a nd phenolic content (Figure 7) highlights that the EAE recovered after IL treatment of the fractions combined severala dvantages:l ower molar masses (M n % 700 gmol À1 ), higher PhOH content (6-12 mmolg À1 ), andh igherr adical-scavenginga ctivity( EC 50 = 0.10-0.17 gL À1 ).
Moreover,i ts hows that the molar mass differencesb etween the fractionsw ere levelled by the IL treatment. These characteristics make them good candidates as antioxidant ingredients for the formulation of plastics or cosmetics or for the synthesis of new biobased polymers and multifunctional molecules. Based on these results, an integrated process could be proposed,and its sustainability was discussed.
Towards as ustainable cascadeintegrated process
Ta ken together,t he results showed that IL treatment could be advantageously applied to lignin fractionsf or different purposes:p roduction of competitive antioxidant extracts, increaseo f lignin free phenol content, and partial dissolution of insoluble residues.I na ll cases, the demethylation and depolymerization induced by the treatmenti sb eneficial. To design ac ascade approach and preliminarily assess its sustainability,i ti sn ecessary to consider the technical performances of the productsa long with the recovery yields andsafety aspects.
Recoveryyields and technical gain
The process proposed in Figure 8a llows the productiono fa total of 67.7 %o fp roductsw ith enhanced antioxidant activity compared with PB1000,i ncluding 35.5 %E A-soluble oligomers enriched in PhOH.T oo ptimize the gain in AOP and PhOH,t he IL treatmentc onditions selected are the most drastic ones (CH40), which on the contrary do not favor the formation of soluble material. However,b ecause the insoluble products exhibit high antioxidant properties, they could be advantageously incorporated directly as fillers in plasticso rf unctionalized by graftingo ft he PhOH. In the first step of the process, af unctional fraction is directlyo btained from PB1000 by EA extraction. In the second step, the residue is submitted to aM EK extraction combined with IL treatment of the extract to recover both EA-soluble and -insoluble highly antioxidant products.I n the last stage, the MEK extraction residue undergoes aM eOH extraction combined with IL treatment to recover EA-soluble oligomers highly enriched in phenol groups togetherwith antioxidanti nsoluble compounds. Owing to its lowest contenti n lower-molar-mass phenolic extractives, the final residue of the process might find applications,for instance, as afiller in materials. [21] Figure 6. DPPHC radical-scavenging capacity of phenolic compoundsp roduced by IL treatment of PB1000 fractions (compounds A-D)c ompared with monolignols and acommercial synthetic antioxidant (BHT). Error < 1%.
Safety-and environment-related aspectsa nd further sustainability considerations
Sustainabilitya ssessment of the developed value chain was furthere xamined for physicochemical hazards and environmental impactsp otentially arisingf rom the key chemicals involved.W ith regard to the fire hazard, the two main critical points of the process in terms of safety are the use of organic solvents (EA, MEK, and MeOH) for the extraction steps and the use of an ew ionic liquid ([HMIM]Br). Of course, the well-estab-lished flammability of the mentioned solvents has to be handled from supply to process end-use, which leads in this particular context to limited issues according to mild operations conditions. Despite their flammability properties, such materials remains till recommendable as extractions olvents, with regard to HSE criteria taken into consideration with some prioritizing rules. [22] In particular, these substances are not mentioned in any list of the EU REACH regulation designating substances of particular concern owing to their adverseh ealth or environmental impacts. As ummary of resultsp ertaining to physical hazards potentially associated to the use of [HMIM]Br is provided in Ta ble 4. Moreover, EA and MeOH could potentially be substitutedw ith biobased recommendeda lternatives, bio-ethyl lactate and bioethanol, respectively (the latter is nowadays totally biosourced). MEK, if not so easily substituted, and despite some toxicityh azard, might at least be produced from biomass in the future,i nacost-effective way as an intermediate to biobutanol. [23] Although often considereda sg reen solvents, ILs remain controversial for safety issues [24] and chiefly requireacase-by-casea nalysisi nt he context of use because safety assessments are not based only on intrinsic properties but essentially on apparatusa nd testing procedure-dependentp roperties( flash point, thermals tability, metal corrosivity). [25] Ap reliminary assessment of the [HMIM]Br safety profile hasb een performed and is providedh erein (with technical details in the Supporting Information, S3), integrating both physicochemical hazards (fire, corrosivity to metals)a nd eco-toxicological properties of the IL, in line with REACH regulation safety data neededf or future registration. This studya lso provides new insights regarding [HMIM]Br thermals tability and fire behavior,s howing in particular ar emarkable resistance to ignition and af lame retardancy property. A first-order evaluation of the corrosivity potentialo f the neat IL has indicated that further investigation on the matter will be required for the appropriate selection of the reactor materialo wing to practical conditions for use of the IL.
Regardingt he environmentalp roperties of the IL, [26] the selected test battery includes the tests required by annex VII of the REACH regulation (substancem anufactured or imported into the European Union in quantities between 1a nd 10 tons per year) and immunotoxicity tests on the three-spined stickleback (Gasterosteus aculeatus). The results (Table5)s howed that [HMIM]Br has low the toxicity for Daphniam agna and Pseudokirchneriella subcapitata (EC 50 > 100 mg L À1 ). No biodegradation was observed in the manometric respirometryt est, leading us to conclude that [HMIM]Br is not rapidlybiodegradable. These results are consistent with those already obtainedo nc ompounds of the imidazolium family,w hich demonstrate that the toxicity increases It was concluded that 75 %o fc leanI Lc an be recovered at the end of the treatment andc ould be used again for similarr eactions for several cycles. [10] All these resultsc ontribute to as afeby-design biorefineryi nvolving the studied value chain.
Conclusions
The possibility to transfer the methyli midazolium bromide ([HMIM]Br)t reatment to technicall ignins was demonstrated, by using alkali grass lignin Protobind 1000 (PB1000) as ac ommercial reference sample. Safety assessment of [HMIM]Br highlighted the effective flame-retardant property of this ionic liquid (IL) and provided data on its eco-toxicological footprint,w hich does not departf rom that of other ILs of the imidazolium family and is useful for future REACH registration. The treatment induced both depolymerization andd emethylation of lignin, leading to the formation of additional free phenols. Based on theser esultsa nd after checking that similar effects were obtained with other commercial technical lignins including Kraft lignin (data not shown), an integrated cascade process combining IL treatment and solvent extractionsw as designed to optimize the recovery yield of ethyl acetate (EA)-soluble extracts with enhanced performance comparedw ith the technical lignin. The first step directly provides af unctional EA extract, whereas the second and third steps combine extractions with IL treatment to further improve functionalities for applicationsa sa ntioxidantso rb uildingb locks. Indeed,t he extracts consisted of free-phenol-rich oligomers with antioxidant properties favorably competing with ferulic acid and tert-butylated hydroxytoluene (BHT). Besides their high antiradical activity,t hese extracts have the advantages of standardized average molar masses( 872-937 gmol À1 ), low polydispersity (1.2-1.3), and high free-phenol content(up to 11.9 mmolg À1 ), which provides opportunities for green biobased innovation in plastics and cosmeticsformulations.
Experimental Section
General materials and methods PB1000 was purchased from GreenValue LLC (USA). [27] [HMIM]Br was synthesized by following ap reviously reported procedure. [28] EA was purchased from Carlo Erba Reagents (France) and used as received. All other reagents as well as compounds A (ref 410659) and B (ref 796883), were purchased from Sigma-Aldrich Chemical Co. (USA) and were used as received. Thin-layer chromatography (TLC) experiments were performed with aluminum strips coated with Silica Gel 60 F 254 from Macherey-Nagel, revealed under UV light (254 nm), then in the presence of a5%w/w ethanolic solution of phosphomolybdic acid. Evaporations were conducted under reduced pressure at temperatures below 35 8Cu nless otherwise stated. Column chromatography (CC) was performed with an automated flash chromatography PuriFlash system and pre-packed INTERCHIM PF-30SI-HP (30 mms ilica gel) columns. 1 Ha nd 13 CNMR spectra were recorded in CD 3 OD at 400 or 100 MHz, respectively,w ith aB ruker Ascend 400 MHz instrument. Chemical shifts are reported in ppm relative to internal references (solvent signal).
Lignin fractionation and EA solubility tests EA extraction (1):P B1000 (1 kg) was fractionated by at hree-step sequential solvent extraction process, according to ap reviously published approach. [8] The following solvents were used sequentially in as emi-continuous process:E A, MEK, MEOH. Lignin was loaded in the first solvent in the glass column. After settlement, EA was pumped by HPLC pump at af low rate of 2-4 mL min À1 into the column. The solubilized fraction was collected at different heights in the column. The solvent was removed by vacuum evaporation, and the final solvent was removed by vacuum drying. The recovered solvent was reused in the process. When the concentration of solubilized lignin was very low,t he second solvent was added by the pump into the column. For each solvent, the procedure was repeated until, after three solvent extractions, the residual lignin fraction was collected from the column. This fraction was dried at amaximum temperature of 40 8C. EA extraction (2):P B1000 (6.5 g) was dissolved in EA (250 mL), and the mixture was stirred at room temperature for 30 min. The resulting solid residue was filtered, and the filtrate was recovered. The procedure was repeated nine times to obtain an exhaustive extraction. The combined EA extracts were concentrated under reduced pressure, and the final solvent was removed by vacuum drying. The residual lignin fraction was collected and dried at am aximum temperature of 40 8C. EA solubility test:L ignin solubility was determined gravimetrically by dispersing lignin (100 mg) in EA (10 mL, room temperature, 30 min), then centrifuging the suspension (20 8C, 20 min, 4000 g), and drying the solid residue at 40 8Cf or 48 h. Solubility was determined in duplicate, based on the amount of solid residue.
[HMIM]Brtreatments
For all treatments, the IL was vacuum-dried at room temperature before use. [HMIM]Br (2 g) were placed in an Anton Paar 30 mL reaction tube equipped with am agnetic stirrer.T he mixture was irradiated with P = 300 W, T max = 110 8C, ramp 30 s, hold 10 s, with full air cooling and stirring. At the end of the reaction, the solid residue was filtered and washed with water (20 mL) and EA (20 mL). The filtrate was recovered, the layers were separated, and the aqueous layer was extracted with EA (2 20 mL). The combined EA extracts were dried over MgSO 4 and concentrated under reduced pressure below 35 8C. The crude soluble mixture was analyzed by 31 PNMR spectroscopy,H PSEC, and thioacidolysis. CH treatment:T he sample (200 mg) and [HMIM]Br (2 g) were placed in an Ace pressure tube equipped with am agnetic stirrer under an inert atmosphere, then flushed with Ar.T he tube was closed, and the mixture was stirred in an oil bath at 110 8C. After 20 or 40 min, the solid residue was filtered, and the same downstream procedure as for MW irradiation was applied to the solid residue and the filtrate.
Synthetic procedure for compounds Ca nd D Acetosyringone (500 mg, 2.5 mmol) and [HMIM]Br (2.5 g, 6equiv.) were placed in an Anton Paar 30 mL reaction tube equipped with am agnetic stirrer.T he mixture was irradiated with P = 300 W, T max = 110 8C, ramp 30 s, hold 10 s, with full air cooling and stirring. At the end of the reaction, water (20 mL) and EA (20 mL) were added. The layers were separated, and the aqueous layer was extracted with EA (2 20 mL). The combined EA extracts were dried over MgSO 4 Chemical analysis of lignin and lignin-derived products GC-MS analysis:E As olutions (20 mL, 1mgmL À1 )p reviously dried with Na 2 SO 4 were silylated with bistrimethylsilyl-trifluoroacetamide (BSTFA, 100 mL) and GC-grade pyridine (10 mL). The silylation was completed within af ew minutes at room temperature. GC-MS analyses were performed in splitless mode with an Agilent 7890A GC coupled to an Agilent 5977B MS, with ap oly(dimethylsiloxane) column (30 m 0.25 mm;Rxi-5Sil, RESTEK), working in the temperature program mode from 70 to 330 8Ca t+ 30 8Cmin À1 ,o ver 20 min, with helium as the carrier gas. The chromatographic system was combined with aq uadrupole MS operating with electron-impact ionization (70 eV) and positive-mode detection, with a source at 230 8Ca nd an interface at 300 8C, and with a5 0-800 m/z scanning range. [29] Quantitative 31 PNMR spectroscopy and sample preparation:D erivatization of the samples with 2-chloro-4,4',5,5'-tetramethyl-1,3,2- dioxaphospholane (TMDP,S igma-Aldrich, France) was performed according to ar eported procedure. [30] Lignin samples (20 mg) were dissolved in am ixture of anhydrous pyridine and deuterated chloroform (400 mL, 1.6:1 v/v). Then, as olution (150 mL) containing cyclohexanol (6 mg mL À1 )a nd chromium(III) acetylacetonate (3.6 mg mL À1 ), which served as internal standard and relaxation reagent, respectively,a nd TMDP (75 mL) were added. NMR spectra were acquired without proton decoupling in CDCl 3 at 162 MHz, with aB ruker Ascend 400 MHz spectrometer.Atotal of 128 scans were acquired with ad elay time of 6s between two successive pulses. The spectra were processed by using To pspin 3.1. All chemical shifts were reported in ppm relative to the product of phosphorylated cyclohexanol (internal standard), which has been observed to give ad oublet at 145.1 ppm. The content in hydroxyl groups (in mmol g À1 )w as calculated on the basis of the integration of the phosphorylated cyclohexanol signal and by integration of the following spectral regions:a liphatic hydroxyls (150. 8 Thioacidolysis:T hioacidolysis of lignins (5 mg) was performed according to al iterature protocol, [31] by using heneicosane (C 21 H 44 , Fluka) as internal standard. Lignin-derived p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) thioacidolysis monomers were analyzed as their trimethylsilyl derivatives by GC-MS (Saturn 2100, Varian) equipped with ap oly(dimethylsiloxane) column (30 m 0.25 mm;S PB-1, Supelco) and by using the following heating program:4 0-180 8Ca t3 08Cmin À1 ,t hen 180-260 8Ca t28Cmin À1 .T he MS was an ion trap with an ionization energy of 70 eV and positive-mode detection. The determination of the thioethylated H, G, and Sm onomers was performed from ion chromatograms reconstructed at m/z = 239, 269 and 299, respectively,c ompared with the internal standard signal measured from the ion chromatogram reconstructed at m/z = (57 + 71 + 85). The molar yield of the detected thioethylated monomers was calculated on the basis of the Klason lignin content of the sample, determined according to a published procedure. [32] Molar mass distribution: M n and M w of the samples were estimated by HPSEC using as tyrene-divinylbenzene PL-gel column (Polymer Laboratories, 5 mm, 100 ,6 00 mm 7.5 mm inner diameter) with ap hotodiode array detector (Dionex Ultimate 3000 UV/Vis detector) set at 280 nm, and by using BHT-stabilized THF (1 mL min À1 ) as eluent. The samples were solubilized in THF and filtered through ap olytetrafluoroethylene membrane (0.45 mm) before injection. The molar mass averages were assessed from the apparent molar masses determined by ac alibration curve based on polyethylene oxide standards (Igepal, Aldrich) and lignin model dimers. [33] Assessmento fantioxidant properties Preparation of the solutions:Alignin sample was weighed into a 2mLm icrofuge tube, and the solvent (90:10 v/v dioxane/water mixture) was added to obtain concentrations between 0.1 and 0.5 mg mL À1 .T he dispersion was homogenized by using av ortex (Heidolph TOP-MIX 94323, Fisher Scientific Bioblock, Vaulx Milieu, France) for 30 sa t2 0000 Hz. The resulting solutions were tested for their radical-scavenging activity. Measurement of the free-radical-scavenging activity by DPPHC test:T he free-radical-scavenging activity of the samples was evaluated by measuring their reactivity toward the stable free radical DPPHC according to ap ublished method. [14] In aq uartz cuvette, the sample dioxane/water solution (77 mL) was added to 3mLo fa6 10 À5 mol L À1 DPPHC solution, prepared daily in absolute ethanol. The absorbance at 515 nm of each sample was monitored by using an UV/Visible double-beam spectrophotometer (Shigematsu Scientific Instrument, USA), until reaching ap lateau. Ab lank was prepared under the same conditions, by using 77 mLo ft he solvent instead of the sample solution. All kinetics were obtained from at least six solutions, prepared from three different lignin preparations. The kinetics of the disappearance of DPPHC were obtained by calculating at each time the difference between the absorbance of the blank solution and the absorbance of the sample. When the absorbance reached ap lateau, the percentage of residual DPPHC was calculated and plotted versus the concentration of soluble lignin in the sample tested. The concentration of antioxidant extract needed to reduce 50 %o ft he initial DPPHC (EC 50 ,w ith EC standing for efficient concentration) was determined from this linear curve.
Safetya ssessment of [HMIM]Br
The overall multicriteria safety analysis has been inspired by the global strategy developed by some of the authors of this manuscript for greener use of ILs in general, as exemplified by Eshetu et al. [34] in the case of energy storage in electrochemical devices. Fire hazard:E xamined by fire calorimetry testing based on the use of the most polyvalent fire calorimeter designated as the Fire Propagation Apparatus, following ISO 12136. See also the Supporting Information for further details on procedures and complementary details on achieved experimental data. Corrosivity screening tests:C arbon and stainless-steel specimens, partially immersed in plastic cells containing neat IL and IL added with 10 %w ater,f ollowing ah ome-made procedure (exposure in an oven regulated at 100 8Cf or 8days), with mass loss determined before and after exposure with ac alibrated balance, inspired by the procedure developed for IL corrosivity assessment by German ILs producer IO-LI-TEC. Ecotoxicity tests:E cotoxicity tests required by annex VII of the REACH regulation (substance manufactured or imported into the European Union in quantities between 1a nd 10 tons per year) have been performed. They are presented briefly in Ta ble 6. In addition to these regulatory tests, fish immunomarker tests were conducted to study possible long-term effects on aquatic ecosystems. The protocol is detailed in aprevious paper. [26] | v3-fos-license |
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} | pes2o/s2orc | Aryl hydrocarbon receptor-mediated potencies in field-deployed plastics vary by type of polymer
Plastic is able to sorb environmental pollutants from ambient water and might act as a vector for these pollutants to marine organisms. The potential toxicological effects of plastic-sorbed pollutants in marine organisms have not been thoroughly assessed. In this study, organic extracts from four types of plastic deployed for 9 or 12 months in San Diego Bay, California, were examined for their potential to activate the aryl hydrocarbon receptor (AhR) pathway by use of the H4IIE-luc assay. Polycyclic aromatic hydrocarbons (PAH), including the 16 priority PAHs, were quantified. The AhR-mediated potency in the deployed plastic samples, calculated as bio-TEQ values, ranged from 2.7 pg/g in polyethylene terephthalate (PET) to 277 pg/g in low-density polyethylene (LDPE). Concentrations of the sum of 24 PAHs in the deployed samples ranged from 4.6 to 1068 ng/g. By use of relative potency factors (REP), a potency balance between the biological effect (bio-TEQs) and the targeted PAHs (chem-TEQs) was calculated to 24–170%. The study reports, for the first time, in vitro AhR-mediated potencies for different deployed plastics, of which LDPE elicited the greatest concentration of bio-TEQs followed by polypropylene (PP), PET, and polyvinylchloride (PVC). Electronic supplementary material The online version of this article (10.1007/s11356-019-04281-4) contains supplementary material, which is available to authorized users.
Introduction
In the early 1970s, plastic particles were detected in marine systems for the first time (Carpenter and Smith 1972). By the late 1990s, more than 180 marine species were recorded to demonstrate ingested plastic particles (Laist 1997). Today, three quarters of all marine debris is plastic, and more than 500 marine and coastal species, including fish, seabirds, marine mammals, marine reptiles, and brackish turtles, are known to be affected by marine debris via ingestion or entanglement (CBD 2016). Considering the increasing global plastic consumption per year, numbers of affected species can be expected to increase. In 2015, worldwide production of plastic was 322 million tonnes, of which the European Union contributed 58 million tonnes (PlasticsEurope 2016). It has been estimated that at least 8 million tonnes of plastics enter the oceans every year from land-based sources (Jambeck et al. 2015).
While the mere physical threat such as entanglement, strangulation, and abrasion that plastic can pose to organisms is obvious, it has also been hypothesized that plastics hold a toxicological risk due to the transfer of pollutants when the particles are ingested (Teuten et al. 2007;Teuten et al. 2009). Numerous studies have demonstrated that a variety of anthropogenic environmental pollutants can be found on different types of marine and beached plastic debris (GESAMP 2016). Additionally, there is mounting evidence for the presence of Responsible editor: Markus Hecker Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-019-04281-4) contains supplementary material, which is available to authorized users. plastic particles in higher trophic level organisms (CBD 2016) as well as in commercially important fish and shellfish species (Romeo et al. 2015;Van Cauwenberghe and Janssen 2014). Trophic transfer of microplastics has also been observed in laboratory studies (Farrell and Nelson 2013;Watts et al. 2014). Besides a possible bioaccumulation of contaminants due to ingested plastics (Teuten et al. 2009), there might be a potential for biomagnification of contaminants throughout the food chain. In some cases, a trophic transfer of contaminants can lead to a biomagnification, this is generally said to happen when the trophic transfer factor (TTF) is greater than 1. The TTF is defined as the concentration of contaminant in consumer tissue divided by the concentration of the contaminant in food (Suedel et al. 1994).
The sorption of chemicals from ambient environments to plastic is influenced by the physical and chemical properties of the type of polymer (Karapanagioti and Klontza 2008;Rochman et al. 2013;Teuten et al. 2007), as well as the surrounding environment (Velzeboer et al. 2014). Furthermore, sorption of chemicals is compound-specific and reflected in varying partition coefficients (Ziccardi et al. 2016). For instance, sorption affinities increase with increasing hydrophobicity (Smedes et al. 2009), while sorption kinetics decrease with increasing hydrophobicity (Endo et al. 2013;Rochman et al. 2013). In general, rubbery polymers such as low-density polyethylene (LDPE) and polypropylene (PP) are expected to have a greater diffusivity than glassy polymers like polyethylene terephthalate (PET) and polyvinylchloride (PVC) (Rochman et al. 2013;Teuten et al. 2009). Due to its great sorptive capacity for hydrophobic organic compounds (HOCs), polyethylene (PE) has been widely used in passive sampling approaches in different media (Zabiegala et al. 2010) and it is well known that there is a good correlation between polyethylene-water partition coefficients (log K PE-W ) and octanol-water partition coefficients (log K OW ) for different HOCs (Adams et al. 2007;Choi et al. 2013). In the marine environment, PE, PP, and polystyrene (PS) are the three most commonly observed types of polymers (Hidalgo-Ruz et al. 2012). A variety of anthropogenic chemicals including polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polybrominated biphenyl ethers (PBDEs) and organochlorine pesticides (OCPs) can be associated with marine plastics. Within these groups of aforementioned HOCs, some chemicals are potentially harmful to humans and wildlife, and can act as mutagens, carcinogens, and endocrine disruptors or cause oxidative stress (Costa et al. 2008;Fonnum et al. 2006;Fry 1995;Zhang et al. 2016). Doubts have been raised about the importance of marine plastics as a vector to transfer potentially hazardous HOCs to organisms upon ingestion, because other sources such as natural prey and marine components, like colloids and sediments, in general hold greater concentrations of HOCs compared to plastics, and therefore potentially predominate the flux of HOCs to marine organisms (Herzke et al. 2016;Koelmans et al. 2016). Other researchers have suggested that the transfer of additives, such as flame retardants and plasticizers, from plastics, could be of greater importance than the transfer of sorbed HOCs, such as PCBs (Rochman et al. 2014;Tanaka et al. 2013;Teuten et al. 2009). Nevertheless, there is limited experimental data available on biomarker of exposure responses on cellular and subcellular levels upon exposure to microplastics containing adsorbed HOCs (Ziccardi et al. 2016), and it is important to understand how the mixture of plastics and sorbed HOCs will interact with biota.
The objectives of this study were to assess the potentials of four types of primary plastics, also referred to as preproduction or virgin plastics, to activate the aryl hydrocarbon receptor (AhR), by use of the H4IIE-luc, in vitro, transactivation assay. The plastic pellets were previously deployed in an urban marine environment (Rochman et al. 2013), and thus contained sorbed HOCs. Simultaneously, contribution of PAHs to AhR-mediated signal transduction was assessed by use of a potency balance analysis. In vitro bioassays are useful to screen for integrated effects of contamination in a variety of matrices (Eichbaum et al. 2014). One benefit of in vitro assays, based on expression of specific reporter genes, is that they integrate the overall potency of a mixture to modulate specific biological activities of an environmental sample as well as correct for interactions between and among constituents and correct for chemical potencies. The herein utilized H4IIE-luc assay (Murk et al. 1996) has been applied as a biomarker to screen for dioxin-like chemicals (DLCs) including co-planar non-di-ortho-substituted polychlorinated biphenyls (DL-PCBs) and some PAHs (Giesy and Kannan 1998;Larsson et al. 2012;Van den Berg et al. 2006). The cytosolic receptor protein, AhR, mediates most of the toxicological effects of specific halogenated aromatic hydrocarbons (HAH) and is also involved in inducing transcription of certain enzymes that catalyze metabolic activation of PAHs to mutagenic derivatives (Hankinson 1995). These effects can include impaired neurobehavioral functions and reproductive and neurochemical alterations such as reduced sperm production and reduced thyroid hormone levels in the brain and body (Brouwer et al. 1995). The Ah receptor evolved very early in the evolution of vertebrates, and thus, exists in a variety of species including fishes (Hankinson 1995).
In a long-term field study, exposing preproduction plastic pellets to the marine system in the San Diego Bay, it was observed that polyethylene plastics sorbed greater amounts of PCBs and PAHs than did PP, PET, and PVC (Rochman et al. 2013). Therefore, it was hypothesized by the authors that PE plastics might pose a greater toxicological risk to organisms when ingested than other polymer types. In the present study, a subset of the samples that were analyzed by Rochman et al. (2013) was used to assess the hypothesis that extracts of PE plastics will elicit a greater AhR-mediated response than PP, PET, and PVC. A potency balance was conducted using concentrations of 42 polycyclic aromatic compounds (PACs) since the previous study demonstrated that PAHs (n = 15) were the predominant AhR agonists found in the samples.
For more information regarding chemical standards, refer to the Supporting Information (SI).
Deployment
Four types of plastic preproduction pellets, PP, PVC, PET, and LDPE, were deployed for 9 and 12 months from docks at three locations ( Figure S1 of the Supporting Information), Shelter Island (SI), Harbor Excursion (HE), and Nimitz Marine Facility (NMF), in the San Diego Bay, CA, USA. Additionally, one blank sample of every polymer type was analyzed before deployment, time zero. Hereafter, samples are named by the polymer acronym, acronym of the site of deployment and number of the months of deployment. PET samples were cylindrical (2 mm diameter, 3 mm long), pellets of LDPE, PP‚ and PVC were spherical (3 mm diameter). All docks were floating except for the Nimitz Marine Facility, which was a fixed-elevation dock. The plastic samples were deployed at a depth of approximately 0.5 m below surface. For more information regarding the deployment, see Rochman et al. (2013). Upon collection, after deployment, plastic pellets were stored at − 20°C until analysis.
Extraction
In total, 26 samples, including blank polymers, were extracted. Samples PVC NMF 9 and PET NMF 12 were not extracted because of insufficient amounts of sample material (Table S1 and Table S2 of the SI). Replicate samples were not available for this study. Samples were weighed into 15 ml brown glass vials and filled up with approximately 10 ml of n-hexane, masses ranged from 0.7 to 2.5 g (Table S1 of the SI). Vials were placed into an ultra-sonication bath for 60 min, and the extracts were then transferred to Erlenmeyer flasks and fresh solvent was added to the remaining samples. The sonication procedure was repeated two times. N-hexane extracts were evaporated with a rotary evaporator to a volume of 1 ml and subsequently split into two aliquots. Pellets with masses equal or less than 1.3 g were not split and those extracts were only used for bioassay evaluations, these samples were PVC SI 9, PVC SI 12, PVC HE 12, and PET HE 12. One aliquot of each extract was further evaporated and solvent exchanged into 25 μl of dimethyl sulfoxide (DMSO) for use in bioassays. The second aliquot was further evaporated and solvent exchanged into 400 μl of toluene for use of quantification of PACs. Toluene extracts were spiked with labeled internal (IS) and recovery (RS) standards, which resulted in 22 extracts for chemical analysis.
H4IIE-luc assay
Sample PVC HE 9 was not available for bioassay analysis, which reduced the total number of samples tested in the bioassay to 25 (Table S1 of the SI). AhR-mediated potencies of extracts were quantified by use of the H4IIE-luc assay, which is a transactivation assay based on a recombinant rat hepatoma cell line, which has been stably transfected with a luciferase reporter gene (Murk et al. 1996). The H4IIE-luc assay was performed as previously described (Larsson et al. 2013). Prior to exposure, dilutions of sample extracts were prepared as fourfold serial dilutions of the initial concentration in culture medium. Six different concentrations were derived and added to the test plates. Initial medium concentrations ranged from 245 mg plastic/ml for the sample PVC HE 12 to 540 mg plastic/ml for PET SI 9. On each test plate, a TCDD standard curve of seven concentrations (0.4-300 pM) and a solvent control of DMSO were tested as well. The concentration of DMSO in test media exposure was 0.4%. Samples, standards, and solvent controls were tested in triplicate wells on each plate. After a 24-h incubation, exposure medium in the wells was discarded and the wells were washed twice with 100 μl of phosphatebuffered saline solution (PBS). Cells were lysed in 25 μl of PBS and 25 μl of steadylite plus™ substrate mix and incubated for 15 min in darkness at room temperature for enzymatic reaction to take place. Cell lysates were transferred to white, 96-well, microtiter plates, and the luciferase activity in each well was measured in a luminescence plate reader (FLUOstar Omega; BMG Labtech). Activity of luciferase was expressed in counts per second (CPS) and is proportional to the potency of AhR agonists in the mixture. Concentration-response curves for TCDD and e x t r a c t s w e re o b t a i n e d b y u s e o f a s i g m o i d a l concentration-response (variable slope) equation (GraphPad Prism® 5.0 software). The cells were microscopically examined for cytotoxicity before and after the exposure.
Chemical analysis
Quantification of 42 polycyclic aromatic compounds, including PAHs, oxy-PAHs, and azaarenes, was performed by use of an Agilent 7890A gas chromatograph, coupled to a 5975C, low-resolution mass spectrometer (GC/LRMS), which was equipped with a ZB-SemiVolatiles column (30 m × 0.25 mm, 0.25 μm film thickness; Phenomenex). The initial oven temperature was 90°C (held for 2 min), ramped by 8°C min −1 to 300°C (held for 10 min). All measurements were performed in single ion-monitoring mode. Identification and quantification of the PACs in extracts were done by use of quantification mixtures including 24 PAHs and 18 oxy-PAHs and azaarenes in addition to IS and RS. Concentrations of PACs were calculated by use of the isotope dilution method.
Data analysis
For data analysis of the bioassay results, the raw data was tested for outliers with a Grubbs' test (alpha = 0.05) using the program QickCalcs outlier calculator by GraphPad Software. Bioassay-derived TCDD equivalents (bio-TEQs) were calculated from concentration-response curves by relating the luciferase induction potency of the samples to that of the TCDD standard as described previously (Larsson et al. 2013). Only plates with a TCDD EC50 value between 6.3 and 14 pM and a maximal induction factor (MIF) between 7 and 14 were used in TEQ calculations. Limit of detection (LOD) in the assay was calculated as the mean of the solvent control (DMSO) plus three times the standard deviation.
Chemically derived TCDD equivalents (chem-TEQs) were calculated as the sum of the product of individual concentrations of PACs multiplied with their H4IIE-luc assay-specific relative potency factors (REP) based on 25% effect concentration (EC25) (Eq. 1). The applied REP values were previously established and published by our laboratory (Larsson et al. 2012(Larsson et al. , 2014. The potency balance was calculated as the percentage of the fraction of quantified PACs (chem-TEQ) that can explain the AhR-mediated potency (bio-TEQ).
Statistical analyses were conducted by using GraphPad Prism® 5.0 software, and statistical significance was defined as p < 0.05.
H4IIE-luc assay
In total, 25 different extracts were tested in the H4IIE-luc assay including four blank samples of each polymer type (LDPE, PP, PET, and PVC) before deployment. The blank samples had low AhR-mediated potencies, which were less than the LOD of the assay. Two of the deployed samples also exhibited potencies that were less than the LOD, those were pellets of PVC and PET deployed for 12 months at Shelter Island and Nimitz Marine Facility, respectively. All other samples had quantifiable AhR-mediated potencies in the H4IIEluc assay. Because some of the analyzed samples had efficacies that were less than 50% of the TCDD maximum induction (TMI), bio-TEQs for all samples were calculated at the 25% effect level (Villeneuve et al. 2000). Bio-TEQs ranged from 2.7 pg/g in PET deployed for 9 months at Nimitz Marine Facility (PET NMF 9) to 277 pg/g in low-density PE deployed for 9 months at Harbor Excursion (LDPE HE 9) (Table S2 of the SI). Among the four different polymers, LDPE samples deployed at the sites Harbor Excursion and Shelter Island exhibited greater AhR-mediated potencies (bio-TEQs) at both durations of deployment, compared to the three other polymers (Fig. 1). The differences among plastic types were, however, not significant (ANOVA, p = 0.041, followed by Tukey's test, p > 0.05), also the three tested locations were not significantly different from each other (ANOVA, p = 0.24). At the third site, the Nimitz Marine facility, PP had the greatest AhR-mediated potency followed by LDPE. Deployed PVC and PET samples exhibited relatively small AhR-mediated potencies with concentrations of bio-TEQ Fig. 1 Comparison of concentrations of bio-TEQs and chem-TEQs in pg/ g for four preproduction plastic pellets at 9 and 12 months of deployment at three sites determined by use of H4IIE-luc assay. Data represent the mean value of n replicates (see Table S2 of the SI) and error bars represent the standard deviation of n replicates. N.d. no detectable response, less than the limit of detection (LOD) two orders of magnitude less than the greatest concentrations of bio-TEQ in extracts of LDPE. AhR-mediated potency of samples was less in most extracts of samples that had been deployed 12 months compared to those of samples deployed for 9 months (Fig. 1).
Chemical analysis
Chemical analysis of 24 PAHs, 18 oxy-PAHs, and azaarenes was conducted on 18 deployed plastic pellets and 4 blank preproduction plastic pellets (Table 1). Concentrations of oxy-PAHs and azaarenes were less than the LOD in all samples, while the 24 selected PAHs were detectable in most of the samples. Blank samples contained measurable concentrations and the sum of detected PAHs was greatest in PP (12 ng/ g), followed by LDPE (2.1 ng/g) and below LOD in PET and PVC (Table 1). Among the polymers, LDPE sorbed the greatest concentrations of PAHs. Concentrations of the sum of 24 PAHs in deployed samples ranged from 4.6 (PVC HE 9) to 1068 ng/g (LDPE HE 12) (Fig. 2) All calculated chem-TEQ values for blank samples were less than the LOD. Chem-TEQ values ranged from 1.5 (PVC HE 9) to 98 pg/g (LDPE HE 12) ( Table S2). The greatest chem-TEQ values were calculated for LDPE compared to the three other plastic pellets, followed by PP, PET, and PVC with the lowest calculated chem-TEQ. Potency balances were between 24 and 170% (Table S2).
Discussion
The results of this study present experimental evidence to corroborate the hypothesis by Rochman et al. (2013) that PE plastics might pose a greater toxicological risk compared to PP, PET, and PVC. In particular, the finding that LDPE had the greatest AhR-mediated potencies compared to the other tested polymers was expected, considering the fact that polyethylene has been recognized as a good sorbent for HOCs in passive sampling devices (Zabiegala et al. 2010). This is of importance since PE is a high-volume production polymer used in, for example, packaging materials (PlasticsEurope 2016) and is frequently found in microplastic fractions in surface waters globally (Hidalgo-Ruz et al. 2012). Interesting for a toxicological assessment of plastic in the marine environment is the fact that six out of eight pairs of the samples were less potent in activating the AhR pathway after 12 months of deployment compared to 9 months. This was independent of the type of polymer but most pronounced in LDPE at Harbor Excursion. The decline in AhR-mediated potency at 12 months illustrated by bioassay results cannot be attributed to a possible desorption and/or degradation of the analyzed PAHs, because most samples illustrated a greater total concentration of PAHs after 12 months compared to 9 months. In the study by Rochman et al. (2013), using more time data points, total concentrations of PAHs adsorbed to LDPE and PP were found to be in equilibrium at 6 months and for PET and PVC equilibrium occurred at 3 months. AhR ligands other than the quantified PAHs could have been desorbed from the plastic pellets or degraded during the period of deployment from 9 to 12 months. Biofilms with a diverse microbial community, such as hydrocarbon-degrading bacteria, can develop on marine plastic and possibly biodegrade the plastic polymer (Zettler et al. 2013). Hence, a reduction of Ah receptor agonists sorbed to the surface of the polymer, other than quantified PAHs and PCBs, might occur and lead to a decreased induction in the bioassay after a longer time of deployment. Therefore, preproduction plastic pellets might potentially become less toxic with time in the aquatic system due to biodegradation. It has been reported from passive sampling devices that fouling can lead to a decrease in the uptake of HOCs compared to not fouled devices (Harman et al. 2009). A longer residence time in the marine environment might also lead to an increased leaching of unreacted monomers and additives from the plastic products to the surrounding environment, which could lead to a lesser AhR-mediated potency in the bioassay when they are AhR agonists. It is also possible that other chemicals, which act as AhR antagonists, sorbed to the pellets with continuing time. These antagonists could have been masking the effects of the Ah receptor agonists and therefore may have led to a decrease in potency in the bioassay from 9 to 12 months. For instance, copper that has been found as a pollutant of concern in the San Diego Bay marina (CA.EPA 2016) could have been sorbed to the preproduction pellets as it has been shown to be present in marine plastics (Ashton et al. 2010;Holmes et al. 2012), and can lead to an inhibition of AhR expression (Darwish et al. 2014;Korashy and El-Kadi 2004). The ability to conclude that the AhRmediated potency decreased with continuing residence time in the marine system was hampered by a limited sample amount of some samples, resulting in the inability to analyze all time points (see Table S2 in the supporting information). The study design by Rochman et al. (2013) included two Rochman et al. (2013). For a better understanding of the potential impact of dioxin-like contaminants adsorbed to marine plastic debris, it is important to investigate whether the finding that AhRmediated potency decreased with extended marine residence time can be applied to even longer weathered microplastics as well. This is particularly of relevance because chemicals with a greater hydrophobicity and higher molecular weight are expected to reach equilibrium later than the lower molecular weight and less hydrophobic chemicals (Muller et al. 2001). It has been estimated that sorption of most HOCs to marine microplastics with a size of 0.5-5 mm will be at equilibrium after 2 years (Koelmans et al. 2016). There is one study so far that demonstrated that weathered plastic pellets had greater distribution coefficients for phenanthrene compared to preproduction PE and PP samples, however, these materials had slower diffusion kinetics (Karapanagioti and Klontza 2008). This would suggest that weathered plastics are able to bind a greater amount of pollutants over time, and thus could lead to greater AhR-mediated potencies than their primary plastic counterparts. The sorption should be further investigated, however, testing for different types of polymers, different pollutants with a range of log K ow values, and importantly for different particle sizes. It has been shown in a model that with decreasing pellet diameter, the sorption of pollutants to plastic pellets will be faster (Endo et al. 2013). Since weathered plastics are more prone to break down into smaller pieces due to embrittlement, those particles might absorb pollutants even faster than their preproduction counterparts.
The quantification of 24 PAHs is consistent with previous findings that reported LDPE to have the highest sorption capacity, followed by PP, PET, and PVC (Rochman et al. 2013). San Diego Bay is a natural harbor and deep-water port with facilities for cargo and cruise ship terminals. The adjacent areas of the bay are highly urbanized and the San Diego International airport is in close proximity to the sampling site Harbor Island. The two other sampling sites are influenced by several recreational marinas as well as commercial fishing fleets. Therefore, it was expected to find chemicals of anthropogenic origin in the plastic samples, especially PAHs from pyrogenic sources formed by incomplete combustion of fossil fuels, e.g., from ships, as was demonstrated in a previous study by Rochman et al. (2013). The prevalence of PAHs was reflected in the present study by the relatively great contribution of PAHs to the AhR-mediated potency in the samples. Fluoranthene was observed to be major component of the total PAH concentrations in all deployed LDPE samples and most of the PP samples. This is in accordance with surface sediments from San Diego marinas in which the higher molecular weight PAHs (4-6 rings) were detected as the major contributors to total PAH concentrations of 36 individual PAHs (Neira et al. 2017). However, for the assessment of AhR-mediated signaling, fluoranthene is negligible because this compound does not activate the Ah receptor following a 24-h exposure (Larsson et al. 2012). In all analyzed PET and PVC samples, fluorene was the main contributor to the sum of the concentrations of individual PAHs. Fluorene is a lower molecular weight PAH and a weak AhR agonist that does not contribute to the AhR-mediated potency in the bioassay, which was also reflected by the low bio-TEQ results for these polymers. In all deployed samples, benzo[b+j]fluoranthenes and benzo[k]fluoranthene were the major contributors to the overall biological efficacy.
The quantified PAHs explained more than 50% of the observed AhR-mediated potency in 12 of the 16 samples for which both bio-TEQ and chem-TEQ values were available. However, in samples with the greatest potential for AhR pathway activation (LDPE HE 9 and LDPE HE 12), there is an unexplained potency of 66% and 44%, respectively. Many other compounds are able to bind to the AhR and activate the AhR pathway including PCBs, polychlorinated dibenzop-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) (Lee et al. 2013;Van den Berg et al. 2006). In fact, the presence of other pollutants besides PAHs have been reported in the San Diego Bay, such as PCBs, copper, mercury, zinc, chlordane, and lindane/hexachlorocyclohexane (HCH) (CA.EPA 2016). The previous study by Rochman et al. (2013) observed measurable levels of PCBs in all of the plastic samples with the greatest PCB concentrations at the sampling site Shelter Island. At this site, LDPE did not reach equilibrium by 12 months, while PET and PVC reached equilibrium by 6 months and PP was at equilibrium at 12 months. The time to B la n k P V C B la n k P P B la n k P E T B la n k L D P E Fig. 2 Total concentrations of PAH in ng/g as the sum of 24 PAHs sorbed to four types of plastic pellets deployed for 9 and 12 months at three locations. Blank samples represent the respective preproduction pellet before deployment reach equilibrium for the total concentration of PCBs sorbed to LDPE at Shelter Island was estimated to be 19 months. Although the total PCB concentrations were noticed to be approximately one to two orders of magnitude less than the measured total PAH concentrations, some PCBs are more potent AhR agonists than PAHs. They are, therefore, able to induce a greater potency for the receptor in a mechanism specific bioassay such as the H4IIE-luc. Reported H4IIE-luc-specific REP values for 11 PCBs (CB#81,77,123,118,114,105,126,167,156,169,and 189) ranged from 1 × 10 −9 to 0.1 (Lee et al. 2013). PCB 126 was observed to have the greatest REP value of 0.1 in the H4IIE-luc assay (Lee et al. 2013), whereas the most potent PAH analyzed in the present study, benzo[k]fluoranthene, had a REP value of 0.002 (Larsson et al. 2012). In order to determine a possible contribution of PCBs to the measured bio-TEQs, theoretical chem-TEQ values were calculated for the polymers LDPE, PP, and PET, based on the previously reported concentrations of individual PCB congeners by Rochman et al. (2013) and the reported REP values by Lee et al. (2013). Overall, it was observed that PCBs did not contribute to the measured bio-TEQs, except in one of the duplicate LDPE SI 9 samples where PCBs contributed about 3% to the bio-TEQ. Considering the theoretically calculated contribution of PCBs and PAHs to the AhRmediated potency in the samples, results suggest that the remaining unexplained potency in the bioassay must be attributed to AhR-activating compounds other than the measured PAHs and PCBs. Dioxins, for example, are more potent AhR agonists than PAHs (Van den Berg et al. 2006). An effectdirected analysis could be a useful tool to implement here in order to identify causative agents. Because the bioassay is integrating the complete potency of chemicals present in a mixture and chemical analysis is only looking for target compounds, it is possible that given only the chem-TEQs, one would over-or underestimate the potency in the bioassay (Table S2). It is possible that the chemicals are interacting with each other in a way that is masking the effect of quantified PAHs present in the mixture, which is reflected in a greater chem-TEQ than bio-TEQ. On the other hand, the chemicals can maybe have additive effects (Giesy and Kannan 1998), and thus elicit a greater bio-TEQ compared to chem-TEQ.
As this is the first study to measure AhR-mediated potency on microplastics deployed in the marine system there is no comparable bioassay data available. Nevertheless, there is data available from sediment toxicity studies. One study of sediment samples from the estuary at the river Elbe in Germany revealed bio-TEQ values ranging from 45 pg/g dry matter (d.m.) to 268 pg/g d.m., determined by use of the H4IIE-luc assay (Otte et al. 2013). These values are similar to the bio-TEQs for the deployed plastic pellets. PAH concentrations of the 16 priority PAHs defined by the US EPA were reported to range from < 20 to 906 ng/g d.m., which is comparable to concentrations found in the present study and underlines the role of plastic pellets as a sorbent for environmental pollutants, such as PAHs, with similar sorptive capacities as sediment.
A study on surface sediments in San Diego Bay marinas revealed concentrations of the 16 priority PAHs to range from 246 to 3452 ng/g d.m. sediment at sampling sites in Shelter Island and 606-19,967 ng/g d.m. at sampling sites in Harbor Excursion East (Neira et al. 2017). The LDPE samples at Harbor Excursion were observed to be within this range with a total 16 priority PAH concentration of 943 and 982 ng/g at 9 and 12 months, respectively. Concentrations of LDPE samples from Shelter Island were found to be less than concentrations of sediment samples, with concentrations of 160 and 195 ng/g by 9 and 12 months. However, PAH concentrations have been shown to vary about one or two orders of magnitude at different sampling sites in the same marina (Neira et al. 2017) resulting in large spatial variation at the same sampling site.
Assessing the risk of AhR-mediated effects due to microplastic exposure is not possible since the intake and bioavailability of AhR agonists from microplastics is unknown. Concentrations of total PAHs in this study, however, were much less than the effect range-low (ERL) of 4022 ng/g d.m. for sediments, established by the US National Oceanic and Atmospheric Administration (NOAA) (Buchman 2008). Even when considering only the16 priority PAHs, the ERL (2968 ng/ g) is almost three times that of the greatest total PAH concentration of 1068 ng/g (LDPE HE 12) detected in this study.
Due to the fact that an exhaustive extraction method was used in this study, a worst case scenario of desorption of pollutants from the plastic pellets was represented. It might be unlikely that the same amount of pollutants would desorb within an aquatic organism from microplastics of the size used in this study during the residence time in the body of an organism, however, this needs to be confirmed with experimental data. Because some sites in the San Diego Bay marina were classified as potentially toxic for benthic organisms (Neira et al. 2017), the body burden of the resident organisms can be expected to be already high, and ingested plastic might not lead to elevated levels of organic pollutants in the bodies. For example, it has been demonstrated that the transfer of pollutants into tissues of lugworms can be higher from sand compared to microplastics (Browne et al. 2013). Therefore, uptake of pollutants from other marine matrices, such as sediments, colloids, and dissolved organic carbon (DOC), might predominate because they are more abundant in most habitats than plastic and also hold a greater percentage of sorbed HOCs than plastic (Koelmans et al. 2016). Additionally, prey as a source of pollutants needs to be considered. Nevertheless, marine plastic is able to travel over long distances and can be transported into less polluted areas. Therefore, it might have a bigger impact on organisms that live in these habitats than in already polluted sites. Furthermore, it has to be stressed that the AhR-mediated potency is given on a per 1 g of plastic basis, which is, for example, ten times greater than the ecological quality objective (EcoQO) of < 0.1 g ingested plastic that should be achieved in 10% of the seabird population of northern fulmars (OSPAR 2008). It might not be the case in a common marine environment that 1 g of plastic will be ingested by an organism, but it could be the case for accumulation zones and other hot spots of plastics. Unfortunately, a vast number of publications report ingested plastic pieces only as number of pieces and do not give the weight or diameter of those pieces. This information could be beneficial in the future to estimate the load of chemicals that can be attached to ingested plastics and ultimately might be able to transfer into marine organisms.
Conclusions
The measurable in vitro AhR-mediated potencies (bio-TEQs) in preproduction plastic pellets, of which 24 selected PAHs contributed more than 50% to the bio-TEQ in 76% of the samples, varied by the type of polymer. Extracts of LDPE demonstrated the greatest potential for AhR pathway activation as well as greatest sorption capacities of PAHs. This reinforces the hypothesis that PE has the potential to hold a greater toxicological risk than PP, PET, and PVC. The background levels of AhR-mediated potencies of marine microplastics are unknown and should be investigated in remote areas. The observed tendency for a decreased AhRmediated potency in some of the 12-month deployed samples compared to 9 months should be further investigated and verified in another set of field deployed samples. In the future, other mechanisms of action such as endocrine receptormediated pathways should be assessed as well in order to get a more complete understanding of the possible impacts of marine microplastics.
Funding information This study was funded by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (223-2014-1064) and by the EnForce research project, funded by the Knowledge foundation.
Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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} | pes2o/s2orc | Evaluation of NO + reagent ion chemistry for online measurements of atmospheric volatile organic compounds
NO chemical ionization mass spectrometry (NO CIMS) can achieve fast (1 Hz and faster) online measurement of trace atmospheric volatile organic compounds (VOCs) that cannot be ionized with H3O ions (e.g., in a PTR-MS or H3O CIMS instrument). Here we describe the adaptation of a high-resolution time-of-flight H3O CIMS instrument to use NO primary ion chemistry. We evaluate the NO technique with respect to compound specificity, sensitivity, and VOC species measured compared to H3O. The evaluation is established by a series of experiments including laboratory investigation using a gas-chromatography (GC) interface, in situ measurement of urban air using a GC interface, and direct in situ measurement of urban air. The main findings are that (1) NO is useful for isomerically resolved measurements of carbonyl species; (2) NO can achieve sensitive detection of small (C4–C8) branched alkanes but is not unambiguous for most; and (3) compoundspecific measurement of some alkanes, especially isopentane, methylpentane, and high-mass (C12–C15) n-alkanes, is possible with NO. We also demonstrate fast in situ chemically specific measurements of C12 to C15 alkanes in ambient air.
Introduction
Volatile organic compounds (VOCs) are central to the formation of ozone and secondary organic aerosol and can have direct human health effects.Attempting to understand the behavior of these species in the troposphere presents sev-eral measurement challenges (Glasius and Goldstein, 2016).First, VOCs are highly chemically diverse.Second, many environmentally important species require measurement precision of better than 100 parts per trillion (ppt).Finally, numerous applications, such as eddy flux analyses or sampling from a mobile platform, require fast in situ measurements, with 1 min or faster time resolution.
H 3 O + chemical ionization mass spectrometry (H 3 O + CIMS), more commonly known as proton-transfer-reaction mass spectrometry (PTR-MS), is a well-established approach to measuring VOCs (de Gouw and Warneke, 2007;Jordan et al., 2009b).In H 3 O + CIMS, air is mixed with hydronium (H 3 O + ) ions in a drift tube region.VOCs are ionized by transfer of the proton from H 3 O + to the VOC.These instruments are capable of VOC measurements that are fast, sensitive, and chemically detailed (Jordan et al., 2009b;Graus et al., 2010;Sulzer et al., 2014;Yuan et al., 2016).
Despite these advantages, H 3 O + CIMS has several limitations related to the reagent ion chemistry.For one, this technique generally cannot distinguish between isomers.For instance, this is a significant limitation when measuring aldehyde and ketone carbonyl isomers, which display very different behavior in the atmosphere.Separation of propanal and acetone with PTR-MS has been explored using collisioninduced dissociation with an ion-trap mass analyzer, but this technique negatively affects the instrument time resolution and sensitivity (Warneke et al., 2005).Additionally, some proton transfer reactions are dissociative.Large hydrocarbons (C 8 and larger) fragment into common small masses, making spectra difficult to interpret (Jobson et al., 2005;Er-Published by Copernicus Publications on behalf of the European Geosciences Union. ickson et al., 2014;Gueneron et al., 2015).Alcohols and aldehydes can lose H 2 O, lowering the sensitivity to the protonated parent mass; their product ion masses then coincide with those of hydrocarbons, making independent measurement difficult (Španěl et al., 1997;Buhr et al., 2002).Furthermore, H 3 O + CIMS is not sensitive to small (∼ C 8 and smaller) saturated alkanes, as their proton affinities are lower than or very close to that of water (Arnold et al., 1998;Gueneron et al., 2015).This is a serious limitation in studies of urban air or emissions from oil and natural gas extractions, where small alkanes can contribute a large fraction to the total gas phase carbon and chemical reactivity (Katzenstein et al., 2003;Gilman et al., 2013).Gas-chromatography (GC) techniques avoid many of these limitations but have much slower time resolution.
Use of NO + reagent ion chemistry may address some of the limitations of H 3 O + .Reaction of NO + with various VOCs has been extensively studied using selected-ion flow tube methods (SIFT-MS).SIFT methods use a quadrupole mass filter between the ion source and ion-molecule reactor, which provides a very pure reagent ion source but limits the primary ion signal.SIFT studies have identified the major products of the reaction of NO + with VOCs representative of many different functional groups (Španěl and Smith, 1996(Španěl and Smith, , 1998a(Španěl and Smith, , b, 1999;;Španěl et al., 1997;Arnold et al., 1998;Francis et al., 2007a, b).Aldehydes and ketones are easily separable: ketones cluster with NO + , forming mass (m + 30) ions, whereas aldehydes react by hydride abstraction, forming mass (m − 1) ions (where m is the molecular mass of the species).Rather than losing H 2 O, as in H 3 O + CIMS, alcohols react by NO + adduct formation or hydride abstraction.And finally, NO + can be used to detect alkanes: small (> C 4 ) branched alkanes and large (> C 8 ) n-alkanes react by hydride abstraction, forming mass (m − 1).
The application of SIFT methods to atmospheric analysis has been limited by relatively poor sensitivity (Smith and Španěl, 2005;Francis et al., 2007b;de Gouw and Warneke, 2007), although better sensitivities have been reported in recent years (Prince et al., 2010).The adaptation of an existing CIMS instrument to use the SIFT technique requires extensive instrument modification or the purchase of an external SIFT unit (Karl et al., 2012).Several groups have experimented with low-cost adaptation of H 3 O + CIMS instruments to use NO + chemistry.Knighton et al. (2009) adapted an H 3 O + CIMS instrument to measure 1,3-butadiene and demonstrated in situ detection of this species in the atmosphere.Jordan et al. (2009a) have developed a hollowcathode ion source capable of switchable reagent ion chemistry, and they demonstrated laboratory measurement with NO + of several aromatics, chlorinated aromatics, and carbonyls, with sensitivities comparable to H 3 O + CIMS.The NO + capability of the Jordan et al. instrument has been used in the laboratory by Inomata et al. (2013) to investigate detection of n-tridecane, by Agarwal et al. (2014) to measure picric acid, and by Liu et al. (2013) to investigate the be-havior of methyl vinyl ketone (MVK) and methacrolein in a reaction chamber.
These studies suggest that an easy, low-cost adaptation of H 3 O + CIMS instruments to NO + chemistry could greatly enhance our capability to measure VOCs in the atmosphere.However, the number of VOC species investigated to date is small and few field measurements have been reported.The ability of a modified H 3 O + CIMS instrument to separate carbonyl isomers in ambient air, and to measure small alkanes both in the laboratory and in ambient air, has not been evaluated.Finally, the lack of fragmentation of n-tridecane reported in Inomata et al. (2013) is intriguing, but the use of an NO + CIMS instrument to measure similar high-mass alkanes in ambient air has not been demonstrated.
Here we evaluate the adaptation of an H 3 O + CIMS instrument to use NO + reagent ion chemistry.We provide specifics on instrument setup and operating parameters.We report the sensitivity and spectral simplicity of NO + CIMS, relative to H 3 O + CIMS, for nearly 100 atmospherically relevant VOCs, including a wide range of functional groups, and provide product ion distributions for several representative compounds.We demonstrate, interpret, and evaluate measurements of separate aldehyde and ketone isomers, light alkanes, and several other species in ambient air.Finally, we investigate measurement of high-molecular-mass alkanes using NO + .We extend the laboratory analysis of high-mass alkanes to C 12 -C 15 n-alkanes and demonstrate fast, in situ measurement of these species in ambient air.
Instrumentation
Two separate H 3 O + CIMS instruments (referred to hereafter as PTR-QMS and H 3 O + ToF-CIMS) were adapted to NO+ chemistry in this work.Both instruments consist of (1) a hollow cathode reagent ion source, (2) a drift tube reaction region, (3) an ion transfer stage that transports from the drift tube to the mass analyzer and allows differential pumping, and (4) a mass analyzer.Both instruments have nearly identical hollow cathode ion sources and drift tube reaction regions, described in detail in de Gouw and Warneke (2007).The PTR-QMS (Ionicon Analytik) uses ion lenses to transfer ions from the drift tube to a unit-mass-resolution quadrupole mass analyzer (Pfeiffer).This instrument is described further by de Gouw and Warneke (2007).The H 3 O + ToF-CIMS uses RF-only segmented quadrupole ion guides to transfer ions from the drift tube to a time-of-flight mass analyzer produced by Aerodyne Research Inc./Tofwerk with a mass resolution of 4000-6000 (Bertram et al., 2011).This instrument is described further by Yuan et al. (2016).A similar PTR-ToF instrument using quadrupole ion guides has also been recently described (Sulzer et al., 2014).ToF-CIMS data were analyzed using Tofwerk high-resolution peak-fitting software (Aerodyne Research Inc./Tofwerk AG).A description of the algorithm is given in DeCarlo et al. (2006).
A GC instrument was used both as an interface to the ToF-CIMS and as a separate instrument using an electron-impact quadrupole mass spectrometer.The GC collects VOCs in a liquid nitrogen cryotrap for a 5 min period every 30 min.VOCs are then injected onto parallel Al 2 O 3 /KCl PLOT and semi-polar DB-624 capillary columns to separate C 2 -C 11 hydrocarbons and heteroatom-containing VOCs.When used as an interface to the ToF-CIMS, the column eluant was directed to the inlet of the ToF-CIMS, where it was diluted with 50 sccm of clean air with controlled humidity.When operated as a separate instrument, the column eluant was directed to an electron-ionization quadrupole mass spectrometer (EIMS) operated in selected-ion mode.The response of this GC-EIMS instrument to various VOCs has been well characterized over a long period of field and laboratory applications, and further operational details have been reported elsewhere (Goldan et al., 2004;Gilman et al., 2010Gilman et al., , 2013)).
Adaptation of H
Ideally, both H 3 O + and NO + reagent ion chemistry can be utilized with a single instrument.The fewest possible number of hardware parameters were changed to facilitate fast switching and instrument stability.
To achieve generation of NO + ions, the water reservoir was replaced with ultra-high-purity air.The source gas flow (5 sccm), the hollow cathode parameters, and the drift tube operating pressure (2.4 mbar) were not changed.To optimize the generation of NO + ions relative to H 3 O + , O + 2 , and NO + 2 , and the generation of the desired VOC + ion products, the voltages of the intermediate chamber plates, V IC1 and V IC2 , and the drift tube voltage V DT were adjusted.An instrument schematic showing the locations of V IC1 , V IC2 , and V DT can be found in the Supplement (Fig. S1).Optimization was performed sampling dry air.
It has been demonstrated that the quadrupole ion guides of the ToF-CIMS can significantly change the measured distribution of reagent and impurity ions (Yuan et al., 2016).The PTR-QMS does not have that issue as strongly (modeled and measured cluster distributions are largely similar, as discussed by de Gouw and Warneke, 2007) and therefore we explored the effect of V IC1 , V IC2 , and V DT on reagent ion distribution using the PTR-QMS.As the PTR-QMS and ToF-CIMS have nearly identical ion source and drift tube design, we assume that ion behavior in these regions is the same for the two instruments.
First, V DT was held constant at 720 V (the original setting of the PTR-QMS instrument), and V IC1 and V IC2 were varied (Fig. 1).The settings of V IC1 (140 V) and V IC2 (80 V) were selected as a compromise between high NO + ion count rate and low-impurity ion count rates.The major impurity ions are H 3 O + , O + 2 , and NO + 2 , and it is desirable to limit the formation of these ions because they react with VOCs, compli- cating the interpretation of spectra.Next, several VOCs with different functional groups were introduced into the instrument, separately, and the drift tube electric potential scanned.A drift tube voltage of 350 V (electric field intensity relative to gas number density E/N = 60 Td) was selected as a compromise between maximizing NO + ion count rate, minimizing H 3 O + , O + 2 , and NO + 2 , maximizing VOC ion count rates, minimizing alkane fragmentation, and promoting different product ions for carbonyls and aldehydes (Fig. 2).This setting results in about 10×10 6 cps (counts per second) of NO + primary ions, while in typical PTR-MS settings we achieve about 30 × 10 6 cps of H 3 O + primary ions.
We note that the E/N of 60 Td used for the NO + CIMS is much lower than that used in typical PTR-MS settings (circa 120 Td).In air, NO + will react with water to produce H 3 O + and HNO 2 (Fehsenfeld et al., 1971).The electric field in the drift tube limits the formation of the NO + • (H 2 O) n intermediaries in this reaction, promoting high NO + count rates and VOC sensitivity.In PTR-MS, the drift field is used to prevent the formation of analogous (Keesee and Castleman, 1986); hence the need for a higher E/N in PTR-MS settings.
The remainder of the work detailed in this paper was performed using the ToF-CIMS with the settings as described here.The ToF-CIMS has the advantages of high mass resolution, fast time resolution, and simultaneous measurement of all masses.Further small adjustments were made to the ToF-CIMS quadrupole ion guide voltages using Thuner software (Tofwerk AG) to promote sensitivity to VOCs and separate carbonyl isomers.The two most important such adjustments decreased the electric potentials immediately upstream of each quadrupole ion guide (Fig. S2).These adjustments re- VOCs from several calibration cylinders (VOCs listed in Table S1 in the Supplement) were diluted with high purity air to mixing ratios of approximately 10 ppbv and introduced into the sampling inlet of the GC interface.Eluant from the column was directed into the ToF-CIMS as described above.A relative humidity of 20 % was used for this experiment.This humidity condition is similar to that expected for ambient measurements discussed in Sect.3.2; this condition was cho-sen to aid interpretation of ambient air data.Humidity effects are discussed in Sect.3.1.5.Several species co-elute with another compound (m-and p-xylenes; myrcene and camphene; 1-ethyl,3-methylbenzene and 1-ethyl,4-methylbenzene); reported sensitivities and product ions are an average of the two co-eluting species.
Each VOC mixture was sampled twice, once with H 3 O + and once with NO + reagent ion chemistry and instrument settings.Based on the results we evaluated the utility of NO + CIMS relative to H 3 O + CIMS using two metrics.The first metric is sensitivity for individual VOCs.To determine the sensitivity (S), the signals (cps) of all product ions were integrated over the width of the chromatographic peak and sensitivities for the measured VOCs using NO + chemistry were calculated relative to the sensitivity using H 3 O + chemistry (S NO+ /S H 3 O + ).For several VOCs, we also calculated the relative sensitivity when only the most abundant product ion (the quantitation ion) is measured (Table 2b).Because only one concentration was sampled, this metric relies on sensitivity being linear with concentration.Linear sensitivity is a reasonable assumption for the NO + and H 3 O + ToF-CIMS because separate multiple-point calibrations for select VOCs showed a linear response (Sect.3.1.4,Fig. S10), H 3 O + CIMS has demonstrated linear sensitivity over a wide range of concentration (de Gouw and Warneke, 2007;Sulzer et al., 2014;Yuan et al., 2016), and the NO + CIMS agrees well with an independent technique over a range of atmospheric concentrations (Sect.3.2.2).
The second metric is the simplicity of spectra.In an ideal instrument, each VOC would produce only one product ion, and each ion mass would be produced by only one VOC.However, using NO + and H 3 O + reagent ions, fragmentation of product ions does occur.As a metric for the complexity of the product ion distribution resulting from particular VOCs, we determined the fraction of the most abundant ion to the total signal from this VOC (F ) and discuss (F NO + ) relative to (F H 3 O + ). Figure S3 contains a comparison of F NO + and F H 3 O + and an example product ion distribution.A larger value of this ratio means that NO + reagent ion chemistry creates a simpler product ion distribution for that particular VOC.This metric does not indicate whether a particular product ion is produced by only one VOC.Uniqueness of product ions is discussed in Sect.3.1.2.The NO + CIMS product ion distributions of 25 atmospherically relevant VOCs are reported in Table 2.
Figure 3 summarizes the comparison between NO + and H 3 O + reagent ion chemistry for the two metrics.On the y axis the spectrum simplicity metric and on the x axis the sensitivity metric are shown.
Branched alkanes and most cyclic alkanes are detected with far greater sensitivity using NO + chemical ionization than with H 3 O + chemical ionization.Aromatics and alkenes are detected slightly more sensitively, and, on average, ketones are detected slightly less sensitively.Alcohols are detected more sensitively, by at least a factor of 2, with the exception of methanol.The lower sensitivity to methanol is consistent with slower reaction kinetics reported in the literature (Španěl and Smith, 1997).Monoterpenes and acetonitrile are detected substantially less sensitively.
In comparing the simplicity of the product ion distribution between H 3 O + and NO + chemistry, most branched and cyclic alkanes, ketones, and monoterpenes have a higher fraction of signal on a single product ion (simpler spectra).We also highlight that many alkyl substituted aromatics fragment substantially with H 3 O + chemistry but do not with NO + chemistry.The few exceptions (notably, benzene) create more complicated spectra because an NO + cluster product is also present (m + 30).
Distribution of product ions
It is somewhat more difficult to predict the ionized VOC products of NO + CIMS compared to H 3 O + CIMS, because NO + has three common reaction mechanisms: charge transfer, hydride abstraction, and cluster formation.Groups of VOCs that have similar charge transfer and hydride abstraction enthalpies tend to react with similar ionization mechanisms (Fig. 4). Figure 4 uses thermodynamic information from Lias et al. (1988), as well as mechanistic information from this work (see Table S1 for a list of species) and from SIFT studies (Španěl and Smith, 1996(Španěl and Smith, , 1998a(Španěl and Smith, , b, 1999;;Španěl et al., 1997;Arnold et al., 1998;Francis et al., 2007a, b).Charge transfer occurs when the reaction enthalpy is favorable, regardless of the hydride transfer enthalpy.When the charge transfer enthalpy is close to zero, then NO + clustering occurs; when charge transfer is not favorable but hydride transfer is, then hydride transfer will occur.In terms Although Fig. 4 provides a general way to predict the possible mechanisms for a particular VOC, it provides no information about the distribution of the signal between different mechanisms or the degree of fragmentation.The distribution depends strongly on instrumental conditions, which include E/N settings in the ion-molecule reaction region (by far the most important effect), fragmentation and clustering in the ion optics, presence of impurity ions such as O + 2 from the converted hollow cathode ion source, and relative humidity (Sect.3.1.5).
In Fig. 5 the product ion distributions of several VOCs determined in this work are compared to three others using NO + .Studies by the University of Leicester used a much higher E/N ratio in the drift tube, leading to higher fragmentation and lower NO + adduct formation compared to this work (Wyche et al., 2005;Blake et al., 2006).Investigation of higher-mass alkanes by Yamada et al. (2015) used similar E/N but achieved lower contaminant O + 2 , which is a likely explanation for the higher degree of fragmentation of tridecane seen in this work.In SIFT-MS studies, without an electric field, fragmentation is minimized and preselection of NO + primary ions eliminates contaminant H 3 O + and O + 2 and therefore SIFT product ion distributions are generally simpler.These differences highlight the importance of selection of drift tube operating conditions and instrument characterization.
Alkane fragmentation
Small (C 4 -C 10 ) branched alkanes cannot be measured by H 3 O + CIMS.With NO + CIMS, these VOCs are detectable but generally fragment to produce several ionic fragments that are common to different species.These masses (for example, m/z 57 C 4 H + 9 ) are produced by many different compounds and are likely not useful for chemically resolved atmospheric measurements.A few masses (e.g., m/z 71 C 5 H + 11 and m/z 85 C 6 H + 13 ) are only produced by a few compounds and were therefore targeted for further investigation in ambient air measurements.Conversely, cyclic alkanes fragment very little.Fig. S4 shows the product ion distributions of several representative aliphatic compounds.We note that the major product ions of cyclic alkanes (M-H) are the same with H 3 O + and with NO + chemistry.However, the mechanism is different: NO + ionizes by hydride abstraction, while H 3 O + ionizes by protonation followed by loss of H 2 (Midey et al., 2003).The H 3 O + ionization mechanism has a secondary channel consisting of protonation followed by elimination of CH 4 or C n H 2n (Midey et al., 2003).The difference in ionization mechanism is a likely explanation for the lower degree of fragmentation observed using NO + chemistry.
Compared to small (C 8 and smaller) alkanes, large (C 12 and higher) n-alkanes show little fragmentation, with at least 50 % of the total ion signal accounted for by the expected parent mass (m − 1; Fig. 6).Additionally, the degree of fragmentation decreases with increasing carbon chain length.It is quite difficult to measure these compounds with H 3 O + CIMS because they fragment extensively and are not detected sensitively (Erickson et al., 2014).NO + CIMS could provide a fast, sensitive, chemically specific measurement of these compounds.It should be mentioned that large n-alkanes (C 10 and larger) are not measurable with the GC interface.Dodecane (C 12 H 26 ), tridecane (C 13 H 28 ), tetradecane (C 14 H 30 ), and pentadecane (C 15 H 32 ) were sampled directly with the NO + ToF-CIMS and product ions were identified by correlation with the expected major product ion (m−1).The NO + ToF-CIMS sensitivity to pentadecane was determined using a permeation source (Veres et al., 2010).Contaminant O + 2 could potentially reduce the measured parent ion ([M-H] + ) through fragmentation; an alkane measurement corrected for O + 2 interference would have higher sensitivity and a simpler product ion distribution (e.g., Yamada et al., 2015).
Instrument response factor for select compounds
A calibration factor was determined for various VOCs by (1) direct calibration, (2) estimation from sensitivity relative to H 3 O + CIMS, or (3) estimation from correlation with GC- In the following discussion we use two metrics of instrument response: cps and normalized cps (ncps).cps is the raw ion count rate of the instrument.Two operations were applied to cps measurements to obtain ncps.First, a duty cycle a Product from residual H 3 O + .b Both product ions can be unambiguously assigned to benzene.We therefore report also the counting statistics and limit of detection for the sum of the two ions.c For technical reasons, pentadecane sensitivity was determined in dry air.
correction was applied (Chernushevich et al., 2001): the ToF and eliminates a mass-dependent sensitivity bias.Then, measurements were normalized to the duty-cyclecorrected NO + (primary ion) measurement, which typically has count rates on the order of 10 6 above that of VOCs: ncps = 10 6 I corr NO + corr . (2) The normalization removes variability due to fluctuations in the ion source and detector.In calculating limits of detection, we use duty-cycle-uncorrected cps, as this best reflects the fundamental counting statistics of the instrument.In reporting ambient air measurements, we use ncps.The ncps measurement reduces several significant instrumental biases and better reflects VOC abundances in air.
Limits of detection at 1 Hz measurement frequency were calculated by finding the mixing ratio at which the signalto-noise ratio (S/N) is equal to 3. The calculation can be expressed by (Bertram et al., 2011;Yuan et al., 2016) where C f is the instrument response factor, in cps per ppb; [X] lod is the limit-of-detection mixing ratio of species X in ppb; t is the sampling period of 1 s; α is the scaling factor of noise compared to expected Poissonian counting statistics; and B is the background count rate in cps.The scaling factor α is generally greater than 1 because high-resolution peak overlap and fitting algorithms create additional noise (Cubison and Jimenez, 2015).For comparison, H 3 O + ToF-CIMS limits of detection, using the same ToF-CIMS instrument, are included where available.Aliphatics and aromatics are generally detected quite sensitively.Aromatics have sub-100 ppt detection limits and are detected slightly more sensitively with NO + CIMS than with H 3 O + CIMS, with NO + detection limits generally about 30 % lower.Aliphatic species are detected with quite low detection limits (less than 50 ppt) and with substantially better sensitivity than H 3 O + : the detection limit of methylcyclohexane using NO + is a factor of 27 lower than with H 3 O + .
Aldehydes and ketones also have detection limits of around 100 ppt or less, with the exception of acetaldehyde (lod = 355 ppt).The higher detection limit of acetaldehyde is due to a somewhat higher instrumental background and a lower response factor that is consistent with reaction kinetics (Španěl et al., 1997).Methanol has a very high detection limit (19 ppb); this is expected from the anomalously low rate constant of the methanol-NO + reaction (Španěl and Smith, 1997).In contrast, ethanol is detected far more sensitively with NO + than with H 3 O + , with a detection limit of 105 ppt (compared to 1600 ppt for H 3 O + ).
Humidity dependence
Humidity-dependent behaviors of primary ions and selected VOCs (acetaldehyde, acetone, isoprene, 2-butanone, benzene, toluene, o-xylene, and 1,3,5-trimethylbenzene) were determined by diluting a VOC calibration standard into humidified air to reach approximately 10 ppb mixing ratio, then sampling directly with the NO + ToF-CIMS.Air temperature was 27 • C. Product ion and signal dependencies on humidity for selected primary ions and VOCs are shown in Fig. 7 (additional species are included in Fig. S6).As relative humidity increases, NO + (m/z 30) remains relatively constant, while protonated water and protonated water clusters (especially m/z 37, H 5 O + 2 ) increase.As the abundance of H 3 O + in the drift tube increases, one might expect to see increased products of VOC reaction with H 3 O + with a corresponding decrease in NO + products.Although an increase of H 3 O + product is seen for some species (e.g., MEK), it is not universally true.For many species, the major effect is that the NO + adduct product increases relative to other NO + product ions.This effect is especially intense for isoprene, where the isoprene-NO + cluster (m/z 98, C 5 H 8 NO + ) increases by a factor of 10 from 0 to 70 % relative humidity.A similar humidity effect, observed during SIFT measurements of alkenes, has been reported previously by Diskin et al. (2002), who attributed the effect to better stabilization of excited intermediary (NO + • R) * ions by H 2 O.A full investigation of this effect is beyond the scope of this paper.In lieu of a complete theoretical understanding of humidity effects, we suggest that an experimental humidity correction could be applied as in Yuan et al. (2016).
GC-NO + CIMS measurements
Measurement of ambient air using the GC interface allowed us to determine which compounds in ambient air produce Aldehydes Ketones which masses.This is the essential link between laboratory measurements of calibration standards and interpretation of ambient NO + ToF-CIMS measurements.Ambient air from outside the laboratory was sampled from 27 to 30 October 2015 through an inlet 3 m above ground level and directed through 10 m of 1/2 in.diameter Teflon tubing at a flow rate of 17 standard L min −1 (residence time approximately 4 s).The GC interface subsampled this stream.Eluant from the column was directed into the NO + ToF-CIMS as described in Sect.2.1.The laboratory is in an urban area (Boulder, CO) and the inlet was located near a parking lot and loading dock.Absolute instrument background (including the GC interface) was determined by sampling zero air at the beginning and end of the 3-day measurement period.Instrument performance and stability, retention times of selected compounds, and instrument background were checked at least once per day by sampling a 56-component hydrocarbon calibration standard.
Figure 8 shows several masses from a typical chromatogram.In this chromatogram, it is clear, for instance, that the majority of signal from m/z 83 (C 6 H + 11 ) can be attributed to one compound (methylcyclopentane).In contrast, m/z 57 (C 4 H + 9 ) is produced from many different compounds with comparable intensities.Aldehydes and ketones appear to be well separated, as expected from the laboratory experiments.Figure 9 summarizes the contributions of different VOCs to several ions (m/z 57, C 4 H + 9 and m/z 83 C 6 H + 11 ) during the entire 3-day measurement period.M/z 57 (C 4 H + 9 ) has contributions from many different VOCs, and the relative proportions are highly variable.Conversely, m/z 83 (C 6 H + 11 ) is mostly attributable to methylcyclopentane during the majority of the 3-day measurement period.M/z 57 (C 4 H + 9 ) does not provide a useful measurement of alkanes, while m/z 83 (C 6 H + 11 ) may possibly provide a useful measurement of methylcyclopentane.Corresponding figures for other masses can be found in the supplemental information (Figs.S7-S9).Table 3 summarizes our assessment of key ions.
NO + CIMS vs. GC-EIMS measurement comparison
Measurements using the GC interface do not provide any information about the fast time response capability of the NO + ToF-CIMS.Additionally, not all compounds detectable by NO + CIMS and present in ambient air can be transmitted through the GC interface.Simultaneous GC-EIMS and NO + ToF-CIMS measurements were conducted to investigate fast NO + measurements, determine whether there are any significant interferences to key NO + masses, and explore NO + CIMS response to VOCs not transmittable through the GC interface.Ambient air was sampled into the laboratory as described in the previous section.The GC-EIMS and the NO + ToF-CIMS were run as separate instruments and subsampled the 17 SLPM flow at the same point.Measurements were taken from 4 to 6 November 2015.The GC-EIMS instrument was operated on a 30 min schedule.GC-EIMS instrument background was determined from zeros taken at the beginning and end of the 3-day measurement period.The 56-component hydrocarbon calibration standard was sampled once per day.The NO + ToF-CIMS measured at 1 Hz frequency.Instru- ment zeros were taken for a 2 min period once every hour (example time series with zeros in Fig. S10).Calibration gas from a 10-component hydrocarbon standard was sampled for 2 min once every 3 h.At the end of the 3-day measurement period, both instruments were disconnected from the ambient air line and sampled air from inside the laboratory for 1.5 h 1 Cross-comparison with independent GC-EIMS not possible due to chromatographic quantitation ion overlap with neighboring peaks. 2 Cross-comparison with independent GC-EIMS not possible due to EIMS quadrupole selected-ion scan window restrictions. 3Concentrations too low in ambient air to determine. 4Winter urban air sampled was likely influenced by local domestic biomass burning; crotonaldehyde may be a smaller fraction of signal in other environments. 5Benzene correlation using sum of m108 C 6 H 6 NO + and m78 C 6 H + 6 . 6With exclusion of single outlier, R 2 = 0.831.
(three GC samples) to investigate the NO + ToF-CIMS response to air with a VOC composition substantially different from urban air.For all comparisons between the two instruments, the 1 Hz NO + ToF-CIMS measurements were averaged over the 5 min GC-EIMS collection period.The NO + ToF-CIMS was calibrated using air with ambient humidity (approximately 20 %) for the 10 species listed in Table 2a, and no further humidity correction was applied.Correlations between independent GC and calibrated CIMS measurements generally show a high correlation coefficient (R 2 > 0.9) and slopes close to 1 (examples in Fig. 10a, b).This demonstrates that an adapted NO + CIMS instrument retains sensitive measurement of atmospherically important species such as aromatics that are often targeted using PTR-MS and in addition can detect compounds such as isopentane, sum of 2-and 3methylpentane, methylcyclopentane, and sum of C 7 cyclic alkanes (Fig. 10c-f) that are usually not detected with PTR-MS.Slopes for calibrated VOCs, and correlation coefficients (R 2 ) for all VOCs investigated, are included in Table 3.The good agreement also indicates that humidity dependence of sensitivity is likely not a severe effect for most species; how-ever, addressing and quantifying this effect should be a priority for future work.
To assess the ability of the NO + ToF-CIMS to separate ketones and aldehydes, we explore measurements of propanal and acetone.The separate measurement of these two species is a good test case because the two peaks are chromatographically well resolved on the GC-EIMS, there are few isomers of C 3 H 6 O (of which acetone and propanal are likely the only atmospherically relevant species), and independent measurements of these two species are interesting for scientific reasons: aldehydes are generally much more reactive with OH than their ketone isomers and may have significantly different behavior in the atmosphere (Atkinson and Arey, 2003).
A time series of propanal and acetone is shown in Fig. 11a.The two compounds have clearly different behavior in the atmosphere: there is fast (seconds to minutes), high variability in the acetone measurement that is not seen in the propanal measurement, and the longer-term (∼ hours) variability of acetone and propanal is not the same.The fast, high spikes in acetone may come from local sources such as exhaust from chemistry labs in the building.The acetone comparison between the GC-EIMS and the NO + ToF-CIMS has a slope of 1.13, a correlation coefficient R 2 of 0.978, and negligible GC-EIMS methylcyclopentane, ppbv offset.The comparison between the GC and CIMS propanal measurements has an R 2 of 0.928 (Fig. 11b, c).12.The episodes show high temporal and compositional variability.The inlet was downwind from a parking lot and next to a loading dock and electric power generator for the building, and it is likely that the elevated C 12 -C 15 alkanes are from any or all of these sources.An ambient air measurement of these species is particularly interesting because they have been implicated in efficient secondary organic aerosol production from diesel fuel exhaust (Gentner et al., 2012).
Summary and conclusions
In summary, an H 3 O + ToF-CIMS (PTR-MS) instrument was easily and inexpensively converted into an NO + CIMS by replacing the reagent source gas and modifying the ion source and drift tube voltages.The usefulness of NO + CIMS for atmospheric VOC measurement was then evaluated by (1) using a GC interface to determine product ion distributions for nearly 100 VOCs and compare the sensitivity and simplicity of spectra to H 3 O + CIMS; (2) measuring ambient air with a GC interface, to map product ions to their VOC precursors and determine which ions may be useful for chem- ically specific measurement; and (3) measuring ambient air directly, to evaluate chemical specificity and investigate fast (1 Hz) time measurement of new compounds.Additionally, the NO + CIMS response to C 12 -C 15 n-alkanes and to vari-able humidity was determined in some detail.Further work is needed to better understand the humidity dependence.
NO + CIMS is a valuable technique for atmospheric measurement because it can separate small carbonyl isomers, it can provide fast and chemically specific measurement of cyclic and a few important branched alkanes (notably, isopentane and methylpentane) that cannot be detected by PTR-MS, it can measure alkyl-substituted aromatics with less fragmentation than H 3 O + CIMS, and it can detect larger (C 12 -C 15 ) alkanes.With NO + CIMS significant fragmentation of most small alkanes does occur, making them difficult to measure quantitatively.There are also interferences on many alcohols (with the exception of ethanol) and butanal.Additionally, it is worth considering that VOC • NO + cluster formation moves certain species into a higher mass range.This may be a drawback because the number of possible isobaric compounds increases with mass, and it may be more difficult for high-resolution peak-fitting algorithms to separate species of interest from isobaric interferences (example in Fig. S11).Finally, because there are three different ionization mechanisms (hydride transfer, charge transfer, and NO + adduct formation), it may be difficult to determine which VOC precursors correspond to particular ions.NO + CIMS may be an extremely useful supplementary approach for specific applications such as studying secondary organic aerosol precursors in vehicle exhaust, investigating emissions from oil and natural gas extraction, identifying additional species in complex emissions such as biomass burning, measuring emissions of oxygenated consumer products and solvents in urban areas, and investigating photochemistry of biogenic VOCs.
The Supplement related to this article is available online at doi:10.5194/amt-9-2909-2016-supplement.
Figure 2 .
Figure 2. VOC and primary product ion dependence on drift tube voltage.Traces are labeled by the nominal product ion m/z in Th.(a) Methyl vinyl ketone; (b) methacrolein; (c) 2,2-dimethylbutane; (d) methylcyclohexane; (e) primary ions and clusters.The dashed line indicates the selected operating voltage.Experiment conducted in dry air (H 3 O + is from residual water in the instrument and in commercial ultra-zero air.)
Figure 3 .
Figure 3.Comparison of production ion distribution and sensitivity of VOCs using NO + and H 3 O + reagent ion chemistry, at a relative humidity of 20 %.
Figure 6 .
Figure 6.Large (C 12 -C 15 ) n-alkane product ion distribution, using relative humidity of 20 %.The expected largest mass resulting from hydride abstraction (m − 1) is highlighted in red.N-octane (C 8 ) is shown for comparison.
Figure 8 .
Figure 8. Example GC-CIMS chromatogram of ambient air sample.Masses have been split between two panels for clarity.Top: select masses corresponding to branched and cyclic alkanes.Bottom: select masses corresponding to aldehydes and ketones.
Figure 10 .
Figure 10.Correlations between VOCs measured with GC-EIMS and NO + ToF-CIMS in ambient air.The 1 Hz NO + ToF-CIMS measurement is averaged to the 5 min GC collection period.Orthogonal least-squares linear best fits (ODR best fit) are shown with dashed lines.The lines appear curved due to log-scale axes.For several compounds (e.g., methylcyclopentane, 2-and 3methylpentane), the single high outlier pulls the best fit slightly away from the data points at low mixing ratios.(a) Toluene.(b) C 8 aromatics: sum of ethylbenzene, o-xylene, m-xylene, and p-xylene.(c) Isopentane.(d) Sum of 2-methylpentane and 3-methylpentane.(e) Methylcyclopentane.(f) C 7 cyclic alkanes: sum of methylcyclohexane, ethylcyclopentane, and dimethylcyclopentane.
Figure 11 Figure 12 .
Figure 11.(a) Time series of acetone and propanal measurements from NO + ToF-CIMS and GC-EIMS in ambient air.Measurements shown include the GC-EIMS measurement (5 min sample every 30 min, circle markers), the NO + ToF-CIMS measurement averaged over the 5 min GC sampling period (cross markers), and the NO + ToF-CIMS measurement averaged to a 5 s running mean.(b) Correlation between NO + ToF-CIMS and GC-EIMS measurement of acetone.(c) Correlation between NO + ToF-CIMS and GC-EIMS measurement of propanal.
Author contributions.P. Veres and C. Warneke obtained CIRES IRP project funding.B. Yuan, A. Koss, C. Warneke, and J. de Gouw developed the ToF-CIMS instrument. A. Koss converted the instrument from H 3 O + to NO + , designed the experiments, collected data, and wrote the manuscript.A. Koss and M. Coggon analyzed data.C. Warneke and J. de Gouw provided guidance on experimental design and interpretation.All authors edited the manuscript.
Table 1 .
VOC species in Fig. and their charge transfer and hydride transfer reaction enthalpies.
Table 2 .
Yuan et al. (2016)detection limits of NO + ToF-CIMS for various VOCs.Additional product ions not used to establish sensitivity are listed in italic.The H 3 O + ToF-CIMS detection limits in the farthest right column are calculated from separate H 3 O + ToF-CIMS calibrations as described inYuan et al. (2016).Species calibrated directly with NO + CIMS; ambient (20 %) relative humidity (Yuan et al., 2016)ermined in previous work(Yuan et al., 2016).These calibration factors were multiplied by the relative peak areas determined in Sect.3.1.1 to obtain estimated NO + ToF-CIMS calibration factors.(An example
Figure 7. Humidity dependence of primary ions and selected VOCs.
Table 3 .
Assessment of significant product ions investigated by GC-NO + CIMS and parallel GC-EIMS and NO + CIMS measurement of ambient air.Masses in bold can be unambiguously assigned to a single VOC or a structurally related, correlated group of VOCs.
A. R. Koss et al.: Evaluation of NO + reagent ion chemistry for online measurements | v3-fos-license |
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} | pes2o/s2orc | Solvent-Free Synthesis of Jasminaldehyde in a Fixed-Bed Flow Reactor over Mg-Al Mixed Oxide
In spite of the rapid developments in synthesis methodologies in different fields, the traditional methods are still used for the synthesis of organic compounds, and regardless of the type of chemistry, these reactions are typically performed in standardized glassware. The high-throughput chemical synthesis of organic compounds such as fragrant molecules, with more economic benefits, is of interest to investigate and develop a process that is more economical and industrially favorable. In this research, the catalytic activity of Mg-Al catalyst derived from hydrotalcite-like precursors with the Mg/Al molar ratio of 3 was investigated for the solvent-free synthesis of jasminaldehyde via aldol condensation of benzaldehyde and heptanal. The reaction was carried out in a fixed-bed flow reactor, at 1 MPa, and at different temperatures. Both Brønsted and Lewis (O2− anions) base sites, and Lewis acid sites exist on the surface of the Mg-Al catalyst, which can improve the catalytic performance. Increasing the reaction temperature from 100 °C to 140 °C enhanced both heptanal conversion and selectivity to jasminaldehyde. After 78 h of reaction at 140 °C, the selectivity to jasminaldehyde reached 41% at the heptanal conversion 36%. Self-condensation of heptanal also resulted in the formation of 2-n-pentyl-2-n-nonenal. The presence of weak Lewis acid sites creates a positive charge on the carbonyl group of benzaldehyde, and makes it more prone to attack by the carbanion of heptanal. Heptanal, is an aliphatic aldehyde, with higher activity than benzaldehyde. Therefore, the possibility of activated heptanal reacting with other heptanal molecules is higher than its reaction with the positively charged benzaldehyde molecule, especially at a low molar ratio of benzaldehyde to heptanal.
Introduction
Jasminaldehyde, which also known as α-amylcinnamaldehyde, is a traditional perfumery chemical with a violet scent. Before the 20th century, perfumes were mainly dependent on natural sources for their ingredients, and due to the limitations in supply, they were expensive to produce [1]. According to the reports, natural sources are used for the production of less than 5% of perfumery molecules [2]. Owing to the lower price of synthetic materials, their higher stability in acidic, basic, and even oxidizing media, these compounds became more attractive to be used in the synthesis of perfumery chemicals [2]. Jasminaldehyde can be synthesized via the cross aldol condensation of heptanal (C 7 H 14 O) and benzaldehyde (C 7 H 6 O) (Scheme 1), and this process has been intensively studied by researchers to enhance the yield of jasminaldehyde [3][4][5][6][7][8][9][10]. In addition to jasminaldehyde, the self-condensation of heptanal resulted in the formation of 2-n-pentyl-2-n-nonenal (C 14 H 26 O) as the main by-product of this reaction. A high molar ratio of benzaldehyde to heptanal is required during the reaction to reduce the In the conventional industrial process, homogeneous inorganic hydroxides such as NaOH and KOH are used for the production of jasminaldehyde. However, the use of theses homogeneous hydroxide-based catalysts has several drawbacks, such as lack of reusability of catalyst, employing a corrosive mixtures, formation of a large amount of liquid waste, separation and disposal of effluent containing sodium or potassium hydroxides [3,5,[12][13][14]. Thus, it is necessary to develop solid base heterogeneous catalysts for the synthesis of jasminaldehyde to provide an industrial process with easy product separation, lower corrosion of the reactor, the possibility of regeneration and reuse of catalysts, and the formation of the product with higher selectivity to jasminaldehyde. The higher reaction rate is achieved using base catalysts, while acid catalysts give slower rates [1]. Different types of heterogeneous catalyst have been used for the synthesis of jasminaldehyde by Wong et al. [15], and a microwave reactor was used for the evaluation of the catalyst performance in batch mode, at 180 °C. It has been reported that the nanocrystalline aluminosilicate F-type zeolite prepared from rice husk ash silica source, was used as a heterogeneous catalyst in microwave-enhanced aldol condensation for the synthesis of jasminaldehyde. The heptanal conversion of 77.1% and jasminaldehyde selectivity of 69.5% was obtained over this catalyst at 180 °C, 40 min, benzaldehyde to heptanal ratio of 5, and 40 min of microwave irradiation time. The reaction was also evaluated in the presence of several types of solvents with different polarity (in E scale), including dichloromethane, acetonitrile, dimethyl sulfoxide, ethanol, and water, with the polarities of 0.309, 0.444, 0.460, 0.654, and 1.000, respectively. It was observed that the polar solvents resulted in better conversion of heptanal, while the selectivity to jasminaldehyde was found to be independent of the type of solvent. However, compared with the solvent-containing reactions, the solvent-free reaction showed a better catalytic performance. The prepared nanocatalysts also showed excellent reusability, and after five consecutive reaction cycles, there was not a significant loss in their activity and selectivity.
Hamza and Nagaraju [7] investigated the synthesis of jasminaldehyde using the amorphous metal-aluminophosphate catalysts (MAlPs), where M is 2.5 mol.% Cu, Fe, Zr, Cr, Zn, or Ce. The aldol condensation reaction was performed in a three-necked round bottom flask with the benzaldehyde to heptanal ratio of 5. The FeAlP catalyst showed the highest catalytic activity (75%) as well as the highest selectivity to jasminaldehyde (71%) after 4 h of reaction at 140 °C. The good catalytic performance of this catalyst could be ascribed to the cooperative role of the optimal amount of acidic and basic sites with weaker strength. In general, because of its monobasic property and the low selectivity to jasminaldehyde, magnesium oxide is not an appropriate catalyst for the synthesis of In the conventional industrial process, homogeneous inorganic hydroxides such as NaOH and KOH are used for the production of jasminaldehyde. However, the use of theses homogeneous hydroxide-based catalysts has several drawbacks, such as lack of reusability of catalyst, employing a corrosive mixtures, formation of a large amount of liquid waste, separation and disposal of effluent containing sodium or potassium hydroxides [3,5,[12][13][14]. Thus, it is necessary to develop solid base heterogeneous catalysts for the synthesis of jasminaldehyde to provide an industrial process with easy product separation, lower corrosion of the reactor, the possibility of regeneration and reuse of catalysts, and the formation of the product with higher selectivity to jasminaldehyde. The higher reaction rate is achieved using base catalysts, while acid catalysts give slower rates [1]. Different types of heterogeneous catalyst have been used for the synthesis of jasminaldehyde by Wong et al. [15], and a microwave reactor was used for the evaluation of the catalyst performance in batch mode, at 180 • C. It has been reported that the nanocrystalline aluminosilicate F-type zeolite prepared from rice husk ash silica source, was used as a heterogeneous catalyst in microwave-enhanced aldol condensation for the synthesis of jasminaldehyde. The heptanal conversion of 77.1% and jasminaldehyde selectivity of 69.5% was obtained over this catalyst at 180 • C, 40 min, benzaldehyde to heptanal ratio of 5, and 40 min of microwave irradiation time. The reaction was also evaluated in the presence of several types of solvents with different polarity (in E N T scale), including dichloromethane, acetonitrile, dimethyl sulfoxide, ethanol, and water, with the polarities of 0.309, 0.444, 0.460, 0.654, and 1.000, respectively. It was observed that the polar solvents resulted in better conversion of heptanal, while the selectivity to jasminaldehyde was found to be independent of the type of solvent. However, compared with the solvent-containing reactions, the solvent-free reaction showed a better catalytic performance. The prepared nanocatalysts also showed excellent reusability, and after five consecutive reaction cycles, there was not a significant loss in their activity and selectivity.
Hamza and Nagaraju [7] investigated the synthesis of jasminaldehyde using the amorphous metal-aluminophosphate catalysts (MAlPs), where M is 2.5 mol.% Cu, Fe, Zr, Cr, Zn, or Ce. The aldol condensation reaction was performed in a three-necked round bottom flask with the benzaldehyde to heptanal ratio of 5. The FeAlP catalyst showed the highest catalytic activity (75%) as well as the highest selectivity to jasminaldehyde (71%) after 4 h of reaction at 140 • C. The good catalytic performance of this catalyst could be ascribed to the cooperative role of the optimal amount of acidic and basic sites with weaker strength. In general, because of its monobasic property and the low selectivity to jasminaldehyde, magnesium oxide is not an appropriate catalyst for the synthesis of jasminaldehyde. Therefore, Fan [3], prepared the acid-base bi-functional magnesium oxide catalysts (MgO-NO 3
-H 2 O 2 )
Catalysts 2020, 10, 1033 3 of 14 using a simple hydrogen peroxide reflux calcination procedure. The catalytic activity of the catalysts for aldol condensation reactor was evaluated in a two-necked round bottom flask placed in an oil bath. Before addition of catalyst to the reactor, the proper amount of benzaldehyde and heptanal with the benzaldehyde to heptanal ratio of 5 was mixed and the mixture heated up to 140 • C. The prepared catalysts showed a high selectivity of 88% and heptanal conversion of 99% after 5.5 h of reaction at 140 • C. By increasing the benzaldehyde to heptanal ratio to 15, the jasminaldehyde selectivity increased to 94%, while the conversion remained unchanged (99%). The investigation of reaction mechanism over this catalyst indicated that this bi-functional magnesium oxide catalyst favored adsorption of benzaldehyde over heptanal, which resulted in a higher ratio of benzaldehyde to heptanal on the surface of the catalyst. Furthermore, the existence of weak Lewis acid sites on the surface of the catalyst caused a partial positive charge on carbonyl group of benzaldehyde, makes it more prone to a nucleophilic attack by the heptanal carbanion. Results revealed that the acid-base bifunctional catalysts are the desired types of catalyst for aldol condensation reaction with high selectivity to jasminaldehyde.
Hydrotalcites (HTCs) are found to be a promising catalyst for this reaction. The chemical composition of hydrotalcites can be shown with the general formula of (M 2+ where M 2+ (e.g., Cu 2+ , Ni 2+ , Zn 2+ , Mg 2+ ) is the divalent metal cation, and M 3+ is the trivalent cation (e.g., Fe 3+ , Al 3+ , Mn 3+ , Cr 3+ ), and A n− is a n-valent anion (e.g., NO − 3 , CO 2− 3 , SO 2− 4 , OH − ). The partial metal cation M 2+ /M 3+ replacement take place, and the additional positive charge is counterbalanced by anions A n− existing in interlayers, along with the water molecules. The x parameter is the surface charge determined by the ratio of two metal cations (x = M 3+ /(M 2+ + M 3+ )), and its value is reported to be in the range of 0.1 to 0.5, however for the pure phases, it is reported to be in the range of 0.2 < x < 0.33 [16][17][18]. Thermal treatment of hydrotalcites could convert them into the well-dispersed mixed metal oxides (MMOs) with a lot of Lewis base sites, high surface areas, and a large number of defects formed as a result of the incorporation of Al 3+ ions into the Mg 2+ lattice [16,17]. Subsequently, rehydration of MMOs, in the absence of carbon dioxide, converts them to the layered structure with hydroxyl anions incorporated in the interlayer area and forming activated HTCs with rich Brønsted-type basic sites [16,17].
The nature and strength of the basic and acidic sites of the hydrotalcites can be tuned by variation of these parameters: (a) nature of the substituting cations in the structures of hydrotalcite, (b) nature of anions existed in interlayer regions, (c) characteristic M 2+ /M 3+ molar ratio, and (d) thermal treatment and activation of layered materials [19,20]. Sharma et al. [8,12] used the Mg-Al hydrotalcite catalyst with the Mg/Al molar ratio of 2.0 to 3.5 via the coprecipitation method. The highest jasminaldehyde selectivity of 86% and heptanal conversion of 98% was obtained for the Mg/Al ratio of 3.5, after 8 h of reaction at 125 • C, and heptanal to benzaldehyde molar ratio of 5. They also studied the effect of reconstruction of Mg-Al hydrotalcite on its performance in the synthesis of jasminaldehyde. The reconstruction was undertaken by stirring the calcined hydrotalcite sample in de-carbonated deionized water under the nitrogen atmosphere, resulted in the reinstatement of the original layered structure of hydrotalcite containing hydroxyl groups as the main compensation anions in the interlayer space instead of carbonate anions [12]. Results revealed that the conversion of heptanal increased to 96% for the reconstructed hydrotalcite, which is higher than those of as-synthesized (51%) and calcined (60%) hydrotalcites. High conversion of almost 96% with the selectivity of 65% to jasminaldehyde obtained at benzaldehyde to heptanal ratio of 3, at 130 • C and 120 min of reaction. The selectivity increased to about 84% by raising the benzaldehyde to heptanal ratio to 10. In another study by Yadav and Aduri [13], the Mg-Al calcined hydrotalcite (CHT) supported on hexagonal mesoporous silica (HMS) (CHT/HMS = 20% (w/w)), was used for the synthesis of jasminaldehyde. The catalyst showed an excellent activity and selectivity in this reaction, at 150 • C and the benzaldehyde to heptanal ratio of 5. The results were ascribed to the bi-functional character of the CHT/HMS catalyst, where the weak acid sites are responsible for the activation of benzaldehyde by protonation of the carbonyl groups, which favors the attack of enolate heptanal intermediate formed on the basic sites of the CHT/HMS catalyst. Vrbková et al. [6] synthesized several layered double hydrotalcite catalysts, with the Mg/Al molar ratio of 2.0 to 4.0, and their activity in aldol condensation reaction was investigated. After 4 h of reaction, conversion of at least 90% was achieved with all catalysts, however, in general, catalysts with Mg/Al ratio of 3 and 4 showed higher activity and selectivity in the solvent-free reaction at 100 • C, and benzaldehyde to heptanal ration of 2. The reaction also studied in the presence of N,N -dimethylformamide (DMF) as the solvent, and it was found that the DMF is not a suitable solvent for this reaction, and resulted in lower reaction rate and selectivity. One of the reasons for the lower activity in the presence of a solvent might be its basicity. The aldehydic hydrogen in benzaldehyde is a bit more acidic than that of heptanal; thus, it could preferentially interact with DMF, and it is less available to react with heptanal. Another reason might be that the different compounds are competing for the same active sites, where solvent also can be adsorbed on both acidic and basic sites. Therefore, the presence of solvent could decrease the possibility of adsorption of reactant molecules. Later, the same research group [5], studied the synthesis of jasminaldehyde over the zinc modified Mg-Al mixed oxide at 80-120 • C, 7 h, and the benzaldehyde to heptanal mole ratio of 2. Comparison of the activity of Mg-Al oxide and the zinc-modified Mg-Al oxides catalysts under the same reaction conditions revealed that the zinc-modified Mg-Al catalysts yielding 15-20% higher jasminaldehyde than Mg-Al catalyst. The higher reaction temperature led to a higher reaction rate as well as a higher selectivity to jasminaldehyde. The presence of DMF solvent was also found to have a negative impact on the reaction rate and selectivity to jasminaldehyde, but it can be suitable for reducing the viscosity of the mixture, which is preferred for the reaction in a flow reactor.
Even though during the last decades, there are rapid developments in synthesis methodologies in different fields, but organic synthesis is still being performed in a very traditional way. These reactions are typically carried out in standardized glassware, and regardless of the type of chemistry, compounds are synthesized in batch-wise. Due to its direct application to the synthesis of organic chemicals such as fragrant molecules, the high-throughput chemical synthesis has been a major field of interest in the past decade, and efforts were made to make them more economical than natural products. Using the continuous-flow processes, in addition to improving the catalytic performance and optimizing the reaction conditions, are considered to be a universal lever to make the process more economical and industrially favorable. Therefore, in the current study, a continuous flow fixed-bed reactor is used for the aldol condensation reaction over Mg-Al hydrotalcite catalyst with the Mg/Al ratio of 3. The Mg-Al hydrotalcite catalysts prepared by the coprecipitation method and their physicochemical properties were characterized through different characterization methods.
Catalyst Characterization
The chemical composition of the prepared Mg-Al hydrotalcite was analyzed by inductively coupled plasma-optical emission spectrometry (ICP-OES). It was found that the Mg/Al ratio of the prepared hydrotalcite was higher than the targeted ratio. The weight percentage of Mg and Al were 25 wt.% and 8 wt.%, respectively, with the Mg/Al molar ratio of 3.45. The differences between the targeted and obtained Mg/Al ratio could be attributed to the use of hydrated nitrate precursors for the synthesis of the catalyst, where the amount of bonded water molecules can vary. Also, there could be some losses during the washing of precipitated solids, which caused some changes in the actual obtained ratio.
Thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) analysis were performed to investigate the transformation process of hydrotalcite layered structure into the mixed oxide. As can be seen in Figure 1a, several phases of weight loss observed for the prepared Mg-Al hydrotalcite, which could be attributed to the thermal decomposition of the catalyst. The 25% weight loss at low temperature (up to 280 • C) and the DTG peak at 200 • C was due to the evaporation of the physically adsorbed and interlayer water molecules. The second phase of weight loss (up to 45%) Catalysts 2020, 10, 1033 5 of 14 at a higher temperature range (280-550 • C) and the DTG peak at around 400 • C were attributed to the removal of the carbonate anions and hydroxyl from the interlayer space, together with interlayer water. These findings are in good accordance with the mass spectroscopy results, where two peaks were observed for the released water at around 240 • C and 420 • C. During the thermal treatment of hydrotalcites, dehydroxylation and decarbonization are overlapped; therefore, performing the mass spectroscopy analysis following TGA could help to identify both reactions. The interlayer carbonate of the hydrotalcite structure was released as CO 2 simultaneously with water around 440 • C. The TG analysis of the synthesized Mg-Al hydrotalcite is in line with the results observed by Kloproggea et al. [21], and the TGA-MS (mass spectrometry) of Mg-Al hydrotalcites confirmed a major loss of H 2 O (dehydroxylation) and CO 2 at around 410 • C. water. These findings are in good accordance with the mass spectroscopy results, where two peaks were observed for the released water at around 240 °C and 420 °C. During the thermal treatment of hydrotalcites, dehydroxylation and decarbonization are overlapped; therefore, performing the mass spectroscopy analysis following TGA could help to identify both reactions. The interlayer carbonate of the hydrotalcite structure was released as CO2 simultaneously with water around 440 °C. The TG analysis of the synthesized Mg-Al hydrotalcite is in line with the results observed by Kloproggea et al. [21], and the TGA-MS (mass spectrometry) of Mg-Al hydrotalcites confirmed a major loss of H2O (dehydroxylation) and CO2 at around 410 °C.
(a) (b) [22][23][24]. After calcination at 450 °C under air atmosphere, the hydrotalcite structure of the sample is decomposed and converted into the mixed oxides of MgO and Al2O3. The peaks corresponding to the MgO phase are observed at 43.2° and 62.7° (JCPDS 77-2179). The diffraction peaks corresponding to the AlOx phase were not observed in the XRD pattern of the calcined catalysts. It could be due to the insertion of Al 3+ into the MgO lattice without any phase separation in the calcined samples [23,25].
The scanning electron microscope (SEM) images of the dried Mg-Al hydrotalcite is displayed in Figure 2. A well-developed layered hydrotalcite structure with visible edge and thin flat crystals observed for this sample, which is in good agreement with the XRD result, where a very clear hydrotalcite crystallized structure was observed for this sample. After calcination of the Mg-Al hydrotalcite at 450 °C, the nanoplates-like feature can still be discriminated, which indicated that the thermal decomposition of the interlayer anions occurred without any phase segregation and sintering, which could be occurred at higher calcination temperature [25].
Nitrogen adsorption-desorption isotherms analysis was carried out to determine the textural properties of the prepared Mg-Al catalysts (Figure 3a). The type IV isotherm and the H3 hysteresis loop was observed for both samples. The characteristic features of type IV isotherm are attributed to its hysteresis loop, which is attributed to the capillary condensation occurring in mesopores, and the limiting uptake over a range of high P/P0. Hysteresis appearing in the multilayer range of physisorption isotherms is generally related to the capillary condensation in the mesopore structures. Generally, the shapes of hysteresis loops have been recognized with the specific pore structures. Type H3 hysteresis loop, which does not show any limiting adsorption at high P/P0, is usually identified with agglomerates or aggregates of plate-like particles giving rise to slit-shaped pores [26]. The isotherm for these catalysts at a relative pressure of 0.45 to 1.0 demonstrated the typical hysteresis loop of mesoporous materials with a wide pore size distribution. The Mg-Al dried sample showed a wide pore size distribution varied from 5 nm to about 30 nm, while the calcined sample showed a The diffraction peaks corresponding to the AlO x phase were not observed in the XRD pattern of the calcined catalysts. It could be due to the insertion of Al 3+ into the MgO lattice without any phase separation in the calcined samples [23,25].
The scanning electron microscope (SEM) images of the dried Mg-Al hydrotalcite is displayed in Figure 2. A well-developed layered hydrotalcite structure with visible edge and thin flat crystals observed for this sample, which is in good agreement with the XRD result, where a very clear hydrotalcite crystallized structure was observed for this sample. After calcination of the Mg-Al hydrotalcite at 450 • C, the nanoplates-like feature can still be discriminated, which indicated that the thermal decomposition of the interlayer anions occurred without any phase segregation and sintering, which could be occurred at higher calcination temperature [25].
Nitrogen adsorption-desorption isotherms analysis was carried out to determine the textural properties of the prepared Mg-Al catalysts (Figure 3a). The type IV isotherm and the H3 hysteresis loop was observed for both samples. The characteristic features of type IV isotherm are attributed to its hysteresis loop, which is attributed to the capillary condensation occurring in mesopores, and the limiting uptake over a range of high P/P 0 . Hysteresis appearing in the multilayer range of physisorption isotherms is generally related to the capillary condensation in the mesopore structures. Generally, the shapes of hysteresis loops have been recognized with the specific pore structures. Type H3 hysteresis loop, which does not show any limiting adsorption at high P/P 0 , is usually identified with agglomerates or aggregates of plate-like particles giving rise to slit-shaped pores [26]. The isotherm for these catalysts at a relative pressure of 0.45 to 1.0 demonstrated the typical hysteresis loop of mesoporous materials with a wide pore size distribution. The Mg-Al dried sample showed a wide pore size distribution varied from 5 nm to about 30 nm, while the calcined sample showed a narrower size distribution, mainly less than 20 nm (Figure 3b). As summarized in Table 1, the specific surface area and pore volume of the sample increased from 73.3 to 241.9 m 2 /g after calcination at 450 • C. These findings are also confirmed by other researchers [16,17], who reported that the calcination of Mg-Al hydrotalcite converted them into well-dispersed mixed oxides with high surface areas, and a large number of defects formed as a result of the incorporation of Al 3+ ions into the Mg 2+ lattice.
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 14 narrower size distribution, mainly less than 20 nm (Figure 3b). As summarized in Table 1, the specific surface area and pore volume of the sample increased from 73.3 to 241.9 m 2 /g after calcination at 450 °C. These findings are also confirmed by other researchers [16,17], who reported that the calcination of Mg-Al hydrotalcite converted them into well-dispersed mixed oxides with high surface areas, and a large number of defects formed as a result of the incorporation of Al 3+ ions into the Mg 2+ lattice. Catalysts 2020, 10, x FOR PEER REVIEW 6 of 14 narrower size distribution, mainly less than 20 nm (Figure 3b). As summarized in Table 1, the specific surface area and pore volume of the sample increased from 73.3 to 241.9 m 2 /g after calcination at 450 °C. These findings are also confirmed by other researchers [16,17], who reported that the calcination of Mg-Al hydrotalcite converted them into well-dispersed mixed oxides with high surface areas, and a large number of defects formed as a result of the incorporation of Al 3+ ions into the Mg 2+ lattice. The CO 2 temperature-programmed desorption (CO 2 -TPD) analysis was used for determination of the strength and amount of the basic sites of the calcined sample. The asymmetric CO 2 desorption peak is centered at around 100-130 • C, together with two shoulders at 150-180 • C and 200-250 • C, corresponding to three types of basic sites including the weak Brønsted OH groups, medium-strength metal-oxygen Lewis pairs, and strong Lewis basic sites-oxygen anions, respectively [27]. The basicity of hydrotalcite-derived mixed oxides is affected by the precursor composition, including the type of anions in the interlayers and the cations in brucite-like layers. The total basicity of the Mg-Al catalyst was calculated through the integration of the CO 2 desorption profile, which was deconvoluted into three Gaussian contributions corresponding to each type of weak, medium, and strong basic sites. The total basicity of the sample was found to be 1193 µmol/g, where the concentration of the weak, medium, and strong basic sites were 22.8%, 50.0%, and 27.2% of total basicity respectively. It has been reported that the higher basic sites with medium and strong strength could be beneficial for aldol condensation of benzaldehyde with heptanal [5]; therefore, the active basic sites in this reaction are mainly M n+ − O 2− pairs.
The strength and density of the acid sites were determined using ammonia temperature-programmed desorption (NH 3 -TPD) analysis (Figure 4b). The NH 3 -TPD profile has two overlapping curves, where the first peak at around 110 • C could be due to the desorption of residual NH 3 weakly bonded to the surface of the catalyst, and could be attributed to the existence of Brønsted acid sites on the surface of catalysts, created by the existence of OHgroups [28]; and the second peak at around 190 • C, might be caused by further desorption of stronger bonded NH 3 species from the Lewis acid sites ascribed to the presence of Al 3+ − O 2− − Mg 2+ species in the structure of the mixed oxides and containing the Al 3+ cations mainly in octahedral sites [28,29]. The CO2 temperature-programmed desorption (CO2-TPD) analysis was used for determination of the strength and amount of the basic sites of the calcined sample. The asymmetric CO2 desorption peak is centered at around 100-130 °C, together with two shoulders at 150-180 °C and 200-250 °C, corresponding to three types of basic sites including the weak Brønsted OH groups, medium-strength metal-oxygen Lewis pairs, and strong Lewis basic sites-oxygen anions, respectively [27]. The basicity of hydrotalcite-derived mixed oxides is affected by the precursor composition, including the type of anions in the interlayers and the cations in brucite-like layers. The total basicity of the Mg-Al catalyst was calculated through the integration of the CO2 desorption profile, which was deconvoluted into three Gaussian contributions corresponding to each type of weak, medium, and strong basic sites. The total basicity of the sample was found to be 1193 µmol/g, where the concentration of the weak, medium, and strong basic sites were 22.8%, 50.0%, and 27.2% of total basicity respectively. It has been reported that the higher basic sites with medium and strong strength could be beneficial for aldol condensation of benzaldehyde with heptanal [5]; therefore, the active basic sites in this reaction are mainly M O pairs.
The strength and density of the acid sites were determined using ammonia temperatureprogrammed desorption (NH3-TPD) analysis (Figure 4b). The NH3-TPD profile has two overlapping curves, where the first peak at around 110 °C could be due to the desorption of residual NH3 weakly bonded to the surface of the catalyst, and could be attributed to the existence of Brønsted acid sites on the surface of catalysts, created by the existence of OHgroups [28]; and the second peak at around 190 °C, might be caused by further desorption of stronger bonded NH3 species from the Lewis acid sites ascribed to the presence of Al O Mg species in the structure of the mixed oxides and containing the Al 3+ cations mainly in octahedral sites [28,29].
Catalytic Performance for the Aldol Condensation Reaction
The catalytic activity for aldol condensation of benzaldehyde with heptanal was investigated in a fixed-bed flow reactor under solvent-free condition with the benzaldehyde to heptanal ratio of 2, at two different reaction temperatures (100 °C, 140 °C), at the reaction pressure of 1 MPa ( Figure 5). The results of the reaction at 100 °C (Figure 5a) showed that after 10 h of reaction the heptanal conversion was around 30%, and after 20 h of reaction, the conversion started to decrease and reached around 16% after 44 h of reaction and then remained constant. In contrast, longer reaction time is favored to the formation of jasminaldehyde and 2-n-pentyl-2-n-nonenal. The jasminaldehyde selectivity was almost 27% after 32 h of reaction, and reached 40% after 44 h of reaction and remained almost constant
Catalytic Performance for the Aldol Condensation Reaction
The catalytic activity for aldol condensation of benzaldehyde with heptanal was investigated in a fixed-bed flow reactor under solvent-free condition with the benzaldehyde to heptanal ratio of 2, at two different reaction temperatures (100 • C, 140 • C), at the reaction pressure of 1 MPa ( Figure 5). The results of the reaction at 100 • C (Figure 5a) showed that after 10 h of reaction the heptanal conversion was around 30%, and after 20 h of reaction, the conversion started to decrease and reached around 16% after 44 h of reaction and then remained constant. In contrast, longer reaction time is favored to the formation of jasminaldehyde and 2-n-pentyl-2-n-nonenal. The jasminaldehyde selectivity was almost 27% after 32 h of reaction, and reached 40% after 44 h of reaction and remained almost constant until the end of the reaction (68 h). The selectivity to 2-n-pentyl-2-n-nonenal also followed the same trend and increased from 35% after 30 h to 52% after 68 h of the reaction. conversion at 100 °C, at same reaction time. However, after 14 h of reaction, the heptanal conversion decreased gradually and reached 36% after 76 h of reaction, which is still 2.2 times higher than the conversion at 100 °C. The selectivity to jasminaldehyde also slightly increased from 30% to 34% by increasing the reaction temperature (after 10 h) and gradually increased to 43% after 76 h of reaction. At 140 °C, the selectivity to 2-n-pentyl-2-n-nonenal was almost the same as jasminaldehyde during the reaction. Selectivity to 2-n-pentyl-2-n-nonenal was lower at 140 °C than its selectivity at 100 °C. The formation of other by-products (long-chain aldehydes) was lower at the higher temperature, especially in the first 32 h of reaction. By increasing the formation of jasminaldehyde and 2-n-pentyl-2-n-nonenal after 32 h of reaction, the formation of other products slightly decreased. Improvement of heptanal conversion and jasminaldehyde selectivity by increasing the reaction temperature was also reported by other researchers [4,5,10,30], which is in line with the results obtained in the current study. Aldol condensation of benzaldehyde with heptanal using the zinc- By increasing the reaction temperature from 100 • C to 140 • C (Figure 5b), after 10 h of reaction, conversion of heptanal significantly increased to about 80%, which is about 2.5 times higher than the conversion at 100 • C, at same reaction time. However, after 14 h of reaction, the heptanal conversion decreased gradually and reached 36% after 76 h of reaction, which is still 2.2 times higher than the conversion at 100 • C. The selectivity to jasminaldehyde also slightly increased from 30% to 34% by increasing the reaction temperature (after 10 h) and gradually increased to 43% after 76 h of reaction. At 140 • C, the selectivity to 2-n-pentyl-2-n-nonenal was almost the same as jasminaldehyde during the reaction. Selectivity to 2-n-pentyl-2-n-nonenal was lower at 140 • C than its selectivity at 100 • C. The formation of other by-products (long-chain aldehydes) was lower at the higher temperature, especially in the first 32 h of reaction. By increasing the formation of jasminaldehyde and 2-n-pentyl-2-n-nonenal after 32 h of reaction, the formation of other products slightly decreased. Improvement of heptanal conversion and jasminaldehyde selectivity by increasing the reaction temperature was also reported by other researchers [4,5,10,30], which is in line with the results obtained in the current study. Aldol condensation of benzaldehyde with heptanal using the zinc-modified Mg/Al oxides was studied by Tišler et al. [5], and they reported that the reaction rate increased by raising the temperature and the selectivity to jasminaldehyde also increased from 57% to 71% by increasing the reaction temperature from 80 • C to 140 • C. According to their findings, this increase in selectivity could be due to the higher heptanal reflux at the higher temperature, and consequently, lower concentration of heptanal in the reaction mixture, which decreased the possibility of self-condensation of heptanal, and increased the selectivity to jasminaldehyde. The same results were reported by Vrbková et al. [4,30], where the aldol condensation of benzaldehyde with heptanal was investigated using Mg-Al mixed oxides, Cs-MCM, and 1.5-K-Al 2 O 3 catalysts. Adwani et al. [10] also reported that with an increase in the temperature from 80 • C to 160 • C the conversion of heptanal and selectivity to jasminaldehyde both increased, when chitosan-hydrotalcite nano-bio composite was used as the catalyst for this reaction. In another study by Fan [3], the aldol condensation reaction was studied using an acid-base bi-functional magnesium oxide catalyst, and they also reported that increasing the temperature from 100 • C to 140 • C, increased the heptanal conversion around 35% to around 94% after 5 h of reaction. The selectivity of jasminaldehyde remained almost unchanged by increasing the reaction temperature. All these aforementioned reported results for aldol condensation of benzaldehyde with heptanal were obtained in the batch mode reactors, and generally, a round-bottomed flask equipped with a condenser was used as the reactor. The aldol condensation in fixed-bed flow reactors has not been investigated in the reported literature.
A possible reaction mechanism for the production of jasminaldehyde via aldol condensation of benzaldehyde with heptanal is shown in Scheme 2a. The main basic sites in the un-calcined hydrotalcites are the Brønsted base sites (OH − groups), while after calcination, Brønsted and Lewis base sites (O 2− anions) exist on the surface of the Mg-Al catalyst. Deprotonation of α-C atoms through the accessible base sites on the catalyst surface resulted in an initially activation of heptanal, and generates carbanion and leads to the formation of enolate anions. Simultaneously, benzaldehyde, by its O atom, is attached to the weak Lewis acid site (Al). The C=O groups of benzaldehyde are interacting with Lewis acid sites to polarize the C=O group and increase the positive charge on that α-C atom. Increasing the positive charge of α-C of benzaldehyde increases the possibility of it being attacked by enolate anions of heptanal. After attacking the α-C atom of benzaldehyde with enolate anion, an intermediate alkoxide is formed, which quickly deprotonates a water molecule and produces hydroxide and aldol product or a β-hydroxyaldehyde. Both acidic and basic sites cooperate for the production of jasminaldehyde [9,13]. Self-condensation of heptanal also resulted in the formation of 2-n-pentyl-2-n-nonenal during the reaction. The possible mechanism for this reaction is depicted in Scheme 2b. First, heptanal molecules are adsorbed on two acid and base sites of the catalyst, and an intermediate formed, which is then transformed into 2-n-pentyl-2-n-nonenal by releasing a water molecule.
In addition to jasminaldehyde and 2-n-pentyl-2-n-nonenal, the formation of another by-product was also observed. After 10 h of reaction at 100 • C, 26% of the other by-product was formed and increased to 40% after 32 h of reaction, then sharply decreased to around 10%. Formation of another by-product at 140 • C also slightly increased after 14 h, and then gradually decreased till the end of the reaction. It has been reported that in the oxidizing atmosphere and the presence of oxygen, oxidation of heptanal and benzaldehyde may result in the formation of heptanoic acid and benzoic acid [3,31]. However, in this study, due to the absence of air or oxygen in the reactor, there is not any possibility for oxidizing of the reactants, and these two acids were not detected in the gas chromatography analysis of product mixture. Heptanal, as an aliphatic aldehyde, is more reactive than benzaldehyde and competes for the acidic site along with benzaldehyde. The activated heptanal can continuously react with other heptanal or 2-n-pentyl-2-n-nonenal molecules. In the current study, it is hypothesized that owing to the presence of active sites on the surface hydrotalcites, heptanal and 2-n-pentyl-2-n-nonenal molecules are adsorbed on two base and acid sites, and the heptanal carbanion attacks the positively charged molecule, and longer chain aldehydes are formed. The formation of by-products could be decreased by increasing the molar ratio of benzaldehyde to heptanal, which can increase the chance of attacking the activated heptanal to positively charged benzaldehyde molecules, which are adsorbed on the Lewis acid sites on the surface of the catalysts.
Catalysts 2020, 10, x FOR PEER REVIEW 10 of 14 heptanal, which can increase the chance of attacking the activated heptanal to positively charged benzaldehyde molecules, which are adsorbed on the Lewis acid sites on the surface of the catalysts.
Catalyst Preparation
The Mg-Al catalyst derived from hydrotalcite-like precursors with the Mg/Al molar ratio of 3 was synthesized by coprecipitation of metal precursors at 60 °C and a constant pH of 9.5. Typically, the catalysts were prepared as follows: aqueous solutions of metal precursors, containing 1 mol/dm 3 of aluminum nitrate Al(NO3)3·9H2O (Lachner, p.a. purity) and magnesium nitrate Mg(NO3)2·6H2O (Lachner, p.a. purity), was precipitated with a mixture of basic solution containing 2 mol/dm 3 potassium hydroxide KOH (Lachner, p.a. purity) and 0.2 mol/dm 3 potassium carbonate K2CO3 (Penta, p.a. purity). The filtered cake was washed with demineralized water until neutral pH of the filtrate, then dried in an oven overnight at 65 °C. Prior to the aldol condensation reaction, the dried sample was calcined in situ at 450 °C for 3 h under nitrogen atmosphere (5 °C/min).
Evaluation of Catalyst
The aldol condensation reaction was performed in a stainless steel fixed-bed reactor, with 700 mm length and an inner diameter of 17 mm. Three thermocouples were located at three different points of the reactor, at the beginning, center, and end of the catalyst bed. The reactor was loaded with a mixture of 5 gr catalyst, with particle size of 224-560 μm, and silicon carbide SiC (0.1 mm) with the catalyst to SiC ratio of 1:1. A layer of SiC (1-2 mm) was also placed above and below the catalyst. Quartz wool plugs separated all individual layers. Before the reaction, the catalyst was calcined in situ at 450 °C for 3 h under nitrogen atmosphere, then the temperature was reduced to 100 °C, and the pressure was fixed at 1 MPa under a flow of N2 (5 L/h).
After activation of the catalyst, the feedstock containing benzaldehyde and heptanal (both Sigma-Aldrich, St. Louis, MO, USA, 97%), with the benzaldehyde to heptanal molar ratio of 2, was pumped into the reactor with the flow rate of 10 g/h. The products of the reaction were analyzed by Scheme 2. The possible reaction mechanism for the production of (a) jasminaldehyde via aldol condensation of benzaldehyde with heptanal, (b) 2-n-pentyl-2-n-nonenal via self-condensation of heptanal.
Catalyst Preparation
The Mg-Al catalyst derived from hydrotalcite-like precursors with the Mg/Al molar ratio of 3 was synthesized by coprecipitation of metal precursors at 60 • C and a constant pH of 9.5. Typically, the catalysts were prepared as follows: aqueous solutions of metal precursors, containing 1 mol/dm 3 of aluminum nitrate Al(NO 3 ) 3 ·9H 2 O (Lachner, p.a. purity) and magnesium nitrate Mg(NO 3 ) 2 ·6H 2 O (Lachner, p.a. purity), was precipitated with a mixture of basic solution containing 2 mol/dm 3 potassium hydroxide KOH (Lachner, p.a. purity) and 0.2 mol/dm 3 potassium carbonate K 2 CO 3 (Penta, p.a. purity). The filtered cake was washed with demineralized water until neutral pH of the filtrate, then dried in an oven overnight at 65 • C. Prior to the aldol condensation reaction, the dried sample was calcined in situ at 450 • C for 3 h under nitrogen atmosphere (5 • C/min).
Evaluation of Catalyst
The aldol condensation reaction was performed in a stainless steel fixed-bed reactor, with 700 mm length and an inner diameter of 17 mm. Three thermocouples were located at three different points of the reactor, at the beginning, center, and end of the catalyst bed. The reactor was loaded with a mixture of 5 g catalyst, with particle size of 224-560 µm, and silicon carbide SiC (0.1 mm) with the catalyst to SiC ratio of 1:1. A layer of SiC (1-2 mm) was also placed above and below the catalyst. Quartz wool plugs separated all individual layers. Before the reaction, the catalyst was calcined in situ at 450 • C for 3 h under nitrogen atmosphere, then the temperature was reduced to 100 • C, and the pressure was fixed at 1 MPa under a flow of N 2 (5 L/h).
After activation of the catalyst, the feedstock containing benzaldehyde and heptanal (both Sigma-Aldrich, St. Louis, MO, USA, 97%), with the benzaldehyde to heptanal molar ratio of 2, was pumped into the reactor with the flow rate of 10 g/h. The products of the reaction were analyzed by a gas chromatograph (Agilent 7890A, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a flame ionization detector (FID), using a GC capillary column HP-5 (length: 30 m, inner diameter: 0.32 mm, film thickness: 0.25 µm).
Characterization of Catalyst
Inductively coupled plasma-optical emission spectrometry (ICP-OES; Agilent 725/Agilent Technologies Inc., Santa Clara, CA, USA) was used for the determination of the bulk metal contents in the prepared catalyst. For the analysis of the catalyst composition, around 500 mg of Mg-Al catalyst was dissolved in 10 mL aqueous solution of sulfuric acid H 2 SO 4 and the solution heated. Then, the temperature of this solution was decreased and it was diluted with demineralized water, and heated to 100 • C for two minutes. Finally, the obtained solution was used for the analysis.
The TGA Discovery series (TA Instruments, Lukens Drive, NW, USA) was used for the thermogravimetric analysis (TGA). About 20 mg of the Mg-Al catalyst was located in an open aluminum crucible and the temperature increased from 50 • C to 900 • C (10 • C/min) under a flow of nitrogen (20 mL/min). An OmniStar GSD320 quadrupole mass detector (Pfeiffer Vacuum Austria GmbH, Vienna, Austria) was used to detect the fragments.
The morphology of the synthesized Mg-Al hydrotalcite was studied by a scanning electron microscope (SEM) (LYRA3 TESCAN; TESCAN ORSAY HOLDING, as, Brno, Czech Republic).
The crystallographic structure of the prepared catalyst was measured using a D8 Advance ECO (Bruker AXC GmbH, Karlsruhe, Germany) with CuKα radiation (λ = 1.5406 Å). The step time and step size were 0.5 s, and 0.02 • , respectively. The Diffrac.Eva software with the Powder Diffraction File database (PDF 4 + 2018, International Centre for Diffraction Data) was used for the analysis of diffractograms.
The Autosorb iQ (Quantachrome Instruments Boynton Beach, FL, USA) was used for N 2 -adsorption/desorption at −196 • C for the determination of surface textural properties (specific surface area, pore size distribution, and pore volume). Before the analysis, the sample was pretreated at 110 • C for 16 h, under vacuum.
The Autochem 2950 HP (Micromeritics Instrument Corporation, Norcross, GA, USA) was used for the CO 2 temperature-programmed desorption (CO 2 -TPD) and ammonia temperature-programmed desorption (NH 3 -TPD) analysis to determine the acid-base properties of the catalysts. Prior to the TPD analysis, about 100 mg of Mg-Al catalyst was pretreated at 450 • C with the heating rate of 10 • C/min for 1 h under flow of helium (25 mL/min), and then cooled down to • C. After pretreatment, the sample was subjected to the 10 vol.% CO 2 /He (for CO 2 -TPD) and 10 vol.% NH 3 /He (for NH 3 -TPD) with the flow rate of 25 mL/min for 30 min at 50 • C. Then, the gas was changed to helium (25mL/min) for 1 h at 50 • C to remove the weak physically adsorbed CO 2 or NH 3 molecules. Finally, the CO 2 -TPD and NH 3 -TPD were carried out by increasing the temperature from 50 • C to 400 • C with the heating rate of 10 • C/min, under helium (25 mL/min). The desorbed CO 2 and NH 3 in the outlet gas were detected by a thermal conductivity detector (TCD).
Conclusions
Solvent-free synthesis of jasminaldehyde via aldol condensation of benzaldehyde with heptanal was investigated in a fixed-bed flow reactor, using the Mg-Al catalyst derived from hydrotalcite-like precursors with the Mg/Al molar ratio of 3. Results showed that the catalytic performance is attributed to the presence of both Brønsted and Lewis (O 2− anions) base sites, and Lewis acid sites on the surface of the Mg-Al catalyst. First, heptanal is adsorbed on the available basic sites on the surface of the catalyst, and an enolate anion is formed by deprotonation of α-C atoms. At the same time, a benzaldehyde molecule is adsorbed on the weak acid site via its O atom; the C=O groups of benzaldehyde interact with Lewis acid sites, and the positive charge on that α-C atom increased by the polarization of the C=O group, which make it more likely to a nucleophilic attack by the heptanal carbanion. Both heptanal conversion and selectivity to jasminaldehyde are increased by increasing the reaction temperature from 100 • C to 140 • C. Simultaneous self-condensation of heptanal also resulted in the formation of 2-n-pentyl-2-n-nonenal. Heptanal as an aliphatic aldehyde is more reactive and competes for the acidic site along with benzaldehyde molecules; consequently, the possibility of the interaction between two heptanal molecules increases, especially at a low molar ratio of benzaldehyde to heptanal. In addition to jasminaldehyde and 2-n-pentyl-2-n-nonenal, other by-products are formed during the reaction. It is assumed that due to the presence of the active sites on the surface of the catalyst, the reactive heptanal molecule and 2-n-pentyl-2-n-nonenal also can be adsorbed on basic and acidic sites; the positively charged molecule is attacked by the heptanal carbanion and longer chain aldehyde, and produces the longer chain aldehyde. Formation of jasminaldehyde could be enhanced by increasing the molar ratio of benzaldehyde to heptanal to reduce the formation of by-products that are forming due to the self-condensation of heptanal molecules, or the interaction of heptanal and 2-n-pentyl-2-n-nonenal molecule that exists in the product mixture. | v3-fos-license |
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} | pes2o/s2orc | Comparative Anticonvulsant Study of Epoxycarvone Stereoisomers
Stereoisomers of the monoterpene epoxycarvone (EC), namely (+)-cis-EC, (−)-cis-EC, (+)-trans-EC, and (−)-trans-EC, were comparatively evaluated for anticonvulsant activity in specific methodologies. In the pentylenetetrazole (PTZ)-induced anticonvulsant test, all of the stereoisomers (at 300 mg/kg) increased the latency to seizure onset, and afforded 100% protection against the death of the animals. In the maximal electroshock-induced seizures (MES) test, prevention of tonic seizures was also verified for all of the isomers tested. However, the isomeric forms (+) and (−)-trans-EC showed 25% and 12.5% inhibition of convulsions, respectively. In the pilocarpine-induced seizures test, all stereoisomers demonstrated an anticonvulsant profile, yet the stereoisomers (+) and (−)-trans-EC (at 300 mg/kg) showed a more pronounced effect. A strychnine-induced anticonvulsant test was performed, and none of the stereoisomers significantly increased the latency to onset of convulsions; the stereoisomers probably do not act in this pathway. However, the stereoisomers (+)-cis-EC and (+)-trans-EC greatly increased the latency to death of the animals, thus presenting some protection. The four EC stereoisomers show promise for anticonvulsant activity, an effect emphasized in the isomers (+)-cis-EC, (+)-trans-EC, and (−)-trans-EC for certain parameters of the tested methodologies. These results serve as support for further research and development of antiepileptic drugs from monoterpenes.
Introduction
Epilepsy has been characterized as a brain disease characterized by certain conditions: at least two unprovoked seizures in a range greater than 24 h; an unprovoked seizure and the likelihood of further recurrent crises after two unprovoked seizures, continuation over 10 years; and diagnosis of epileptic syndrome [1]. Epileptic patients present recurrent spontaneous seizures [2], and some 30% of them are considered resistant to drug therapy, without adequate response to treatment. Often, surgical removal of the epileptic focus is the only option that actually controls the emergence of seizures [3]. Degradation of cognitive functions (due to factors such as seizure etiology, and type), psychosocial problems [4], and possible psychomotor impairment [5] are clinically observed in these patients.
It is known that the existing clinical anti-epileptic drugs are not good anti-epileptogenics, since they only increase the onset threshold for seizure [6]. Many patients have seizures despite access to the complete pharmacological arsenal [7]. When considering the development of new antiepileptic drugs from 1981-2002, it is important to emphasize the role of natural products, since most of the synthetic drugs developed have had a natural product as their model. This reinforces the role of nature as a source for anticonvulsant agents [8].
Essential oils and their constituents are natural products known for their biological effects [9], including anxiolytic [10], spasmolytic [11], antinociceptive [12], and anticonvulsant [13] activities. Structural diversity in the constituents of essential oils is responsible for the variety of biological effects already observed; this is especially true for the monoterpene class, which has shown interesting anticonvulsant activity in animal models [14]. Besides their functional groups, monoterpenes have a range of optical isomers of specific compounds; these isomers may have different properties involving differing pathways [15], with synergistic and/or antagonistic actions [16], and membrane models [17].
Epoxycarvone is a monoterpene found in essential oils of plants such as Carum carvi [18], Kaempferia galanga [19], and others [20], or it is obtained by organic synthesis [21]. Epoxycarvone has demonstrable antimicrobial activity against Staphylococcus aureus and Candida albicans [22], and pharmacological effects on the central nervous system [23], with antinociceptive [24], and anticonvulsant activities [25]. The compound has four isomers, whose psychopharmacological stereo-selectivity is still unknown. These stereoisomers can be obtained using the enantiomers of carvone as starting materials; they also have differences in anticonvulsant activity due to the influence of their stereogenic center configuration [26]. This study aims to comparatively verify the anticonvulsant potential of the four stereoisomers of epoxycarvone in animal models of chemically and electrically induced seizures.
Preparation of Epoxycarvone Stereoisomers
The enantiomer trans-carvone oxides 9 and 10 were prepared via stereoselective reduction of the carbonyl group of carvones 1 and 2, stereospecific hydroxyl-assisted epoxidation of the allylic alcohols 3 and 4 and oxidative regeneration of the carbonyl group from epoxy alcohols 5 and 6 (Scheme 1).
Pilocarpine-Induced Seizures Test
The results for the behavioral changes induced by administration of pilocarpine (400 mg/kg) in animals treated with isomers (300 mg/kg), vehicle, and diazepam (4 mg/kg) are shown in Table 3. The values represent the mean˘SEM (n = 8 in each group). a p < 0.05; b p < 0.001 for the epoxycarvone stereoisomers group (300 mg/kg) as compared to the control group (ANOVA One way/Tukey's test). Fisher's exact test was used to analyze the percentages of animals with peripheral cholinergic signs, stereotyped movements, tremors, seizures, status epilepticus and the mortality rate.
All groups showed peripheral cholinergic signs, but the animals treated with (9: 50%) and (10: 50%), both 300 mg/kg, showed fewer percentages of animals with these signs. The control and experimental groups had convulsions (100%), while the standard group showed a lower percentage for this parameter (12.5%). The death percentage was highest in animals treated with vehicle (100%) and (8: 100%). The animals that received diazepam (4 mg/kg) did not die during the experiment. All of the animals had tremors, but in animals treated with (9: 50%) and (10: 50%), the parameter was smaller. Regarding emergence of status epilepticus, the group treated with (7: 0%) did not score, while 100% of the control groups did. Pilocarpine (400 mg/kg) induced stereotypical movements in all groups, except the standard group, which received diazepam (0%). Latency to the first seizure (Table 3)
s).
A similar result was observed for latency to death in the animals (Table 3), where those treated with (9: 2895.7˘343.3 s) and (10: 2345.0˘478.0 s), both at 300 mg/kg, increased latency compared to the control group (817.6˘22.3 s). There was a significant difference between the groups treated with (8: 1135.9˘68.1 s) and (9: 2895.7˘343.3 s), and no significant increase for this parameter in animals from groups 7 and 8 (300 mg/kg).
Strychnine-Induced Seizure Test
No significant differences were observed in latency to the first seizure in animals treated with the EC isomers (300 mg/kg) or the vehicle, in the strychnine-induced seizures model. However, there was a significant increase in latency to death of the animals treated (7: 306.5˘55.3 s) and (9: 396.5˘61.5 s), both 300 mg/kg, as compared to the control group (53.0˘0.6 s). All animals had seizures after strychnine administration, and there were no survivors except for the group treated with diazepam (4 mg/kg) ( Table 4). The values represent the mean˘SEM (n = 8 in each group). a p < 0.001; b p < 0.01; c p < 0.05 regarding the epoxycarvone stereoisomers group as compared to the control group (ANOVA One way/Tukey's test).
Fisher's exact test was used to analyze the percentages of animals with seizures, and the mortality rate.
In this study, the anticonvulsant activity of the stereoisomers (+)-cis-EC (7), (´)-cis-EC (8), (+)-trans-EC (9), and (´)-trans-EC (10) were analyzed comparatively in four methodologies that induced seizures chemically and electrically. Pentylenetetrazole-induced seizure test is one of the standard methods of anticonvulsant screening [31]. In this experiment, the isomers 7, 8, 9, and 10 (300 mg/kg) increased the latency to onset of seizure. This finding emphasizes the anticonvulsant potential attributed to the 4 isomers. The protection also extended to the animal mortality rate; there were no deaths in groups treated with the isomers, or in the standard group (diazepam). Anticonvulsants such as diazepam and phenobarbital have the ability to open GABA A channels, inhibiting seizures induced by PTZ [32], a substance that acts as an antagonist of the GABA A receptor [33]. The activation of GABAergic neurotransmission inhibits or attenuates seizures, while inhibition of this neurotransmission increases convulsions [34]. Idris and collaborators [35] evaluated the anticonvulsant profile of three isomers (30 mg/kg) of N-benzyl-3-[(methylphenyl)amino]propanamide, and all demonstrated excellent anti-convulsant properties in the pentylenetetrazole-induced seizure test.
Another study by Wieland [36] compared the effect of two neuro-active endogenous steroid isomers, 3α-hydroxy-5α-pregnan-20-one and 3α-hydroxy-5β-pregnan-20-one, for possible behavioral differences in this methodology. The resulting anticonvulsant activity was similar, which reinforces the findings of the present study. Seizure inhibition in the PTZ test by all four isomers, suggests possible interference in GABAergic neuro-transmission.
Maximal electroshock-induced seizures (MES) testing was also performed. This model is widely used to study anticonvulsant agents [37]. In the MES test, drugs that suppress tonic-clonic seizures have the ability to prevent the spread of seizure discharge through neuronal tissue, and thus increase the seizure threshold [38]. There were differences in the EC isomers effects, although all of the isomers significantly reduced the duration of the seizures compared to the control group. Substances 9 and 10 showed greater reductions. In other studies, constitutional isomers of valproic acid amide derivatives were comparatively tested for anticonvulsant activity in the MES test, with negligible modulations occurring [39]. trans-1-Propenyl-2,4,5-trimethoxy-benzene, also known as α-asarone, showed anticonvulsant property in the above test [40]. As previously reported, the monoterpene α,β-epoxycarvone prevented tonic seizures induced in this test [25], as was seen with the four isomers. Certain enantiomers of neurosteroids [41] also show remarkable anticonvulsant effects in the MES test, supporting the results of this research.
In both the PTZ and MES test all of the isomers showed significant protection (increased time to onset of convulsions). These data emphasize their anticonvulsant potential, and suggests that further studies regarding the possible participation of pathways involving the GABA A receptor, must be performed. A study by Almeida and collaborators [25] showed that the anticonvulsant effect of monoterpene α,β-epoxycarvone was not reversed by pretreatment with flumazenil, a selective GABA A receptor antagonist. Since α,β-epoxycarvone was the isomer analyzed in the comparative study and knowing this information about its mechanism of action, it was suggested that the possible role in GABAergic receptors might occur in other subunits involved with epileptogenesis in both animal models [42] and in humans [43].
Epilepsy pathophysiology has been understood using animal models, which induce seizures. One model that reproduces human epilepsy components is the pilocarpine-induced seizures test [44]. Pilocarpine is a non-selective agonist of muscarinic acetylcholine receptors; and brain structures with a high density of muscarinic receptors play an important role in the emergence of seizures induced by this chemical agent [45]. Following the seizures induced by pilocarpine administration there is a latency period preceding spontaneous and recurrent focal seizures. Neuropathological changes, such as hippocampal sclerosis occur with status epilepticus (induced by pilocarpine), similar to the temporal lobe epilepsy in humans [46]. The animals treated with the EC isomers (300 mg/kg) showed signs of peripheral cholinergic activity; however, greater protection was afforded by isomers 9 and 10, reducing data percentage of cholinergic signs. Regarding the emergence of status epilepticus, 7 was the most protective. The decrease in the tremors (as seen with 9 and 10, the increase in time to onset of seizures (as seen with all isomers), and protection against death (observed in the groups treated with 7, 9 and 10 are of great interest for developing new antiepileptic drugs. Significant differences in latency to death were observed (for the first time) between isomers, where 9 was more protective than 8. The results suggest in this methodology, that isomer 9 was found to be the most promising, since it was significantly effective in increasing the latency to onset of seizures. Protection in latency to death of the animals was also observed. Substance 7, as well as 8, showed significant results only in increased latency to onset of convulsions, and not for latency to death. The animals treated with 7 and 8, though increasing time to the first seizures, showed no protective effects for latency to death. As a comparative study of test substances, the results presented stereoisomer 9 as the most promising. These initial findings with this methodology are not enough to suggest muscarinic antagonism as the source of their anticonvulsant action, but do reinforce the potential of stereoisomers 7, and especially 9, and 10 for future research for the treatment of epilepsy. It was seen that all of the isomers are effective in relation to anticonvulsant activity, showing only slight differences between them in certain parameters. Stereochemical differences between the isomers did not significantly affect their pharmacodynamics in the tested methodologies. A constitutional isomer of valprimide has been shown effective in the pilocarpine-induced seizures test [47]. Another study of chiral derivatives of pyrrolo[1,2-α] pyrazine (in the pilocarpine-induced seizures test) noted one derivative as the more active among the series in preventing status epilepticus [48], differing from the previous findings where all tested substances with structural similarity, also had similar pharmacological effects.
In the strychnine-induced seizures test, protection in animals treated with isomers regarding the latency to onset of convulsions was not observed, presenting the possibility that the compound does not act on spinal cord glycine receptors [49]. Strychnine, an important post-synaptic inhibitory transmitter, acts as a convulsing agent by competitive antagonism with glycine receptors, [50]. In contrast, it was found that the isomers 7 and 9 (at 300 mg/kg), although possibly not affecting glycine receptors, showed an increase in latency to death by still unknown mechanisms. The structural isomers and enantiomers of pinene, a monoterpene with phytotoxic action, manifested differences in physiological parameters as related to germination and corn growth, probably due to differing mechanisms of action [51]. Studies like this, involving isomeric comparisons, are important since the characteristics of the substances are evaluated, allowing the future, the development of new drugs.
Electrophysiological experiments don't conducted to corroborate the effectiveness of EC isomers, although in a study conducted by Almeida and collaborators [25], only (+)-cis-EC was tested, which reduced neural excitability. The use of other isomers for this purpose may confirm (or not) involvement of the same in voltage-gated Na + channels blockade. What we can safely say about these stereoisomers is that behavioral seizures are affected, as seen in the pharmacological experiments. These data are preliminary and require further study of the mechanism of action to indicate how such neuroprotection occurs.
Despite the fact the dose tested in these methodologies (300 mg/kg) is relatively high, previous studies conducted by Sousa [23] showed a high LD 50 for one of the isomers of the epoxycarvone used in this work. Due to structural similarity between four substances, this data suggests a possible low toxicity and safety at the chosen dose for all stereoisomers. Research shows that the use of high doses are not necessarily related to high toxicity, as other studies that also have reports of substances with low toxicity, high doses with pharmacological activity and similarity to the isomers [52][53][54][55][56][57].
Preparation of Epoxycarvone Stereoisomers
The oily compounds were prepared in our laboratory according to the literature [27][28][29][30]. The 1 H-and 13 C-NMR measurements were obtained with a Mercury-Varian spectrometer (Palo Alto, CA, USA) operating at 200 MHz (for 1 H), and 50 MHz (for 13 C). The infrared spectra were recorded on a Bomen Michelson model 102 FTIR (Bomen, Chicago, IL, USA) and the most intense or representative bands reported (in cm´1). Optical rotations were measured on an Optical Activity AA-10 automatic polarimeter (Optical Activity Limited, Ramsey, UK) at ambient temperature. (2) Sodium borohydride (2.5 g, 66.1 mmol) was added at 20˝C to a solution of 1 or 2 (10 g, 67 mmol), and CeCl 3¨7 H 2 O (25 g, 148.5 mmol) in methanol (500 mL). The mixture was stirred for 5 min. Then, diethyl ether (100 mL) and water (100 mL) were added. The organic layer was separated, and the aqueous layer was extracted with diethyl ether (3ˆ100 mL). The organic layers were combined and dried over Na 2 SO 4 , and filtered. The filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography on silica gel using a mixture of ethyl hexane and ethyl acetate (8:2) as eleuent. (´)-cis-Carveol (3) and (+)-cis-carveol (4) were obtained with 80% (53.46 mmol) and 78% (52.0 mmol) yields, respectively [27,28].
Animals
Swiss albino male mice (Mus musculus), 2-3 months old, weighing from 25 to 35 g were used. The animals were housed in polyethylene cages and maintained under controlled temperature (21˘1˝C), with a 12 h light/dark cycle (lights on at 6:00 a.m.), with food (Purina commercial pellet feed, Paulínia, São Paulo, Brazil), and water available ad libitum until 1 h prior to tests. The tests were performed in the period between 12:00 and 17:00; mice were placed in polyethylene cages with 4 animals each and moved to the place of testing at least 1 h before tests to minimize eventual behavioral alterations. Each animal was used only once and euthanized at the end of the test.
All experimental procedures were analyzed and previously approved by the Ethics Committee on Animal Use (CEUA) UFPB, under Certificate No. 0109/11.
Statistical Analysis
The results presented were obtained using ANOVA, followed by Tukey's post-hoc test. Data were analyzed using the GraphPad Prism program version 5.00 (GraphPad Sotware Incorporated, San Diego, CA, USA), and the values obtained, were expressed as the mean˘standard error of mean. Fisher's exact test was used to analyze the percentages of animals with peripheral cholinergic signs, stereotyped movements, tremors, seizures, status epilepticus and the mortality rate. Differences were considered statistically significant when p < 0.05.
Conclusions
The results indicate that the four epoxycarvone stereoisomers tested are active in several seizure models, effectively decreasing tonic seizures, and also in increasing the animal survival rate after induced seizures. The differences in isomer stereochemistry exerted little influence on pharmacological activity in some of the animal models used. Further studies should be conducted to characterize the mechanisms of action of these four epoxycarvone isomers, and to clarify the structure-activity relationships of the tested compounds. | v3-fos-license |
2021-06-27T05:24:23.226Z | 2021-06-01T00:00:00.000 | 235645340 | {
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} | pes2o/s2orc | TGF Beta Induces Vitamin D Receptor and Modulates Mitochondrial Activity of Human Pancreatic Cancer Cells
Simple Summary TGFβ is a proinflammatory molecule produced by the tumor and active in its microenvironment. Its influence on cancer metabolism might explain its wide range of effects, but it is still poorly investigated. This study for the first time analyzes the TGFβ/vitamin D interplay in human pancreatic cancer cells and the effects of these two antagonistic molecules on the epithelial–mesenchymal transition, which is an early step of cancer progression. Data collected in this work also reveal the long-term metabolic effects of TGFβ, which are also relevant in the development of pancreatic cancer in vivo. The results of this research suggest that good levels of vitamin D can prevent the early stages of tumor transformation; on the contrary, chronic exposure to the inflammatory cytokine has irreversible consequences since it supports a prometastatic metabolism, confirming TGFβ as a crucial therapeutic target in pancreatic cancer. Abstract The inflammatory cytokine TGFβ is both a tumor suppressor during cancer initiation and a promoter of metastasis along cancer progression. Inflammation and cancer are strictly linked, and cancer onset often correlates with the insufficiency of vitamin D, known for its anti-inflammatory properties. In this study, we investigated the interplay between TGFβ and vitamin D in two models of human pancreatic cancer, and we analyzed the metabolic effects of a prolonged TGFβ treatment mimicking the inflammatory environment of pancreatic cancer in vivo. We confirmed the induction of the vitamin D receptor previously described in epithelial cells, but the inhibitory effects of vitamin D on epithelial–mesenchymal transition (EMT) were lost when the hormone was given after a long treatment with TGFβ. Moreover, we detected an ROS-mediated toxicity of the acute treatment with TGFβ, whereas a chronic exposure to low doses had a protumorigenic effect. In fact, it boosted the mitochondrial respiration and cancer cell migration without ROS production and cytotoxicity. Our observations shed some light on the multifaceted role of TGFβ in tumor progression, revealing that a sustained exposure to TGFβ at low doses results in an irreversibly increased EMT associated with a metabolic modulation which favors the formation of metastasis.
Introduction
Transforming growth factor β (TGFβ) is a pleiotropic cytokine with crucial activity in the regulation of inflammatory responses, which must be accurately restricted in a spatiotemporal way in order to avoid autoimmunity diseases or immune suppression [1]. Indeed, any deregulation of the immune response, such as an uncontrolled condition of chronic inflammation, can be lethal and promote tumorigenesis [2]. In this context TGFβ is both a mediator of the inflammatory response and a well-known inducer of tumor initiation, development, and metastasis formation [3]. TGFβ overexpression has been detected in most types of cancer, and it is a significant marker of aggressive tumor that the metabolic effects of TGFβ are reverted by the vitamin D signaling pathway [10], which has a central role in maintaining the differentiated state of tissues. By binding the vitamin D receptor (VDR), the active form of vitamin D opposes the EMT response. In fact, it induces the expression of numerous antitumoral genes, increasing the levels of E-cad and cystatin D, while contrasting the Wnt/β catenin pathway. Moreover, for the first time, Ricca et al. [10] demonstrated the reciprocal regulation between TGFβ and the vitamin D/VDR activity in human models of EMT. The results of this study demonstrated that TGFβ is able to induce the expression of VDR, while VDR exerts a negative feedback on TGFβ signaling by inhibiting TGFβ-mediated metabolic effects. Indeed, activation of the VDR signaling pathway triggered by TGFβ suppresses mitochondrial energy metabolism, ROS production, and cell migration.
Considering the heterogeneous effects of the cytokine, a more comprehensive analysis of TGFβ activity in tumoral cells is required, and the TGFβ-VDR feedback must be further demonstrated in cancer. For this purpose, in the present study, we investigated two human pancreatic cancer cell lines and we found them sensitive to the TGFβ-VDR interplay, corroborating the relevance of these signaling pathways in tumor prevention. Moreover, we explored the metabolic effects of a prolonged exposure to TGFβ with the intent of elucidating its contrasting activities in cancer cells, which are surrounded in vivo by an inflammatory microenvironment. The results of this work reveal that a prolonged exposure to TGFβ at low doses induces EMT together with a metabolic modulation which favors the motility of cancer cells.
The Response of Two Human Pancreatic Cancer Models to TGFβ and Vitamin D and the Modulation of Vitamin D Receptor
We selected two human pancreatic cancer cell lines widely exploited by in vitro tumoral studies [24,25]: PANC-1 and CAPAN-2 cells. They were both derived from primary tumors and they are representative of a different tumoral aggressiveness. In this regard, CAPAN-2 cells are characterized by a less aggressive phenotype, since they are well differentiated and do not induce metastasis; on the other hand, the poorly differentiated and strongly metastatic PANC-1 cell line is representative of an aggressive phenotype of human pancreatic cancer [26]. Many cancers (indeed, pancreatic cancer is no exception) are resistant to the antiproliferative and differentiating properties of vitamin D, and pancreatic cancer cells often show low levels of its receptor [27]. The sensitivity of the two cell lines to TGFβ and to the active form of vitamin D (1,25(OH) 2 D 3 , henceforth named vitamin D) was tested after five days of treatment. CAPAN-2 and PANC-1 were incubated with 100 nM 1,25(OH) 2 D 3 or 10 ng/mL TGFβ, which are the standard conditions usually employed for in vitro studies of EMT and signal transduction [28][29][30]. We confirmed the tumorsuppressive activity of TGFβ on this cancer model, because treatment with the cytokine reduced the number of cells as detected by crystal violet staining, due to cytotoxicity or inhibition of growth. On the other hand, the antiproliferative effect of vitamin D was modest and not significant, as shown in Figure 1A,B, thus suggesting a resistance to the hormone. Next, we investigated the expression of vitamin D receptor (VDR), since its epigenetic silencing could explain the lack of effect of the treatment with vitamin D. In our previous studies, we demonstrated the expression of VDR in total, mitochondrial, and nuclear fractions of several cell models [31][32][33]. In pancreatic cancer cells, in accordance with the poor sensitivity to vitamin D, neither cell line expressed the receptor when untreated, and the absence was evident in both the nuclear and the mitochondrial compartments in which the receptor is active [31,32] (Figure 1C). Most interestingly, we discovered the induction of VDR in both compartments after stimulation with TGFβ ( Figure 1C, quantified in Figure 1D); this observation, novel in the pancreatic model, confirmed the interplay between 1,25(OH) 2 D 3 and TGFβ, whereby TGFβ induces VDR expression, previously demonstrated by our studies on human lung epithelial cells [10]. The analysis of protein expression levels revealed that the treatment with TGFβ effectively drove the cells toward previously demonstrated by our studies on human lung epithelial cells [10]. The analysis of protein expression levels revealed that the treatment with TGFβ effectively drove the cells toward EMT, as suggested by the reduction in E-cadherin ( Figure 1C, quantified in Figure 1D). Original blots are shown in Figures S1 and S2 (Supplementary Materials).
Altogether, these data led to the identification of two models of human pancreatic cancer responsive to TGFβ and possibly sensitive to vitamin D, but only as a consequence of the treatment with the cytokine, due to VDR induction; these models can be used to further corroborate the 1,25(OH)2D3/TGFβ interplay in tumoral cells, and we explored this aspect in further experiments. treatment, the Western blot analysis revealed the expression of total, mitochondrial, and nuclear VDR, and the expression of the epithelial marker E-cadherin. Actin, VDAC, and PARP were used as loading controls for total, mitochondrial, and nuclear extracts, respectively. Original Western blots are shown in Figures S1 and S2 (Supplementary Materials). (D) Bands were quantified and normalized by loading control bands on the same blot. Data expression is relative to control. Graphs represent the means ± SEM. ** p < 0.01 vs. ctrl; *** p < 0.001 vs. ctrl. Western blot analysis revealed the expression of total, mitochondrial, and nuclear VDR, and the expression of the epithelial marker E-cadherin. Actin, VDAC, and PARP were used as loading controls for total, mitochondrial, and nuclear extracts, respectively. Original Western blots are shown in Figures S1 and S2 (Supplementary Materials). (D) Bands were quantified and normalized by loading control bands on the same blot. Data expression is relative to control. Graphs represent the means ± SEM. ** p < 0.01 vs. ctrl; *** p < 0.001 vs. ctrl.
Altogether, these data led to the identification of two models of human pancreatic cancer responsive to TGFβ and possibly sensitive to vitamin D, but only as a consequence of the treatment with the cytokine, due to VDR induction; these models can be used to further corroborate the 1,25(OH) 2 D 3 /TGFβ interplay in tumoral cells, and we explored this aspect in further experiments.
Vitamin D Opposes the Epithelial-Mesenchymal Transition Induced by TGFβ
It is well known that vitamin D contrasts EMT in normal and cancer cells, by inducing cell differentiation and increasing cell adhesion structures [34]. Since we discovered that TGFβ was able to induce the expression of VDR, we wondered whether vitamin D could be effective in reducing TGFβ-triggered transition, even in our cancer models insensitive to the antiproliferative action of the hormone. Therefore, we verified the effects on EMT of 1,25(OH) 2 D 3 and TGFβ, alone or combined, by measuring the transcription of EMT markers: epithelial E-cadherin and mesenchymal fibronectin. In this set of experiments, the induction of VDR by TGFβ was confirmed at the transcriptional level, as shown in Figure 2. Moreover, we found that vitamin D increased E-cadherin transcription, and that TGFβ decreased E-cadherin and increased fibronectin transcription, as expected. Interestingly, when vitamin D was added to TGFβ treatment, it reduced the effects of the cytokine; as shown in Figure 2, the cotreatment brought the transcription of E-cadherin back to levels similar to the control (difference not significant between D + T and ctrl). Moreover, the cotreatment reverted the increase in the mesenchymal marker (significant difference between D + T and T for fibronectin).
Vitamin D Opposes the Epithelial-Mesenchymal Transition Induced by TGFβ
It is well known that vitamin D contrasts EMT in normal and cancer cells, by inducing cell differentiation and increasing cell adhesion structures [34]. Since we discovered that TGFβ was able to induce the expression of VDR, we wondered whether vitamin D could be effective in reducing TGFβ-triggered transition, even in our cancer models insensitive to the antiproliferative action of the hormone. Therefore, we verified the effects on EMT of 1,25(OH)2D3 and TGFβ, alone or combined, by measuring the transcription of EMT markers: epithelial E-cadherin and mesenchymal fibronectin. In this set of experiments, the induction of VDR by TGFβ was confirmed at the transcriptional level, as shown in Figure 2. Moreover, we found that vitamin D increased E-cadherin transcription, and that TGFβ decreased E-cadherin and increased fibronectin transcription, as expected. Interestingly, when vitamin D was added to TGFβ treatment, it reduced the effects of the cytokine; as shown in Figure 2, the cotreatment brought the transcription of E-cadherin back to levels similar to the control (difference not significant between D + T and ctrl). Moreover, the cotreatment reverted the increase in the mesenchymal marker (significant difference between D + T and T for fibronectin). These data are in line with previous considerations based on our studies and others [10,30], demonstrating that TGFβ promotes the EMT phenotype and vitamin D counteracts the transition. For the first time, we demonstrated that, due to the induction of VDR, vitamin D was able to oppose the EMT induced by TGFβ in a pancreatic cancer model.
Characterization of TGFβ Metabolic Effects in Pancreatic Cancer Cells: Dose and Time Dependency
In the first experiments, we confirmed the short-term transcriptional activity of TGFβ in EMT, unveiling its important link with the vitamin D axis. Next, we sought to investigate the metabolic effects of the cytokine which could contribute to cancer progression. Considering that a chronic inflammatory microenvironment promotes pancreatic cancer in vivo [35], we decided to explore both short and long exposure to TGFβ. We tested different doses of the cytokine and we considered both short and long treatments. We analyzed the mitochondrial metabolic changes in response to TGFβ treatment starting from an evaluation of the mitochondrial activity rate. For this purpose, we performed a cytofluorimetric analysis with JC-1 dye to measure the mitochondrial membrane potential, which is proportional to mitochondrial respiratory activity. As shown in Figure 3A, we observed a drastic decrease in mitochondrial membrane potential when PANC-1 cells were treated with 10 ng/mL TGFβ (T10) for 48 h, compared to the untreated control, which suggested the cellular condition of stress caused by the acute treatment. As reported in Figure 3A, the sensitivity of mitochondrial activity to TGFβ treatment was dose- These data are in line with previous considerations based on our studies and others [10,30], demonstrating that TGFβ promotes the EMT phenotype and vitamin D counteracts the transition. For the first time, we demonstrated that, due to the induction of VDR, vitamin D was able to oppose the EMT induced by TGFβ in a pancreatic cancer model.
Characterization of TGFβ Metabolic Effects in Pancreatic Cancer Cells: Dose and Time Dependency
In the first experiments, we confirmed the short-term transcriptional activity of TGFβ in EMT, unveiling its important link with the vitamin D axis. Next, we sought to investigate the metabolic effects of the cytokine which could contribute to cancer progression. Considering that a chronic inflammatory microenvironment promotes pancreatic cancer in vivo [35], we decided to explore both short and long exposure to TGFβ. We tested different doses of the cytokine and we considered both short and long treatments. We analyzed the mitochondrial metabolic changes in response to TGFβ treatment starting from an evaluation of the mitochondrial activity rate. For this purpose, we performed a cytofluorimetric analysis with JC-1 dye to measure the mitochondrial membrane potential, which is proportional to mitochondrial respiratory activity. As shown in Figure 3A, we observed a drastic decrease in mitochondrial membrane potential when PANC-1 cells were treated with 10 ng/mL TGFβ (T10) for 48 h, compared to the untreated control, which suggested the cellular condition of stress caused by the acute treatment. As reported in Figure 3A, the sensitivity of mitochondrial activity to TGFβ treatment was dose-dependent, because the decrease in TGFβ concentration corresponded to an increase in mitochondrial Cancers 2021, 13, 2932 6 of 17 potential, until it was quite similar to the untreated control. Considering these data, we selected the concentration of 2 ng/mL (T2) as the safest but still effective dose of TGFβ and we used this concentration to evaluate the effects of a prolonged treatment while minimizing toxicity. The exposure of PANC-1 at this dose for 8 days produced a strong increase in mitochondrial respiratory activity. In agreement with these results, after the short-term exposure (10 ng/mL TGFβ for 48 h), CAPAN-2 cells also decreased their mitochondrial membrane potential, which was instead drastically boosted in response to the long-term exposure (2 ng/mL for 8 days) compared to the control ( Figure 3B). To verify the effects of TGFβ on cell viability and its potency, we calculated the EC 50 after both 48 h and 8 days of treatment. In PANC-1 cells, we estimated a value of 35 ng/mL when the EC 50 for TGFβ was calculated after 48 h of treatment and an EC 50 of 8 ng/mL when the cytokine was used for 8 days.
Cancers 2021, 13, x 6 of 17 dependent, because the decrease in TGFβ concentration corresponded to an increase in mitochondrial potential, until it was quite similar to the untreated control. Considering these data, we selected the concentration of 2 ng/mL (T2) as the safest but still effective dose of TGFβ and we used this concentration to evaluate the effects of a prolonged treatment while minimizing toxicity. The exposure of PANC-1 at this dose for 8 days produced a strong increase in mitochondrial respiratory activity. In agreement with these results, after the short-term exposure (10 ng/mL TGFβ for 48 h), CAPAN-2 cells also decreased their mitochondrial membrane potential, which was instead drastically boosted in response to the long-term exposure (2 ng/mL for 8 days) compared to the control ( Figure 3B). To verify the effects of TGFβ on cell viability and its potency, we calculated the EC50 after both 48 h and 8 days of treatment. In PANC-1 cells, we estimated a value of 35 ng/mL when the EC50 for TGFβ was calculated after 48 h of treatment and an EC50 of 8 ng/mL when the cytokine was used for 8 days. These results persuaded us that the approach of a diverse treatment could provide important information about the metabolic impact of TGFβ depending on the context. Therefore, we decided to proceed with the investigation by evaluating both the short exposure to a high concentration (T10) commonly used in EMT studies in vitro [28][29][30] and the prolonged exposure to the lower concentration (T2) that allows longer treatments without damaging the cancer cells. These results persuaded us that the approach of a diverse treatment could provide important information about the metabolic impact of TGFβ depending on the context. Therefore, we decided to proceed with the investigation by evaluating both the short exposure to a high concentration (T10) commonly used in EMT studies in vitro [28][29][30] and the prolonged exposure to the lower concentration (T2) that allows longer treatments without damaging the cancer cells.
Because the mitochondrial metabolic effects triggered by TGFβ were opposite depending on context, we verified that TGFβ maintained its transcriptional activity even at low doses after prolonged exposure. We found that this was the case; indeed, the induction of VDR and the transcriptional control of EMT markers was identical, even potentiated, at day 8 compared to 48 h ( Figure 3C-E). In fact, the effect of T2 at 8 days was stronger than T2 and T10 at 48 h. We did not test the higher T10 dose after 8 days because the EC 50 assay revealed a cytotoxicity of this concentration incompatible with a correct evaluation of metabolic parameters or gene expression. Although after 48 h vitamin D was able to refrain the efficacy of TGFβ (Figure 2), the hormone failed to revert the transcriptional effects of TGFβ when it was added at the end of long-term incubation ( Figure 3C-E). We concluded that vitamin D lost its power to revert the transition when it was added in the last phase of TGFβ exposure, and this was not surprising because it confirmed similar observations obtained in a previous investigation on epithelial cells [10].
The Cytotoxic Production of ROS Triggered by TGFβ Is Abated at Low Concentration of the Cytokine
A reduction in mitochondrial potential is one of the most evident signs of cellular stress; therefore, we hypothesized that high doses of TGFβ could cause cell damage. For this reason, we evaluated the cytotoxicity caused by TGFβ at different conditions using the MTT viability assay and lactate dehydrogenase (LDH) release assay. As expected, the viability test demonstrated the cytotoxicity of T10 at 48 h and even more after 8 days, whereas T2 never affected cell integrity ( Figure 4A). The toxicity of T10 was confirmed by the LDH release assay; in fact, PANC-1 cells responded to the acute treatment (T10 for 48 h) with a relative LDH release higher than the untreated control. On the contrary, the prolonged treatment (T2 for 8 days) was well tolerated and was characterized by a release similar to the control ( Figure 4B). The difference was confirmed in CAPAN-2 cells, which were even more sensitive to the acute toxic TGFβ exposure ( Figure 4C).
Given the toxicity of TGFβ treatment at high doses, we sought to investigate the cellular process mainly involved. In this regard, it has been widely demonstrated that toxicity is related to an excessive ROS production [9]; indeed, the production of ROS triggered by TGFβ is well known and it occurs through many mechanisms, such as the impairment of the mitochondrial function and the suppression of antioxidant enzymes [36]. Considering the above, we measured the production of ROS in PANC-1 and CAPAN-2 cells under acute (10 ng/mL for 24 h) and prolonged (2 ng/mL for 8 days) TGFβ treatment. As shown in Figure 4D, we found a strong augment in ROS levels in response to the acute exposure, compared to control. Interestingly, ROS production was not modulated by the long-term exposure to TGFβ. The same results were obtained in CAPAN-2 cells ( Figure 4E).
An increased production of ROS could have a mitochondrial origin; for example, it could be elicited by a deregulated respiratory activity. Indeed, it has been reported that TGFβ can modulate the transcription of the electron transport chain (ETC) [37]; therefore, we evaluated the transcriptional effects of TGFβ on mitochondrial respiration. By RT-PCR, we assessed the expression levels of two mitochondrially encoded mRNAs of PANC-1 cells: cytochrome C oxidase subunit 2 (COX-2) and ATP synthase membrane subunit 6 (MT-ATP6). Compared to the control, the short-term exposure triggered a fivefold induction of COX-2 at T10 and a twofold increase at T2, although the latter was not significant after statistical analysis. We also found a mild induction (threefold) after chronic exposure at T2 ( Figure 4F). T10 was not tested after 8 days because of its toxicity. As for the MT-ATP6 transcript, we detected a relevant increase in response to the acute TGFβ treatments, but not after a prolonged incubation ( Figure 4G). viability test demonstrated the cytotoxicity of T10 at 48 h and even more after 8 days, whereas T2 never affected cell integrity ( Figure 4A). The toxicity of T10 was confirmed by the LDH release assay; in fact, PANC-1 cells responded to the acute treatment (T10 for 48 h) with a relative LDH release higher than the untreated control. On the contrary, the prolonged treatment (T2 for 8 days) was well tolerated and was characterized by a release similar to the control ( Figure 4B). The difference was confirmed in CAPAN-2 cells, which were even more sensitive to the acute toxic TGFβ exposure ( Figure 4C). These data supported the conclusion that the toxicity of the acute treatment with TGFβ, detected both by viability assay and by LDH release, associated with the decrease in mitochondrial potential, was due to the increased ROS production, whereas the prolonged TGFβ treatment at low doses did not damage the human pancreatic cancer cells because ROS levels did not change. Moreover, we observed that ROS production occurred together with a striking transcriptional induction of respiration, whereas ROS levels did not change when the transcriptional modulation was modest. Altogether, these data reveal the drastic effects exerted by TGFβ on cellular metabolism and health, because the cytokine affects mitochondrial activity with a dose-dependent efficacy, leading to a proportional production of ROS and a consequent impact on cell survival.
The Long-Term TGFβ Treatment at Low Doses Promotes Cell Migration
As the last step of our analysis, we decided to investigate the impact of short-and long-term exposure to a different concentration of TGFβ on another crucial marker of cancer progression, which is cancer cell migration. PANC-1 and CAPAN-2 cells were analyzed by the wound healing cell migration assay after treatment with TGFβ at high doses (10 ng/mL) for 24 h and at low doses (2 ng/mL) for 8 days. As shown in Figure 5, for both cancer cell lines, the wound closure after short-term treatment was modest and similar to the control, despite the induction of EMT demonstrated by the first experiments of this study; however, after prolonged exposure, the treated cells showed a remarkable ability to migrate ( Figure 5), significantly higher than the movement observed in untreated TGFβ treatment in promoting cancer cell motility and, for the first time, show that the response is dose-and time-dependent. Figure 5. The long-term treatment with TGFβ at low doses promotes cell migration. PANC-1 (A) and CAPAN-2 (B) cells were incubated with TGFβ at 10 ng/mL for 24 h or at 2 ng/mL for 7 days, and then the treatment was continued for another 24 h to carry out the wound healing assay. The effects on cell migration at time 0 (T0) and after 24 h from scratch (T24) were evaluated using a wound-closure assay. The figure presents the empty areas in the wound-closure assay under different experimental conditions; these areas were measured and expressed on the graph as a percentage of wound closure. Data are displayed as the means ± SEM. * p < 0.05; *** p < 0.001.
Discussion
TGFβ is a central player in inflammation. Its protumoral effects can explain why the inflammatory status can predispose to the onset of cancer. It must be emphasized that the Figure 5. The long-term treatment with TGFβ at low doses promotes cell migration. PANC-1 (A) and CAPAN-2 (B) cells were incubated with TGFβ at 10 ng/mL for 24 h or at 2 ng/mL for 7 days, and then the treatment was continued for another 24 h to carry out the wound healing assay. The effects on cell migration at time 0 (T0) and after 24 h from scratch (T24) were evaluated using a wound-closure assay. The figure presents the empty areas in the wound-closure assay under different experimental conditions; these areas were measured and expressed on the graph as a percentage of wound closure. Data are displayed as the means ± SEM. * p < 0.05; *** p < 0.001.
Discussion
TGFβ is a central player in inflammation. Its protumoral effects can explain why the inflammatory status can predispose to the onset of cancer. It must be emphasized that the activity of the cytokine has multiple, even contrasting consequences, depending on the context. While signaling pathways and transcriptional activity have been described in detail and are largely studied, little is known about the metabolic effects of the cytokine. The modulation of cellular metabolism can lead to a context-dependent outcome; therefore, the investigation of the metabolic effects exerted by TGFβ can shed some light on the conflicting results of its activity.
In this study, we investigated the effects of a chronic inflammatory stimulus on two models of pancreatic cancer, and we described the impact of vitamin D and the relevance of TGFβ metabolic activity on cancer progression. Vitamin D shows differentiating and anti-inflammatory properties [38,39], and its efficacy on cancer could depend on its influence on TGFβ signaling. We chose pancreatic cancer as the target of our analysis for several reasons. First of all, it is a cancer that develops through a mesenchymal transition phase with a deadly metastatic spread [40,41]; thus, it allows considering the influence of TGFβ on both aspects of the progression. Moreover, it is a highly fibrotic cancer, as the result of the abundant production of TGFβ by cancer cells and due to its autocrine and paracrine activity [42,43]. The extensive fibro-inflammatory stroma of pancreatic ductal adenocarcinoma plays an active role in its progression and therapeutic resistance, highlighting the importance of acquiring more details on TGFβ activity in this type of cancer. Furthermore, vitamin D deficiency is often associated with cancer development, and it may play a role in both incidence and survival from pancreatic cancer [44,45]; therefore, revealing the efficacy of the hormone in contrasting TGFβ activity could explain the link. Indeed, the first findings of this study confirmed the interplay between TGFβ and vitamin D previously reported only on epithelial cells [10]. For the first time, we demonstrated in human pancreatic cancer the induction of VDR and we revealed that the upregulation of VDR mediated the feedback mechanism via which vitamin D can counteract the EMT induced by TGF beta. Interestingly, we found that vitamin D lost its efficacy when added after a prolonged incubation with TGFβ, in agreement with previous conclusions that the beneficial power of vitamin D is related to the early stages of TGFβ-driven transition [10]. The findings of the present study are relevant considering that proper levels of vitamin D could be important for pancreatic cancer prevention; in fact, it is conceivable that vitamin D could act as a tamer of the excessive activity of TGFβ and could avert cancer.
In this study we found that the metabolic effects of TGFβ were related to dosage and exposure time. In our in vitro models of pancreatic cancer, the cytokine was cytotoxic at the highest doses of 10 ng/mL and especially after prolonged exposure, due to the strong stimulation of respiratory chain associated with the overproduction of ROS, which in turn damaged mitochondrial membrane potential. It is of note that these concentrations are normally used in in vitro studies exploring EMT [34][35][36]. Our observation that the high concentration of TGFβ affects viability on long-term treatments is not in contrast with data from other studies, because the most common experimental setup in EMT investigation is the treatment with 10 ng/mL TGFβ only for short EMT induction (up to 48 h in pancreatic cancer [46][47][48]), with a lower concentration for longer induction (for example, breast cancer [49][50][51][52] and melanoma cancer [53]). To our knowledge, the effects of prolonged treatments at high doses have never been reported. In our model, we estimated an EC 50 of 35 ng/mL and 8 ng/mL when TGFβ was used for 48 h and 8 days, respectively. On the other hand, the cancer cells adapted to lower doses, and, after a prolonged exposure, the metabolic effect of TGFβ as an inducer of respiratory activity became evident. In fact, the low levels of TGFβ stimulated a moderate induction of the respiratory chain, which did not trigger the production of ROS and led to an increase in mitochondrial membrane potential, thus leaving the cell healthy and supported in its motility. The mitochondrial synthesis of ATP is of paramount importance for migration taking place in EMT, as several studies have demonstrated [54][55][56]; therefore, we concluded that the moderate, nontoxic enhancement of mitochondrial activity supported cancer spread. Again, our analysis of cancer cell motility revealed that the response to TGFβ was dose-dependent. In fact, in agreement with previous studies [46], the most used treatment with high dose and short time had little effect on pancreatic cancer motility, despite inducing EMT markers; however, upon prolonged exposure to a lower concentration, the motility was strongly increased; this different dose-dependent response has never been underlined before.
The transcriptional control exerted by TGFβ was evident at both high and low concentration of the cytokine, which activated the nuclear program driving the EMT in all conditions. However, the different impact on metabolism could, at least in part, explain the heterogeneous response of cancer cells to the cytokine. Actually, other studies have described TGFβ as both an inhibitor of mitochondrial metabolic activity [15,57] and an inducer of respiratory oxidative metabolism [58,59]; similarly, the dual role of TGFβ in tumors is also well known, since it acts as both a suppressor and a promoter of cancer progression. The evidence found in our study supports both roles for the cytokine, depending on a context-dependent influence on mitochondrial metabolism. Moreover, the metabolic effect exerted on cancer-associated fibroblasts and the consequent increased production of ROS would lead to collagen secretion and fibrosis; this consideration warrants further investigation. The data obtained in this work highlight the dual impact of TGFβ on mitochondrial metabolism and production of ROS, which can oppose or sustain EMT induced at the transcriptional level; such conclusions are depicted in Figure 6. had little effect on pancreatic cancer motility, despite inducing EMT markers; however, upon prolonged exposure to a lower concentration, the motility was strongly increased; this different dose-dependent response has never been underlined before. The transcriptional control exerted by TGFβ was evident at both high and low concentration of the cytokine, which activated the nuclear program driving the EMT in all conditions. However, the different impact on metabolism could, at least in part, explain the heterogeneous response of cancer cells to the cytokine. Actually, other studies have described TGFβ as both an inhibitor of mitochondrial metabolic activity [15,57] and an inducer of respiratory oxidative metabolism [58,59]; similarly, the dual role of TGFβ in tumors is also well known, since it acts as both a suppressor and a promoter of cancer progression. The evidence found in our study supports both roles for the cytokine, depending on a context-dependent influence on mitochondrial metabolism. Moreover, the metabolic effect exerted on cancer-associated fibroblasts and the consequent increased production of ROS would lead to collagen secretion and fibrosis; this consideration warrants further investigation. The data obtained in this work highlight the dual impact of TGFβ on mitochondrial metabolism and production of ROS, which can oppose or sustain EMT induced at the transcriptional level; such conclusions are depicted in Figure 6. The consequences of TGFβ activity on mitochondrial respiration can be either tumor suppression or fibrosis when ROS production is high, whereas tumor progression takes place when ROS levels are checked. In the investigated model of pancreatic cancer extensively treated at low doses of TGFβ, metabolic and nuclear effects of the cytokine together support EMT and then migration.
The approach of investigating in vitro the activity of TGFβ considering the long-term effects of the cytokine has the merit of assessing the results of a chronic stimulation, which mimics the inflammatory condition considered in vivo a protumorigenic stimulus. TGFβ is produced by both cancer cells and stromal tissue, in addition to the molecules of systemic origin. Therefore, measuring its levels in the tumoral site in vivo is complex and The approach of investigating in vitro the activity of TGFβ considering the longterm effects of the cytokine has the merit of assessing the results of a chronic stimulation, which mimics the inflammatory condition considered in vivo a protumorigenic stimulus. TGFβ is produced by both cancer cells and stromal tissue, in addition to the molecules of systemic origin. Therefore, measuring its levels in the tumoral site in vivo is complex and untested. Moreover, cancer cells are often resistant to TGFβ action, and the final effect of the cytokine in vivo could be comparable to that observed in vitro at low doses. In our in vitro model, we started from the standard dose used in most studies of EMT, and we found that the lower concentration of 2 ng/mL was the best compromise between efficacy and lack of toxicity after prolonged exposure; however, long-term incubation could be carried out at different dosage in other cellular models. The recent and very elegant work of Schworer et al. demonstrated that TGFβ at the same concentration of 2 ng/mL induces mitochondrial respiration and collagen synthesis in fibroblasts, without production of ROS [58]. The response of cancer cells to inflammatory signals could be influenced by their mutation status; depending on its genetic background, each tumor can respond to external stimulation differently. As for TGFβ, we showed that the production of ROS could explain the anticancer properties of high doses of the cytokine; therefore, the efficiency of the intracellular antioxidant defenses could determine the results of TGFβ activity. Mutated K-RAS increases glutamine utilization, and glutamine metabolism is used by cancer cells to produce NADPH essential in antioxidant defense [60]. Moreover, glutamine can also be diverted toward proline biosynthesis and, again, this alternative pathway represents an antioxidant strategy [58]. Pancreatic cancer characterized by mutated K-RAS is, therefore, protected from oxidative stress and takes advantage from the transcriptional activity of TGFβ driving EMT. Other mutations or specific differentiation status of different pancreatic cancer subpopulations could have similar protective effects from the anticancer activity of TGFβ.
Cell Proliferation Assay
PANC-1 and CAPAN-2 cells were seeded in 96-multiwell plates and were incubated with 10 ng/mL TGFβ and 100 nM 1,25(OH) 2 D 3 . After the treatment, they were fixed with 11% glutaraldehyde for 15 min, and then cells were stained with 0.1% crystal violet solution for 20 min, followed by solubilization with 10% acetic acid solution [10]. The absorbance of crystal violet was recorded at 595 nm, and the data were collected as the average of three independent experiments for each experimental condition.
Extract Preparation and Western Blotting Analysis
The subcellular fractionation was carried out as previously reported [10,31,32]. Cells were scraped from the culture dishes and homogenized in lysis buffer containing 10 nM Tris-HCl at pH 7.4, 0.33 M saccharose, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM proteases inhibitor Cocktail set III (Calbiochem), 1 nM Na 3 VO 3 , and 1 mM NaF. Lysates were subjected to differential centrifugation to isolate the nuclear and mitochondrial fraction. Western blot analysis of 30 µg of total lysate, as well as of mitochondrial and nuclear fractions, was carried out after separation by SDS-PAGE at 10%. Proteins of interest were detected with the following antibodies: mouse monoclonal antibodies anti-VDR (sc-13133) and anti E-cadherin (sc-21791) purchased from Santa Cruz, CA, USA. The loading controls were checked on the same membranes and detected by antibodies anti-VDAC (monoclonal anti-porin 31HL, Calbiochem), anti-actin (mouse monoclonal sc-8432 Santa Cruz), and rabbit antibody anti-PARP (sc-7150, Santa Cruz). The bands relative to actin, VDAC, and PARP were chosen as proteins enriched in total, mitochondrial, and nuclear fractions, respectively, and were used as references to correct the quantification of the proteins of interest. The correct band corresponding to VDR was identified in past studies by molecular weight and silencing experiments [31,32], and it corresponds to the lower band when a doublet band is present, whereas the upper band is due to unspecific labeling. The signal that detects VDR is indicated in all figures.
The housekeeping gene ribosomal subunit protein S14 was used as an internal control. The PCR required one denaturation cycle at 95 • C for 2 min, 40 amplification cycles with denaturation at 95 • C for 15 s, and then 30 s of annealing/extension at 60 • C. Data were analyzed according to the 2 −∆∆Ct method.
The assay was carried out as previously described [10]. Briefly, treated cells were harvested by trypsinization and loaded with JC-1 (2 µg/mL final concentration). Intracellular JC-1 was quantified at 530 nm (FL-1 green fluorescence) and 590 nm (FL-2 red fluorescence) by flow cytometry. The ratio of FL2/FL1 was evaluated to determine ∆Ψm. Experiments were performed in triplicate and repeated three times.
Intracellular ROS Production
With the aim of evaluating a general production of radical species as the byproducts of mitochondrial respiratory stimulation, we employed the most widely used probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA), which is used to investigate redox signaling mechanisms and the generation of oxidants [61]. DCFH-DA is a membranepermeable probe which is transformed into DFCH by cytoplasmic nonspecific esterases and is then oxidized by reactive oxygen species (ROS), becoming the fluorescent dichlorofluorescein (DCF). The assay was carried out as previously reported [10]; briefly, after treatments, cells were incubated with 10 µM (DCFH-DA, Sigma) for 15 min. Fluorescence intensities were measured (excitation wavelength of 504 nm and emission wavelength of 529 nm) by a Packard EL340 plate reader (Bio-Tek Instruments, Winooski, VT, USA); fluorescent values were normalized to the protein content and expressed as values relative to control. Experiments were performed in triplicate and repeated three times.
Cell Viability Assay
Cells were treated with TGF beta at different concentrations, from 0.1 ng/mL to 100 ng/mL, or left untreated as control for either 48 h or 8 days. Then, cells were incubated with 500 µg/mL methylthiazolyldiphenyl-tetrazolium bromide (MTT, Merck, Milan, Italy) in serum-free DMEM for 2 h at 37 • C according to Mosmann [62]. Finally, the formazan crystals formed in the bottom of the plate were dissolved with DMSO and the absorbance at 550 nm was measured in a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The 50% effective concentration (EC 50 ) value was determined using GraphPad Prism software (version 6.01, San Diego, CA, USA).
Toxicity Test (LDH Release)
After treatments, the cellular damage was measured by lactate dehydrogenase (LDH) release in the culture medium, as previously described [63]. The enzymatic activity of the culture medium was measured by spectrophotometry as absorbance variance at 340 nm, and it was expressed as µmol of NADH oxidated/min/mg of cellular proteins in order to normalize the extracellular activity to the number of cells. Data were plotted relative to the untreated controls.
Wound Healing Assay
The assay was carried out as previously described [64], with some modifications. PANC-1 and CAPAN-2 cells were incubated with the 10 ng/mL or 2 ng/mL TGFβ for 48 h and 8 days, respectively. Then, 24 h before the end of treatments, confluent cells were starved overnight and a wound line was generated with a sterile pipette tip, followed by further treatment for 24 h. Images were obtained at 0 and 24 h using a light microscope at 20× magnification with a digital camera under bright field illumination. The area of the wound was measured in the central part of each well using the ImageJ software. The measurements were then converted into a percentage of wound closure as follows: 100 − [(area at t 24 /area at t 0 ) × 100].
Band Quantification and Statistical Analysis
Scanning digital densitometry by ImageJ software analysis (ImageJ version 1.29, Sun Microsystems Inc., Palo Alto, CA, USA) was used for the quantification of bands from protein electrophoresis. All data were submitted to ANOVA statistical analysis with Tukey's post hoc correction. A p-value < 0.05 was considered significant and indicated. Data were given as the means ± SEM of three independent experiments.
Conclusions
Our study revealed two important novel aspects of TGFβ signaling. For the first time, the interplay with vitamin D activity was unveiled in cancer cells, and it might explain the link between vitamin D deficiency and cancer risk. Moreover, we shed some light on how, in addition to the nuclear control of EMT, the different modulation of mitochondrial metabolism can tip the balance in favor of either the antitumoral or the prometastatic effects of TGFβ. A better understanding of the conditions that sustain the protumoral activity of TGFβ is of paramount importance, together with efforts to find strategies blocking the cytokine. This approach would decrease metastatic spread and reduce fibrosis, which hampers the efficacy of chemotherapy. The attempts to use anti-TGF-β-based therapies for metastatic pancreatic ductal adenocarcinoma demonstrate the importance of understanding the role of TGFβ in pancreatic cancer progression [65]. | v3-fos-license |
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} | pes2o/s2orc | Fat Replacers in Baked Food Products
Fat provides important sensory properties to baked food products, such as colour, taste, texture and odour, all of which contribute to overall consumer acceptance. Baked food products, such as crackers, cakes and biscuits, typically contain high amounts of fat. However, there is increasing demand for healthy snack foods with reduced fat content. In order to maintain consumer acceptance whilst simultaneously reducing the total fat content, fat replacers have been employed. There are a number of fat replacers that have been investigated in baked food products, ranging from complex carbohydrates, gums and gels, whole food matrices, and combinations thereof. Fat replacers each have different properties that affect the quality of a food product. In this review, we summarise the literature on the effect of fat replacers on the quality of baked food products. The ideal fat replacers for different types of low-fat baked products were a combination of polydextrose and guar gum in biscuits at 70% fat replacement (FR), oleogels in cake at 100% FR, and inulin in crackers at 75% FR. The use of oatrim (100% FR), bean puree (75% FR) or green pea puree (75% FR) as fat replacers in biscuits were equally successful.
Introduction
Dietary fat has an important role within food matrices beyond basic nutrition. It contributes to many sensory and quality properties of a food including physical, textural and olfactory factors which all influence overall palatability. Many snack foods, in particular, rely on dietary fat to fulfil these palatable qualities in order to maintain consumer acceptance and consumption. The World Health Organisation [1], along with many national health authorities [2-5], recommends decreasing consumption of discretionary snack foods due to their poor nutritional content. Excess dietary fat intake, notably from discretionary snack foods, is one of the key contributors to excess energy intake and therefore weight gain [6]. Prevalence of overweight and obesity is rising worldwide [7,8] which is cause for concern as obesity is associated with increased risk of cardiovascular disease [9], type 2 diabetes mellitus [10], and some cancers [11].
Despite consumer awareness and product labelling [12,13], consumption of snack foods is relatively high with little compensation for the increased energy intake [14][15][16]. Many promoters have been attributed to increased snack food intake, such as convenience, taste, marketing and pricing [16,17]. In order to respond to these recommendations and consumer demands, manufacturing companies are increasingly developing snacks which are more nutrient dense than traditional snacks such as chips and cakes, which are typically high in added fat, sugar and sodium. Some examples of these types of innovative snacks include yoghurts, bars, puddings, crackers and chips which contain popular health foods (or superfoods) such as seeds, nuts, ancient grains, other wholegrains, dietary fibres, legumes, fruits and vegetables. While many of these snacks may be high in protein and dietary
Application of Fat Replacers in Baked Products
Fat replacers are defined by the American Dietetic Association as "an ingredient that can be used to provide some or all of the functions of fat, yielding fewer calories than fat" [24]. A wide range of products in the food industry uses fat replacers, some of which include meat, dairy and baked products [24]. It is important for product developers and food technologists to understand how different fat replacers influence the sensory and physical quality of snacks in order to guide the development of healthier alternative products. For example, in cakes, fat can contribute to increased leavening, tenderness and a finer crumb through a combined effect of trapping air cells during the creaming process [26]. This structure is then set during baking due to starch gelatinisation and coagulation of egg proteins [26]. Fat is typically used in biscuits to lubricate and coat the flour granules to prevent water absorption, and the development of starch and gluten in order to achieve a fine crumb (crumbly texture) and soft, tender mouthfeel [27]. Fat also contributes other important functions to cakes, biscuits and crackers such as flavour delivery and shelf life which is achieved through delaying water absorption by starch granules [28][29][30][31].
Fat replacers can be ingredients which are of carbohydrate, protein or fat origin, with many different types of fat replacers with different structures and functions within each group. We have not differentiated fat substitutes and fat mimetics in this review as the majority of fat replacers used in baked food products are fat mimetics. Instead, we have categorised fat replacers in this review as complex carbohydrate powders, gums and gels, whole food purees and products, or a combination thereof. This categorisation is based on their functional and industrial applications rather than their chemical properties.
(a) Complex carbohydrates are typically successful fat replacers due to their ability to bind water to form a paste which can mimic the texture and viscosity of fats in food products through providing lubricant or flow properties similar to fat in some food systems [28,32]. Examples of carbohydrate based fat replacers include inulin, maltodextrin and plant fibres. (b) Gums and gels work similarly in function to complex carbohydrates, in that they bind with water to form gels which mimic the texture and viscosity of fats [28]. While some gums and gels are made up of complex carbohydrates, this is not specific as there are some protein-and fat-based gums and gels. Examples of gums and gels used as fat replacers include pectins, oleogels and whey protein. (c) Whole foods are complete or partial food matrices that are included in a food product as fat replacers. Recently, many products have utilised whole foods such as fruits and vegetables, legumes or cereal based ingredients as fat replacers. These foods are typically successful due to their highly creamy texture when mashed or processed. Foods such as avocado can achieve this due to its oil composition, banana for its high starch content, and legumes for their high starch and protein contents. (d) Combinations of the above fat replacers are useful as they can potentially replicate multiple sensory qualities of dietary fat. In addition, complexes formed from these combinations, such as emulsions and esters, may have a greater fat replacing effect than the sum of their parts.
Summary of the Current Fat Replacers Used in Baked Products
Complex carbohydrate fat replacers range from digestible starches to non-digestible plant fibres (Table 1). It should be noted that the replacement of dietary fat with complex carbohydrates reduced energy density of all the food products in Table 1, regardless of fibre status, due to complex carbohydrate being less energy dense than fat. The use of fibres instead of starches could have an advantage on the market, as foods may meet criteria for fibre content claims. Inulin, a non-digestible dietary fibre typically derived from chicory root, was observed to have the greatest success in replacing dietary fat in baked products, where a fat replacement (FR) level of up to 75% in legume crackers and cake (1:1, inulin: water; and 1:2 inulin: water, respectively) was able to reduce total energy without any changes in consumer acceptance [33,34]. It should be noted that the addition of inulin did change the textural and physical properties of the cracker and cake products. While acceptance was not measured for the use of inulin in muffins, 50% FR had the least sensory and physical changes compared to 75% and 100% FR [35]. In addition, Zahn et al. tested the use of four commercial inulin formulations in muffins which varied in inulin to water ratio and solubility, but the outcomes for each were similar [35]. Maltodextrin was also successful at 75% fat replacer level in legume crackers and at 66% FR in muffins, although there were changes noted in aroma, appearance, taste and texture [33,36]. Total FR of inulin or maltodextrin (100%) had a significant decrease in consumer acceptance, so it is not recommended to fully replace fat in a baked food product. Results were also promising for inulin used as a fat replacer in biscuits, although there was some notable changes to textural and physical properties [33][34][35][37][38][39]. Other complex carbohydrates used as fat replacers in biscuits included lupine extract, maltodextrin, corn fibre, and rice starch, although all of these had significant effects on sensory properties of the biscuits except rice starch [33,36,[40][41][42][43]. Rice starch has no significant effects on sensory properties, but was only tested at 20% FR. All complex carbohydrate fat replacers had a significant effect on the physical properties of doughs and their baked products, with significant increases in density, toughness, breaking strength, moisture, and decreases in volume for nearly all tested products [33][34][35][36][37][38][39][40][41][42][43][44][45][46].
Of all the complex carbohydrate fat replacers, inulin had the greatest success at reducing total fat and energy of the food product, with the least impact on sensory qualities and consumer acceptance, particularly in the legume crackers. Long chain inulin has the ability to form microcrystals which in turn aggregate together, interact with water, and eventually agglomerate creating a gel network [47]. To some extent, this gel network seems to have the ability to mimic the functions of fat in baked products such as being able to lubricate dry ingredients (through surrounding starch and protein), assisting in maintaining a shortening effect. Maltodextrin was also a successful fat replacer in legume crackers, although it was not as successful at replacing fat in biscuits compared to inulin. Inulin is also a good source of fibre, has promising gut health properties due to its prebiotic nature, and may increase absorption of nutrients such as calcium [48]. Moreover, inulin may benefit from marketing with fibre content claims, which may be appealing to consumers. Therefore, we recommend inulin as a reasonably high level fat replacer in crackers, cakes, biscuits and muffins [48]. Gum and gel fat replacers, while mostly being carbohydrates, also include lipid-based and protein-based gums and gels ( Table 2). Some of these fat replacers may also increase suitability for nutrition content claims, such as sources of fibre or protein. Guar gum and xanthan gum had relatively little effect on the physical properties of the cake product when used as fat replacers [49]. While sensory measures were not compared to a control, both types of cakes were rated as acceptable with a greater acceptance in the cake containing xanthan gum, and 50% FR was considered ideal [49]. Oatrim (a tasteless white powder derived from oats, comprised of amylodextrins and 5-10% β-glucan soluble fiber; incorporated as a powder or gel), caused significant changes to the physical properties of cake, croissants and biscuits [50][51][52]. However, this did not appear to have any impact on the sensory properties of these foods, even at 100% FR. Pectin also caused significant changes to the physical properties of cake, croissants and biscuits, specifically increasing the hardness and reducing the volume of these foods, which was paralleled by increased perception of hardness and reduced flavour from sensory evaluations [42,46,53,54]. This is notable as pectin was tested at a relatively low FR level in cake and biscuits (10-30%), suggesting it is not an ideal fat replacer in baked products. Hydroxypropyl methylcellulose (HPMC) had significant effects on physical and sensory properties of crackers and biscuits, even at relatively low FR levels [33,55]. While consumer acceptance was not tested on crackers due to being considered unacceptable by a focus group [33], HPMC in biscuits was considered significantly less acceptable compared to control biscuits containing 18% canola oil suggesting it is also not an ideal fat replacer in these foods.
Oleogels are products of solidifying vegetable oils using natural wax esters [56][57][58][59]. The oleogelation process forms waxy crystal structure which hold liquid oil within a solid matrix, which allows the use of liquid vegetable oils in place of shortening. While this does not necessarily reduce the total fat content of a food product, it is useful in reducing saturated fat content. It should be noted that all oleogels studies reviewed in this paper did reduce overall fat content of their tested foods [56][57][58][59]. However, oleogels were not successful as fat replacers in these studies as they made biscuit and cake denser and harder. Sensory properties seemed to be promising with an increase in taste and no difference in acceptance compared to the control foods. Lastly, whey protein was also not an ideal fat replacer for biscuits as it resulted in a decrease in overall flavour and acceptance [45,46].
Overall, gums and gels were not very successful as fat replacers in baked goods. Oatrim appeared to be the most successful as there were no significant changes to the sensory properties of cake and biscuits, although there were a large range of physical changes to these foods which might have an impact on industrial applications. Xanthan gum and guar gum might potential be useful fat replacers in cake as they had little impact on physical properties, although more robust sensory evaluations are needed in future studies. The interest in using whole food fat replacers has increased in recent years. These fat replacers are beneficial as they have a range of carbohydrates, lipids and proteins that may aid in the rheological properties of baked products, making them potentially more suitable than simple extracts and isolates. Overall, whole food fat replacers had the least effect on the physical and sensory properties of baked products, and in some cases increased the consumer acceptance (Table 3). Apricot kernel flour was a successful fat replacer with little impact on the physical and sensory properties of biscuits at a maximum of 50% FR [60,61]. Chia seed mucilage also had little impact on physical properties of cake and bread up to 100% FR [62,63], although sensory properties were not tested in these studies.
High oleic sunflower oil (HOSO) did not significantly decrease the amount of total fat in biscuits, but did reduce the saturated fat content [64]. However, the use of HOSO as a fat replacer was not considered successful as it has significant impact on the volume, colour and texture of the biscuits. The use of avocado puree as a fat replacer in cake and biscuits was successful at 50% FR, as it did not impact consumer acceptance [51,65]. However, at 75-100% FR, acceptance of the low-fat cake decreased compared to the control cake containing shortening [65]. Apple puree or pomace was the only whole food fat replacer to result in a reduction in sensory quality and consumer acceptance, even at low FR levels (10%) [66,67]. Therefore, apple puree is not recommended as a fat replacer in biscuits. Bean puree and green pea puree had very similar effects on the sensory properties of biscuits with increases in sensory qualities at 25-75% FR [68,69]. The use of green pea puree at FR of 25% in biscuits was considered ideal, whereas a FR of 100% resulted in reduced consumer acceptance [69]. Lastly, a high β-glucan product derived from oats or oat bran had significant impact on texture, colour and moisture of biscuits [45,54,70]. Although sensory properties were not tested in these studies, this suggests that the high β-glucan product was not a successful replacer for shortening in biscuits.
Whole foods may be the most suitable candidates for fat replacers in baked foods as they appeared to have the least impact on physical and sensory properties. In addition, they may also be beneficial as they may contain phytochemicals and micronutrients which could increase the health benefits and marketing potential of baked foods products, leading to novel functional foods. Lastly, consumer are more likely to accept foods with ingredients or additives that are made from natural, whole food products [71]. Bean and pea purees were the most successful fat replacers for biscuits at 25-75% FR, and avocado puree was successful at reducing fat in cake at 50% FR. However, more studies on whole food fat replacers in biscuits and bread is needed before they can be recommended as reliable fat replacers. Fat replacers in combination with additional ingredients may provide better fat-like qualities as the additional ingredients are usually designed to supplement the unwanted effects of individual fat replacers, as seen above (Tables 1-3). These additional ingredients are usually other types of fat replacers, but can also be enzymes or emulsifiers. Few studies have assessed combined fat replacers in baked products, although the results appear promising (Table 4). Polydextrose and guar gum were successful fat replacers in biscuits at a relatively high level of FR (70%), with an increase in perceived taste, flavour and consumer acceptance [72]. Maltodextrin and xanthan gum yielded increased moisture, hardness and chewiness in 66% FR muffins, but sensory analysis was not conducted in these samples [36]. Kel-Lite BK, a commercial fat replacer containing xanthan gum, guar gum, cellulose gel, sodium stearoyl lactylate, gum Arabic, dextrin, lecithin, and mono-and diglyceride, resulted in increased bitterness and, oddly increased both crumb firmness and softness in biscuits at 33%, 66% and 100% FR [54]. HOSO and inulin were also successful fat replacers in biscuits at 100% FR [64,73], although HOSO does contain lipids so the biscuits only had reduced saturated fat rather than total fat. However, HOSO and inulin resulted in decreased appearance, flavour, odour, texture, and consumer acceptance in cakes, croissants and muffins [73]. Therefore, HOSO and inulin may only be suitable for use as fat replacers in biscuits. HOSO and β-Glucan may also be a useful fat replacer at 100% FR as this had little impact on physical properties in biscuits, although sensory evaluations were not conducted [64]. A combination of emulsion filled gel based on inulin and extra virgin olive oil (EVOO) has also been trialed as a fat mimetic in biscuits [74]. At 50% FR, there were no changes to the physical properties and the overall consumer acceptance of the biscuit compared to the control biscuit containing 20% butter, although there was a decrease in overall flavour. However, consumer acceptance was not maintained at 100% FR. Inulin, lipase and a commercial emulsifier ("Colco"; a type of alpha-gel emulsifier containing glycerol monostearate and polyglycerol esters of fatty acids) had little impact on physical properties of cake at 50-70% FR, although no sensory evaluation was conducted for this combined fat replacer either [75]. One study assessed the double, but not triple, combinations of corn fibre, maltodextrin and lupine extract in biscuits, each at 30-40% FR [40]. All combinations had little impact on the physical properties of the biscuits compared to the control biscuit containing 33% margarine. However, consumer preference for corn fibre and lupine extract was significant lower than the control, whereas corn fibre and maltodextrin was significant higher than the control [40]. This suggests that the combination of corn fibre and maltodextrin may be an ideal fat replacement in biscuits at a moderate FR level. Tapioca dextrin, tapioca starch and resistant starch as a combination fat replacer had an impact on a wide range of sensory properties in biscuits [76]. However, overall consumer acceptance decreased, even at relatively low FR levels (10-20%), so we do not recommend the use of this combination fat replacer in biscuits.
Overall, combination fat replacers may be potential candidates for ingredients in low-fat baked products. The use of polydextrose and guar gum appears to be a reasonably effective fat replacer in biscuits. However, with the limited evidence currently available, recommendations cannot be made for the use of combination fat replacers in other baked products.
Industry Recommendations and Conclusions
It should be noted that there is limited literature on the use of fat replacers in low-fat baked products. Many of the reviewed fat replacers have only been assessed once, and also only in one type of food. There is a need for additional replicate studies using a variety of recipes. Also, while we have reviewed the current literature here, we cannot compare physical and sensory properties between studies. Therefore, while we can summarise which fat replacers were successful within a certain baked product, it is difficult to determine which fat replacer is best. In addition, the use of fat replacers in bread, muffins and croissants were only assessed in few studies each. Therefore, there is not enough information to make a recommendation of the best type of fat replacer for these products. Below is our recommendations for the best currently assessed fat replacers in a range of baked food products: Biscuit-Oatrim was the most successful fat replacer in biscuits as it was able to retain most sensory properties of a traditional biscuit even at 100% FR, although there was a decrease in hardness and brittleness [51,52]. However, it should also be noted that both bean puree and green pea puree were able to increase the sensory qualities and consumer acceptance of biscuits at 75% FR with less of an impact on the physical properties compared to oatrim [68,69]. Legume purees might also have an advantage over oatrim as they may aid the marketability of food products due to potential nutrition claims such as vegetable and protein content. However, legume purees should not be used at 100% FR. Overall, we recommend the use of either oatrim or legume purees as fat replacers in biscuits.
Cake-Oleogels appeared to be the most successful fat replacer in cake, with no changes to the sensory qualities at 100% FR [57][58][59]. However, there were significant changes to the physical properties of cake when using oleogels at FR levels ≥50% [58] which might lead to difficulty during cake production. An alternative could be avocado puree which was only successful at 50% FR but had less of an impact on the physical properties of cake [65], or inulin which was successful up to 75% FR but had an impact on the physical and textural properties of cake [34].
Cracker-While there was only one study on the use of fat replacers in crackers [33], it assessed and compared a range of fat replacers in the one study. Inulin appeared to be the most successful fat replacer in these crackers, reaching an acceptable level of FR at 75%. The additional benefits of using inulin is that it may aid the marketability of food products due to potential high fibre claims. | v3-fos-license |
2019-07-16T23:01:17.864Z | 2019-06-18T00:00:00.000 | 196678276 | {
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} | pes2o/s2orc | Robust Normalization of Luciferase Reporter Data
Transient Luciferase reporter assays are widely used in the study of gene regulation and intracellular cell signaling. In order to control for sample-to-sample variation in luminescence arising from variability in transfection efficiency and other sources, an internal control reporter is co-transfected with the experimental reporter. The luminescence of the experimental reporter is normalized against the control by taking the ratio of the two. Here we show that this method of normalization, “ratiometric”, performs poorly when the transfection efficiency is low and leads to biased estimates of relative activity. We propose an alternative methodology based on linear regression that is much better suited for the normalization of reporter data, especially when transfection efficiency is low. We compare the ratiometric method against three regression methods on both simulated and empirical data. Our results suggest that robust errors-in-variables (REIV) regression performs the best in normalizing Luciferase reporter data. We have made the R code for Luciferase data normalization using REIV available on GitHub.
Introduction
Transient reporter assays are an important and widely used tool in the study of gene regulation [1][2][3][4], intracellular cell signaling [5][6][7], and other areas of molecular, cellular, and developmental biology [8][9][10]. In a reporter assay, the activity of a promoter, potentially in combination with an enhancer, is measured by placing an inert reporter gene such as luciferase or lacZ under the control of the promoter. luciferase is commonly used as the reporter gene since the assay has both high sensitivity and a very high dynamic range [11].
An important technical challenge in the analysis of Luciferase reporter data is that transfection efficiency can vary significantly from sample to sample, especially in hard to transfect cell types such as primary cells. Other sources of random experimental error, such as the amount of transfected DNA, number of cells assayed, cell viability, and pipetting errors further compound transfection efficiency variation to yield Luciferase luminescence data that can vary over an order of magnitude [4,8,12]. For example, a reporter of CCAAT/Enhancer binding protein, α (Cebpa) promoter expression exhibits ∼3-fold variation in luminescence when assayed in PUER cells [13][14][15], a myeloid cell line with low transfection efficiency (Figure 1; Section 3.1). The prevalent method of correcting for sample-to-sample luminescence variation [3,8] is to co-transfect an independent control reporter, such as Renilla luciferase expressed from a constitutive promoter, along with the promoter/enhancer reporter being assayed. Since transfection efficiency and other experimental errors would affect both constructs equally, the activity of the experimental promoter relative to the constitutive promoter can be determined by taking the ratio of firefly and Renilla luminescence. Let there be i = 1, · · · , N samples and F i and R i represent firefly and Renilla luminescence in the ith replicate. Then, the relative activity is estimated as Although it is in common use, "ratiometric" normalization is statistically unsound since it weights low-and high-luminescence replicates equally even though the former produce less reliable estimates of normalized reporter activity. This problem is especially acute in cell types having low transfection efficiency and, consequently, high variability in luminescence measurements.
Here we propose an alternative approach to the normalization of Luciferase reporter data that is more robust than the ratiometric method. Firefly luminescence is expected to be proportional to Renilla luminescence, and therefore, the relative activity A can be estimated as the slope of the best fit line using linear regression ( Figure 1). Ordinary least-squares finds the best fit line by minimizing the errors in the dependent variable (F), that is, the distances between the line and the data points in the vertical direction. Ordinary least-squares (OLS) regression places a higher weight on high-luminescence points and thus avoids the main weakness of ratiometric normalization.
We also considered two alternatives to OLS to find the method best suited for the normalization of Luciferase data. OLS assumes that the values of the independent variable, Renilla luminescence in our case, are known exactly and do not include random errors. Since Renilla luminescence is itself a random variable in transient assays, we evaluated errors-in-variables (EIV) regression (Section 3.4.3; [16]). In contrast to OLS, which minimizes the vertical error, EIV regression minimizes the perpendicular distance of each data point from the line, leading to the minimization of errors in both the independent and the dependent variables. Lastly, we considered robust errors-in-variables (REIV) regression (Section 3.4.4; [17]). Luciferase luminescence data contain potential outliers (Figure 1; [4,8]), which can unduly influence activity estimates. In REIV, the estimation of the slope and intercept is rendered insensitive to outliers by utilizing a bounded loss function. This means that the contribution of any point to the error stops growing with distance if it lies far enough from the line. Thus, outliers, points that lie far away from the rest, are prevented from having an undue influence on the slope and intercept of the best fit line.
In this study we compared the four methods described above, ratiometric, OLS, EIV, and REIV, in two ways. First, we applied each method to simulated data and assessed its ability to recover the "true" activity used to generate the synthetic data. Second, we tested the methods on empirical measurements of Cebpa promoter and enhancer activity in PUER cells under two different cytokine treatments. Our results show that while ratiometric normalization performs poorly on high variability data from low transfection efficiency experiments and can lead to erroneous biological conclusions, REIV performs the best and robustly estimates activity under a variety of different conditions.
Results and Discussion
We evaluated the performance of four methods, ratiometric, OLS, EIV, and REIV, on simulated data, where the "ground truth" is known. Reporter assays in cells with low transfection efficiency yield activity data with a high coefficient of variation, making normalization more challenging. Other sources of experimental error impact normalization similarly, and accurate normalization becomes more challenging with increasing standard deviation of the experimental error. We aimed to evaluate the methods' ability to recover the known promoter activity at different levels of transfection efficiency and experimental error.
We simulated sample-to-sample variation in transfection efficiency (t) (Section 3.5) by sampling from the Beta distribution. The coefficient of variation of the Beta distribution is inversely related to mean transfection efficiency [16] and consequently synthetic data from low transfection efficiency simulations have greater variability, mirroring experimental reality. Mean transfection efficiency (t) was varied to assess how the methods perform as sample-to-sample variability increases. Random experimental errors, modeled with a Gaussian mixture model [17], were introduced into both Renilla and firefly simulated data (Section 3.5). The Gaussian mixture model includes low-frequency contamination by errors with a higher standard deviation and allowed us to simulate outliers ( Figure 1). We varied the standard deviation of Renilla errors (σ 11 ) to assess the performance of each method at different levels of experimental variability. Robustness of each method to outliers was assessed by varying the standard deviation of the contaminating errors (σ 12 ). We also varied the sample size (N) and "true" activity level to investigate how they impact normalization.
Simulated data were generated for each combination of activity, transfection efficiency, sample size, and error standard deviations. Each normalization method was used to estimate activity from the exact same set of simulated data. The simulations were repeated M = 300 times and the performance of the methods was assessed using three different measures. Let the known or true activity be A and the activity estimated in the kth simulation be A k . We utilized the relative absolute difference between the true and estimated values, as a measure of the relative bias in the estimation. Second, we computed the relative median absolute deviation (MAD) of the estimated activities A k as a measure of precision. Although readily interpretable, activity based measures are less reliable when the true activity is high. Since the activity is the slope of the regression line, small deviations of the regression line from the true line lead to large deviations in activity when the true activity is large. For this reason, we also utilized a measure developed by Zamar (1989) [17], The "Zamar criterion" is independent of the activity level since it is invariant under rotations of the xy-plane [17] and serves well to compare different methods. The Zamar criterion takes values between 0 and 1, the former implying perfect recovery of the true activity.
In the first set of simulations, we assessed the ability of the methods to infer the activity from simulated data lacking outliers (σ 12 = σ 11 ) at different levels of mean transfection efficiency. The performance of all methods improved with increasing transfection efficiency (Figure 2a), however the regression-based methods, OLS, EIV, and REIV, performed better at lower transfection efficiencies as compared to the ratiometric method. At transfection efficiencies less than 25%, the ratiometric method produces rather inaccurate estimates of activity, and the 90th percentile of relative bias is ∼300% at 10% transfection efficiency. Next, we investigated whether the accuracy of the estimates could be improved by increasing sample size (N). Since the main difference between the ratiometric and the other methods arises at low transfection efficiencies, we set the mean transfection efficiency to 25% for all subsequent simulations. Whereas the performance of the regression based methods improved with increasing sample size, the bias in the activity inferred by the ratiometric method did not decrease with sample size (Figure 2b). These set of simulations suggested that the ratiometric method of normalization results in biased activity estimates that do not improve even at very large sample sizes.
In the third set of simulations, we investigated the effect of the activity level on the normalization (Figure 2c). The performance of all the methods deteriorated at activity levels close to 1. An activity level of 1 implies that firefly luminescence is comparable to Renilla luminescence, which only occurs for exceptionally weak promoters since it is common practice to transfect the firefly plasmid in a 10-to 200-fold excess over the Renilla plasmid. Next we investigated the effect of experimental error in Renilla luminescence ( Figure 3a). As one would expect, the performance of all methods deteriorates as the magnitude of experimental error is increased. Furthermore, the ratiometric method had the worst performance amongst all the methods. The errors-in-variables methods, EIV and REIV, were the most resilient to errors in Renilla (Figure 3a; Zamar criterion) since OLS does not attempt to minimize errors in the independent variable. Finally, we investigated the performance of the methods in the presence of outliers. So far, the simulations had been carried out without outliers since the standard deviation of the contaminating errors was the same as the Renilla experimental error (σ 12 = σ 11 ). We increased the standard deviation of the contaminating errors (σ 12 ) to model outliers. REIV had the best performance (Figure 3b; Zamar criterion) of all the methods in the presence of outliers. These simulations suggest that errors-in-variables methods, especially REIV, perform best when normalizing transient transfection data.
Having investigated the performance of the four methods on simulated data, we sought to test them on empirical data. We utilized a dataset of 85 measurements of firefly luminescence driven by the Cebpa promoter paired to Renilla measurements driven by the CMV promoter, acquired in 8 different experiments carried out over a 6 month period (Figure 4a). The Luciferase assays were performed in PUER cells (Section 3.1), which have a transfection efficiency of ∼30%. The data show that despite the variability in firefly and Renilla luminescence, they are linear with respect to each other. We sampled the Cebpa promoter luminescence dataset 1000 times with replacement at sample sizes N = 3, 6, 10, 20, 30. Figure 4b shows boxplots of the normalized activity. The three regression based methods converge to a median activity of ∼13 as sample size is increased. Mirroring the results of the simulations, the ratiometric method estimates activity as ∼16 and this bias is not ameliorated by increasing the sample size. Suspecting that this bias arises from low-luminescence measurements, we sampled a subset of the data with Renilla luminescence greater than 8 relative luminescence units (RLU). The activity inferred by the regression based methods was unchanged as compared to the larger dataset but those inferred by the ratiometric method now agreed with the other three methods (Figure 4c). This result implies that while ratiometric estimation can be biased by low-luminescence measurements, the regression based methods are robust to such data. The bias in the ratiometric estimates of activity can result in erroneous biological conclusions. We illustrate this with an example from our analysis of the regulation of Cebpa enhancers [4,15]. We tested the functional role of binding sites of Gfi1 and C/EBP family transcription factors (TFs) in an enhancer (Figure 5a) found ∼7 kb downstream of the Cebpa transcription start site [4]. We assayed the activity of three reporter vectors in PUER cells cultured in either IL3 without 4-hydroxy-tamoxifen (OHT) or in GCSF 24 h after induction with OHT. OHT induction in the presence of GCSF causes PUER cells to differentiate into neutrophils over a 7-day period [4,14]. The activity of the CMV promoter was not affected by cytokine treatment in PUER cells (Supplementary Materials Figure S1; Welch two sample t-test, N = 10, p = 0.93), allowing for a valid comparison between treatments. Cebpa(0) contained the proximal promoter, Cebpa(7) contained the enhancer in addition to the proximal promoter, and Cebpa(7m1) contained the enhancer in which the C/EBP sites had been mutated. Analysis with REIV (Figure 5b; left panels) shows that the enhancer upregulates promoter activity 4-6 fold in both conditions and that mutating the C/EBP sites reduces activity by about 50% in IL3 conditions, which is consistent with the known role of C/EBP TFs as activators [18] of Cebpa. In the data analyzed using REIV, mutating the C/EBP sites does not have an effect in GCSF conditions. Analysis of the same exact data with the ratiometric method however detects a statistically significant upregulation of Cebpa(7m1) in GCSF conditions (Figure 5b; right panels). The ratiometric method would thus lead one to the incorrect conclusion that C/EBP TFs repress Cebpa in GCSF conditions, which is inconsistent with their well-known role as activators. 11 . Firefly errors were scaled with activity: σ 21 = σ 22 = 10σ 11 (b). Performance plotted against increasing severity of outliers. The standard deviation of the contaminating Renilla errors σ 12 was varied to simulate outliers. True activity: A = 3. Renilla errors: σ 11 = 3. Firefly errors: σ 21 = 9. The standard deviation of firefly contaminating errors was scaled with activity: σ 22 = 3σ 12 . The other parameters are the same as in panel (a). The Zamar criterion of the ratiometric method was greater than the upper limit of the y-axis.
Our results suggest that the commonly used ratiometric method performs adequately on reporter data from cells having transfection efficiency of at least 75% but can provide inaccurate estimates in low transfection efficiency experiments. The regression-based methods in contrast provide accurate inference irrespective of transfection efficiency. Our simulations show that OLS performs poorly when random errors in Renilla luminescence are large (Figure 3a) and both OLS and EIV perform poorly when the data have large outliers (Figure 3b). REIV was the most robust to all of these potential problems. In our tests on reporter data from PUER cells (Figure 4), the three regression based methods, OLS, EIV, and REIV, gave nearly identical estimates. This implies that the particular data used here neither have large random errors in Renilla luminescence nor a significant outlier problem. It cannot be assumed, however, that all datasets are free of these problems. The safest choice therefore would be to favor REIV over the other methods. The main cost of REIV is that it is more computationally intensive than the other methods. This is not a problem in practice, since REIV takes ∼30 s on modern desktop hardware. Use of REIV regression for normalization could enable studies in hard-to-transfect cell lines that were not possible before. We have made R code to analyze reporter assay data using EIV and REIV available on GitHub (Sections 3.4.3 and 3.4.4). A detailed protocol for using the code to analyze dual-Luciferase reporter data is provided in Section 3.6. (7), and Cebpa(7m1) inferred by either the REIV (left panels) or the ratiometric (right panels) methods in uninduced IL3 (progenitor) or induced GCSF (neutrophil) conditions. The activity has been normalized against the activity of the proximal promoter (Cebpa(0)). Error bars are standard errors of the mean. The ratiometric method spuriously detects an activation of Cebpa(7m1) in GCSF conditions (bottom right panel; Welch two sample t-test, N = 10, p = 0.03).
Construction of the Luciferase Reporter Plasmid
The Cebpa promoter [4,15] was cloned into a pGL4.10luc2 Luciferase reporter vector (Promega, E6651) between the XhoI and HindIII sites. The enhancers were inserted between BamHI and SalI sites downstream of the SV40 late poly(A) signal. The promoter was amplified from genomic DNA of C57BL/6J mice with primers TGG CCT AAC TGG CCG GTA CCT GAG CTC GCT AGC CTC GAG AAC TCC TAC CCA CAG CCG CG (Fwd) and TCC ATG GTG GCT TTA CCA ACA GTA CCG GAT TGC CAA GCT TCA GCT TCG GGT CGC GAA TG (Rev), which include 40 bp of sequence homologous to pGL4.10luc2. PCR amplification was carried out using Q5 High-Fidelity 2X Master Mix (NEB, M0492L) following the manufacturer's instructions. The following PCR cycling conditions were used: initial denaturation of 30 s at 98 • C, 30 cycles of 30 s at 98 • C, 30 s at 60 • C, and 60 s at 72 • C, and a final extension for 10 min at 72 C. Gibson Assembly (GA) reactions [19] were carried out using 0.06 pmol of digested vector and 0.18 pmol of insert, for 60 min at 50 • C. NEB high-efficiency competent cells (NEB, E5510S) were transformed according to manufacturer's instructions. See Repele et al. [4] for the construction of the reporter vectors having the wildtype and mutant enhancers.
Transfection and Luciferase Assays
PUER cells were transfected with Cebpa promoter reporter vector and Renilla control vector pRL-CMV (Promega, E2261) in a 1:200 ratio using a 4D-Nucleofector (Lonza). Cells were transfected with 2.26 µg total plasmid DNA in SF buffer (Lonza, V4SC-2096), using program CM134 and incubated for 24 h prior to luminescence measurement. Enhancer/promoter activity was assayed in the neutrophil differentiation by placing cells in medium containing GCSF and OHT after nucleofection. After incubation, firefly and Renilla luminescence were measured using the Dual-Glo Luciferase activity kit (Promega, E2920) and the DTX 880 Multimode Detector (Beckman Coulter) according to manufacturer's instructions. Transfections were performed in at least 10 replicates.
Ratio
For each measurement, the ratio of firefly and Renilla luminescence was computed and averaged over the samples (Equation (1)).
Ordinary Least-Squares Regression
The normalized activity of the construct was determined as the slope of the line F = AR, where F and R are firefly and Renilla luminescence respectively, using ordinary least-squares (OLS) regression. The regression was performed using the lm function of R.
Errors-in-Variable Regression
The normalized activity of the construct was determined as the slope of the line F = AR using errors-in-variables (EIV) regression [16]. EIV regression is implemented as the function eiv in the code on GitHub (https://github.com/mlekkha/LUCNORM).
Robust Errors-in-Variable Regression
The normalized activity of the construct was determined as the slope of the line F = AR using robust errors-in-variable regression (REIV) [17]. Since the REIV algorithm is not available in any R package, we implemented it in R as follows.
A was estimated by minimizing the loss function where R i and F i are individual replicates of Renilla and firefly luminescence measurements, is Tukey's loss function with c = 4.7, and S is an estimate of the scale of the residuals. The argument of Tukey's function is the orthogonal distance of the point (R i , F i ) from the regression line. Tukey's function is bounded for large values of t, which limits the contribution of outliers to the loss function and ensures that the slope estimate is robust to outliers. The value of S was estimated by solving the equation where κ = 0.05 and χ(t) is Tukey's loss function with c = 1.56. The minimization problems were solved by the sequential least-squares quadratic programming (SLSQP) algorithm of the NLOPTR package of R, with parameters xtol_rel and maxeval set to 10 −7 and 1000 respectively. 95% confidence intervals were estimated by bootstrapping using the R package BOOT. 999 replicates were subsampled using the ordinary simulation and the function boot.ci was used determine confidence intervals using the basic bootstrap method. REIV regression is implemented as the function robusteiv in the code on GitHub (https://github.com/mlekkha/LUCNORM).
Generation of Simulated Data
We generated simulated luminescence data that take into account sample-to-sample variation in transfection efficiency, random errors in both firefly and Renilla luminescence from other sources, and potential outliers. For each simulated experiment, we generated i = 1, · · · , N samples. The transfection efficiency of each sample was drawn from the Beta distribution, so that it varied between 0 and 1. The mean transfection efficiency of an experiment ist = α/(α + β). We simulated experiments with different mean transfection efficiencies ( Figure 2) by varying α and β (Table 1). The Renilla luminescence was computed by multiplying t i with the maximal Renilla luminescence R and introducing errors drawn from a Gaussian mixture model where CN(σ 2 11 , σ 2 12 , γ) = (1 − γ)N(0, σ 2 11 ) + γN(0, σ 2 12 ) are random errors with variance σ 2 11 contaminated with errors with higher variance σ 2 12 . The Gaussian mixture model simulates outliers occurring with a frequency of 5%.
Firefly luminescence is computed similarly, with the maximal firefly luminescence given by the product of the "true" relative activity A and R.
In the simulations presented here, we assumed that the standard deviations of firefly luminescence scale with the activity so that σ 21 = Aσ 11 and σ 22 = Aσ 12 . Once the CSV file has been prepared in this manner, the data are ready to be analyzed by REIV.
3.6.4. Importing Input Data into the R Workspace and Preparing It 1.
In the R Console, read the file into a variable.
(Optional) If any of the construct/condition names are numeric, they must be set as categorical variables. This may be accomplished as follows.
1.
Once the data have been saved in a variable, REIV can be used to compute normalized Luciferase activity and its confidence interval for each combination of construct and condition.
norm_activity <-calcSlopesCIs(<data_variable_name>, alpha = <alpha_value>, regmethod = robusteiv, cim = "boot_positive_ci", ignore=c("Luc", "Ren")) Here, alpha is the significance threshold. For example, setting alpha to 0.05 will compute 95% confidence intervals. regmethod is the normalization method. Use regmethod = eiv for EIV normalization. cim is the method used for computing the confidence intervals. Use cim = "gleser_ci" for EIV normalization. ignore specifies which columns/variables are not conditions. If there are other columns in the CSV file that are not conditions, such as annotations, they should be included in the ignore vector.
The normalized activities may be saved to a CSV file for further analysis or visualization using the write.csv function. | v3-fos-license |
2017-08-06T06:10:22.285Z | 2017-04-05T00:00:00.000 | 36601023 | {
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} | pes2o/s2orc | Thermodynamic projection of the antibody interaction network: the fountain energy landscape of binding
The complexity of the adaptive immune system in humans is comparable to that of the central nervous system in terms of cell numbers, cellular diversity and the network of interactions between components. While the application of molecular biological methods and bioinformatics has brought about an ever deepening and sharpening static description of the molecular and cellular components of the system, a unifying theoretical understanding of the laws governing the dynamics of the system is still lacking. We have recently developed a quantitative model for the description of antibody homeostasis as defined by the dimensions of antigen concentration, antigen-antibody interaction affinity and antibody concentration. In this paper we develop the concept of a novel thermodynamic representation of multiple molecular interactions in a system, the fountain energy landscape of binding. We show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network projected onto an energy landscape defined by conformational entropy and free energy of binding. We demonstrate that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the binding fountain energy landscape concept allows a systems biological, thermodynamic perception of the functioning of the clonal humoral immune system.
We have recently developed a quantitative model for the description of antibody homeostasis as defined by the dimensions of antigen concentration, antigen-antibody interaction affinity and antibody concentration. In this paper we develop the concept of a novel thermodynamic representation of multiple molecular interactions in a system, the fountain energy landscape of binding. We show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network projected onto an energy landscape defined by conformational entropy and free energy of binding. We demonstrate that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the binding fountain energy landscape concept allows a systems biological, thermodynamic perception of the functioning of the clonal humoral immune system.
Blood is a massive and critically important extracellular space of multicellular organisms. It is a fluid tissue with cellular, macro-and small molecular components that perfuses the whole multicellular organism, being in direct contact with vascular endothelial cells and blood cells. Its components are potentially derived from any cell of the organism via secretion and leakage (Anderson & Anderson, 2002). Such a hugely diverse molecular pool needs to be regulated with respect to the quality and quantity of its components. One of the mechanisms of regulation is the generation of antibodies by the humoral adaptive immune system (Prechl, 2017a(Prechl, , 2017b. Considering the diversity of antibodies and the diversity of molecular targets, the interaction landscape of humoral immune system is presumably the most diverse in an organism. In this perspective article we approach antibody homeostasis from the thermodynamic point of view, depicting antibody-antigen interactions in a novel energy landscape model. The currently used funnel energy landscape model is suitable for the description of folding and binding of one or a few molecules but it would require landscapes of intractable sizes to depict a whole system, like adaptive immunity. We introduce the fountain energy landscape, a projection of the multidimensional binding landscape of antibodies to the dimensions of entropic penalty and energy of molecular interactions, to accommodate the vast range of interactions of antibodies.
Energy landscape and antibody binding
Molecular interactions can be described by examining structural, kinetic and thermodynamic properties of the binding. Structural approaches aim to define the relative spatial positions of the constituting atoms of the interacting partners in the bound and unbound forms of a molecule. The advantage of the structural approach is the high resolution visual rendering of molecular structure that helps human perception. Systematic analysis of protein structures gives insight into the evolution of protein complexes and the dynamics of assembly and disassembly (Marsh & Teichmann, 2015). Structural information can also reveal networks of protein interactions (Kiel et al , 2008). Kinetic studies follow temporal changes of association and dissociation of interacting partners. These observations are easily applicable to a simple system with a few components only but it is difficult to describe complex systems and crowded molecular environments (Schreiber et al , 2009;Zheng & Wang, 2015). Thermodynamics examines the changes in free energy that accompany a binding event; providing statistical descriptions of enthalpic and entropic components of the interaction. Energy landscape theory resolves some shortcomings and integrates these approaches by assuming the presence of many different conformations that converge to thermodynamically stable forms, the route taken to obtain this conformation dictating the kinetics of the events (Bryngelson et al , 1995). The intramolecular interactions of proteins lead to the emergence of the functional protein conformation, a process called folding. The energy landscape of folding is assumed to be funnel shaped, the stable form of the protein being at the bottom of the funnel with the lowest free energy state (Wolynes, 2015;Finkelstein et al , 2017).
The process of protein folding is obviously strongly dependent not only on general physical parameters, such as temperature and pressure, but on the quality and quantity of molecules present in the system. Water is the solvent of life and interactions with water molecules (Fogarty & Laage, 2014) are of key importance in all molecular interactions associated with life. Additionally, the concentration of hydrogen ions (pH), cations and anions and small molecules in water modulate interactions. Macromolecules influence interactions not only by taking part in the interactions but also by the excluded volume effect, restricting diffusional freedom (Zhou et al , 2008). An exact definition of the binding environment in the examined system is therefore indispensable for a realistic depiction of the binding energy landscape. Defining the antibody binding landscape in blood would therefore require at least a complete list of all constituents, and better involve abundance of each molecule.
Antibodies are globular glycoproteins secreted into the blood and other biological fluids by plasma cells (Nutt et al , 2015). Antibodies are actually a family of oligomeric proteins, with distinct constant regions that qualify them into classes and subclasses and with distinct variable domains that determine their binding specificity (Schroeder & Cavacini, 2010). While most of us think of antibodies as molecules with a well-defined specificity, in fact the majority of the circulating antibodies (especially of the IgM class) is not monospecific (specific to one target) but rather poly-specific and cross-reactive (Seigneurin et al , 1988;Kaveri et al , 2012). Any comprehensive systems approach to describe antibody function therefore must account for the presence of both highly specific and poly-specific antibodies. Our quantitative model of antibody homeostasis indeed attempts to provide a unified framework for the whole clonal humoral immune system (Prechl, 2017b). Antibodies are secreted by plasmablasts and plasmacells, descendants of B cells that had been stimulated by antigen. B cells are thus raised in an antigenic environment, the function of the immune system being the selection and propagation of B cells, which can respond to the antigenic environment. The essence of humoral immunity is therefore the definition and control of this antigenic environment by regulating molecular interactions. In thermodynamically terms, the aim of humoral immunity is to generate and maintain a binding energy landscape of antibodies suitable for sustaining molecular integrity of the host organism.
The binding fountain energy landscape
The funnel energy landscape is a theoretical approach used for the depiction of conformational entropy and free energy levels of one particular molecule (Bryngelson et al , 1995). Besides the description of intramolecular binding (folding) it can also be applied for the interpretation of homo-or heterospecific binding, such as aggregation or ligand binding (Zheng & Wang, 2015). If we tried to capture the network of antibody interactions by the binding funnel energy landscape we would face two interconnected problems, one deriving from antibody heterogeneity and the other from target heterogeneity. Antibody variable domains constitute the most diverse repertoire of all the proteins present in the organism, estimates being in the range of 10^9-10^11 different primary structures at any particular time of sampling, the hard upper limit being the number of B cells in a human body, around 10^12 cells (Bianconi et al , 2013). Even if the tertiary structures show orders of magnitude of lower diversity we still face an immense variability. On the other side, poly-specific antibodies bind to a multitude of targets, with upper limits to the number of targets being set only by experimentation. A combination of these two factors implies that the binding funnel approach would not allow a clearly comprehensible yet thorough description of antibody-antigen binding. To resolve this issue here we develop the concept of a binding fountain energy landscape model.
Free energy changes associated with molecular interactions can be resolved into components that act against or act in favor of binding. The loss of conformational entropy, a.k.a. entropic penalty, acts against binding while energies of non-covalent bonds (enthalpic component), hydrophobic effect (conformational entropy of water molecules) contribute positively. The net difference between these events determines binding energy and protein stability (Figure 1.). Conformational entropy loss of the antibody molecule thereby sets a minimum energy level that needs to be exceeded for any binding event to be stable. First, let us virtually collect all antibody binding events taking place in our system under examination, blood, and sort these events according to the entropic penalty of binding. For the sake of simplicity let us only consider entropic penalty of the variable domains of the antibodies. Second, let us plot free energy changes against conformational entropy. Since entropic penalty sets a minimum, all stable binding events should appear below the theoretical line representing a gradually increasing entropic penalty (Figure 1.). We can also set arbitrary limits for the free energy decrease, as the range of equilibrium constants for reversible antibody binding are known (Figure 1.B) and we can obtain Δ G from K eq by the equation: The resulting plot will show the distribution of binding energies against conformational entropy loss. This latter entity is itself associated with the number of atoms at the binding interface and the buried surface area (Marillet et al , 2017). Experimental evidence suggests that reversible binding is characterized by a range of energies, limits observed both for maximal and minimal values, which are dependent on the magnitude of the interacting surface, whether characterized by the number of atoms or by buried surface area (Brooijmans et al , 2002;Smith et al , 2012).
A binding funnel energy landscape focuses on the one native conformation that can be reached via various conformational routes as represented by a hypersurface of conformation, entropy and energy. Instead, we would like to focus on the several different conformational routes taken by several different antibodies while binding to different targets. To this end we assume that all native unbound antibodies enter our landscape at the top of the energy landscape plot. Here conformational entropic penalty is minimal, represents only that associated with folding. Conformational entropy of the antibody, which is the number of different microtrajectories its developing binding surface can take is the highest here. Moving down along any path will lead to loss of free energy and loss of conformational freedom. In order to get a better resolution of the binding landscape let us spin our two-dimensional plot around the energy axis at the maximal entropy to obtain a conical hypersurface in three dimensions ( Figure 2). Native unbound antibody molecules entering our landscape will move down along a path while interacting with their targets with an increasing binding energy. This gradual increase in Δ G is accompanied by an increasing involvement of the binding site, called antibody paratope. All stable binding events will take place under the theoretical conical surface generated from the stability barrier line. A binding path ends when the antibody finds its lowest state of energy, corresponding to binding to a target with the highest affinity. Where this point is located depends both on the antibody and the nature of its target (e.g. size, chemical characteristics). The hypersurface of conformations in the space of conformational entropy and free energy generated by this approach we shall call a binding fountain energy landscape.
While the conical surface enclosing stable binding events is a theoretical surface, we can obtain descriptors of real binding events by looking at subsets of events of the interaction space. By cutting the binding fountain horizontally at a given Δ G value we obtain the isoenergetic rim ( Figure 2B). The isoenergetic rim is the collection of binding events with identical Δ G and a range of corresponding Δ S. Thus, its Δ S distribution shows the range of entropic penalties that give rise to binding at the given Δ G in our system of study. By cutting the skirt of the cone at a given Δ S value we obtain the isoentropic rim ( Figure 2B). The isoentropic rim is the collection of binding events with identical Δ S and a range of corresponding Δ G values. Thus, its Δ G distribution shows the range of free energy changes and corresponding affinity values that give rise to binding at the given Δ S in our system of study. It shows how enthalpy and hydrophobic effects exceed entropic penalty. Please note that as these lines are derived from a hypersurface the lines are theoretical hyperlines themselves, comprising high-dimensional data that cannot be properly visualized in a simple 2D plot.
Projections of the fountain energy landscape
We have so far worked out an energy landscape interpretation tool, which helps map all the binding events that occur in a molecularly complex environment, such as blood. We assumed that antibodies secreted into the blood gain their native unbound conformations then engage in binding events of various energies until they reach their specific target. The path leading to thermodynamic equilibrium can be rugged, caused by less specific contacts, or smooth, with few intermediate binding states (Figure 3A). It is important to note, however, that blood is the most heterogenous biological fluid, comprising potentially all molecules found in the organism (Anderson & Anderson, 2002). Besides a huge number of secreted molecules any leakage from tissues, debris of cell death and foreign molecules may be present in blood. This vast molecular diversity generates a binding site diversity that we may assume to approach a randomized structural space, representing a huge number of variants of an antibody binding site that covers up to 3000 Å 2 (Marillet et al , 2017). Such a diverse binding space should approach a power law distribution of binding partners, with decay of partners as we increase binding energy or affinity ( Figure 3B) (Zheng & Wang, 2015). A rugged start is therefore expectable for all antibodies, with the path smoothing out depending on the paratope properties and the content of the binding landscape. As we approach higher energy and higher entropy loss regions the epitope "sharpens", as Irun Cohen termed (Cohen & Young, 1991) the gradually increasing affinity of antibodies ( Figure 3C). This sharpening involves both a gradually increasing buried surface area and better fitting surfaces and various combinations of these components. It is also apparent that sectors of conformational entropy contain structurally related binding sites, since sharpening reveals more details of epitopes that appear identical at lower resolution ( Figure 3D), later maturing into distinct conformational entities. This relationship also reflects the clonal relationship of antibodies going through affinity maturation, gaining sharper but constrained vision of targets by improving their fit (Kang et al , 2015).
Interpretation of antibody function as a system of regulated binding landscape
The binding landscape is the set of all potential interactions in a given fluid with given constituents, each interaction being positioned according to the entropic penalty, conformation and free energy decrease. In the binding fountain representation we can trace the fate of a particular antibody in time as a binding path ( Figure 4A) or display several different antibodies at an imaginary thermodynamic equilibrium ( Figure 4B). Owing to the fact that blood is a highly heterogenous fluid with a vast diversity of potential binding sites the frequency of low energy interactions is very high. At the tip of the fountain antibodies are "surfing" along the ripples of low affinity interactions. Moving down the surface they encounter interaction partners with gradually improved fit, spending more and more time in an interaction, until the target with best fit, that is highest free energy decrease and largest entropic penalty, is found ( Figure 4A).
Interaction in the blood cannot reach thermodynamic equilibrium; molecules are continuously entering and leaving this compartment. On the other hand, because of the constant turbulent mixing the distribution of molecules is constantly approaching homodispersity. Thus, we may display antibodies at an imaginary equilibrium where their position reflects their potential energy minimum in the system. This is where actually target antigen-bound antibody molecules are accumulating ( Figure 4B). Registering the position of all the copies of a given antibody species should show a distribution of bound forms determined not only by Δ G but also by the availability of the target molecules, that is antigen concentration [Ag]. The disappearance of the target ([Ag]≈ 0 M) will lead to the disappearance of the low energy position in the landscape. As a consequence, the antibody will accumulate in the interaction with the next available energy level albeit the ratio of bound to free form will be lower as dictated by the higher KD value. Alternatively, the antibody can search the neighboring conformational space along the isoenergetic rim for a binding site with similar Δ G. High concentrations of the target ([Ag]>>KD) will deplete antibody resulting in the potential overflow of related antibodies from the neighboring conformational space. The distance ΔΔ G between any two interactions has three components: a free energy component, a conformational component and an entropic penalty component. These components are perceptible from the side view, top view and both views of the binding fountain, respectively ( Figure 4B).
As suggested above, besides the presence of targets with a given Δ G the actual concentrations of both Ab and Ag determine the frequency of their interactions and the development of the imaginary equilibrium. To appreciate these factors we can project the interactions of a binding fountain into a space where the distance of the interactions is defined by ΔΔ G and the availability of antibody is expressed as the ratio of free antibody to the dissociation constant ([Ab]/KD). This value can be visualized as the radius of the circle representing the interaction ( Figure 4C). Please note that this value corresponds to [AbAg]/[Ag], the ratio of bound and free antigen concentrations. This is the network representation of antibody-antigen interactions as we recently described (Prechl, 2017b). A node with a larger radius also implies that the antibody is available for binding to targets in the conformational space in the vicinity of its cognate target molecule. The total combined volume of the nodes of the antibodies under investigation represents the binding capacity of these antibodies in the epitope conformational space.
The immune response as a regulated binding landscape
The adaptive immune system responds to an antigenic stimulus by the production of antibodies reacting with the eliciting antigen. In our binding landscape antigenic stimulus appears as an impression on the hypersurface representing antibody interactions, the position of the impression being determined by both the conformation of antigen and the conformation of fitting antibodies. The fact that an antigen can stimulate the humoral immune system implies that secreted antibodies that could efficiently bind to the antigen are not present. The antigen therefore binds to the membrane antibodies (B-cell receptors, BCR) of specific B cells ( Figure 5). If BCR engagement reaches a threshold the affected B cells proliferate, differentiate and secrete antibodies (Prechl, 2017a). Depending on the nature of the antigen, the route of entry into the host, the presence of costimulatory signals, the ensuing response can proceed basically in two forms. A thymus independent (TI) response will result in the generation of antibodies with binding properties identical to the parental B cell, since there is no affinity maturation. The structure of the binding site does not change, conformation, entropic penalty and Δ G of binding will be identical to the original interaction ( Figure 5A). These interactions take place in regions with moderate conformational entropy loss and high interaction frequency, meaning that of the huge repertoire of BCRs several will respond. The response appears as a standing wave, the appearance of antigen showing as the development of the impression, the response of antibody secretion as the disappearance of the impression as free antigen is replaced by bound antigen and immune complexes are removed. This kind of response seems suited for keeping concentrations of target molecules stable. We can think of the response as a closely knit elastic net that regains its original shape after applying pressure to a point ( Figure 5A). Thymus dependent (TD) responses will involve the affinity maturation of the antibody binding site, the sequential generation of antibodies with increasing affinity. As the binding site matures the entropic penalty and Δ G increase. The interactions will take place at different positions of the binding landscape ( Figure 5B). The response appears as a propagating wave sweeping down the slope of the binding fountain energy landscape. This wave is taking along the antigen, resulting in the efficient elimination of antigenic molecules.
It is important to note the relative identity of binding partners in this landscape: an antibody can bind to antigens but can also be the target of another antibody. The unique binding site of an antibody, the paratope that determines idiotype (identity as a binder), is itself part of the binding landscape. This can be especially important for antibodies with high intrinsic specificity rate (Zheng & Wang, 2015) that are eager to bind and reach their conformation with lowest energy level. We suggest that in the absence of antigen these high affinity binders could be refrained from non-specific binding by engaging their binding sites in lower affinity interactions.
Proposal
Our current systems biological approach to adaptive immunity can be basically divided into sequence and structure based methods. Next generation sequencing has made it possible to catalogue B-cells as defined by their rearranged heavy and light chain sequences. This approach has very high resolution, allowing the identification of hundreds of thousands of different cells in a sample. The pairing of the chains can also be identified by microfluidic and genetic techniques. Sequencing, in combination with immunogenetic databases and bioinformatic prediction tools, can therefore be used to follow resolve clonality and monitor diversity. The structural and binding properties of reconstituted recombinant antibodies can also be studied albeit on a much smaller scale. One inherent limitation of sequencing, besides intrinsic technological weaknesses, is the sampling bias: we cannot study a whole organism without destroying it, just a sample. In humans this is usually blood. As demonstrated above, the thermodynamic balance of humoral immunity works on a systemic level. Antibodies are produced throughout the body, not just by plasmablast circulating and captured in a blood sample. A systematic study and understanding of humoral adaptive immunity therefore needs different approaches as well.
The second, structural approach is the cataloguing of interactions. Databases devoted to collecting binding data exist but lack quantitative information. The proposed thermodynamic view of the humoral immune system, the network of antibody interactions, suggests that systems level understanding is only achievable by making rigorous quantitative measurements of binding and cross-reactivity. This will require novel technologies and the standardization of antibody and antigen validation. As for technologies, proteins will inevitably follow nucleic acids as subjects of high throughput, high resolution technologies. Standardization of antibody validation is a central issue in life sciences now, and it comes hand-in-hand with antigen standardization. Peptides and mimotopes are the likely candidates for these technologies because of their stability and reproducibility. Exploration of the lower, high-energy binding regions will also require native antigens, since replacement of these would require exact replicas of large interaction surfaces.
The benefits of a systems biological understanding, physical modeling of adaptive immunity would be far reaching. Besides the theoretical beauty observing an evolving cellular system, we expect practical applications in the biomedical fields, from vaccination through autoimmunity and allergy to cancer prevention and treatment.
Summary
Blood carries potentially all the molecules expressed in the host along with those originating from the environment. To ensure that all these molecules find their intended binding partners a regulated binding landscape evolved: the clonal immune system. The clonal humoral immune system generates a regulated binding landscape by constantly sampling the molecular environment via a huge repertoire of B-cell receptors and by the generation of antibodies with a wide range of specificities and affinities. To allow the thermodynamic representation of this multitude of interactions we show here that this landscape can be visualized as a binding fountain, in an analogy with the folding funnel energy landscape. The binding fountain landscape is an anchored conformation landscape with the conformational entropic penalty of binding anchoring the axis of free energy. Binding sites appear as impressions of a hypersurface, which represents thermodynamically favorable binding events with negative Δ G values. This landscape can be further projected into a multidimensional space of the antibody-antigen interaction network. Systemic perception and interpretation of antibody function is expected to help reveal how the immune system actually functions as a whole, a thermodynamic network of interactions, taking us closer to the understanding of so far underappreciated and less characterized functions of the clonal humoral immune system.
Conflicting interests
The author declares no conflicting interest. Figure 1 Energy landscape of binding.
Legends to figures
By anchoring the axis of free energy change at zero entropic penalty we normalize binding events. Any antibody entering the binding landscape appears at the top can search for binding partners with favorable thermodynamic characteristics (A). These binding events take place below the stability barrier, the line representing equality of entropic penalty and energies favoring binding. Theoretically we can collect and position all antibody interactions in this energy landscape (B).
Figure 2
Generation of the binding fountain energy landscape.
By spinning the former representation in figure 1 around the anchored axis we obtain a quasi conical surface. This surface encloses all stable binding events of an antibody molecule and is suitable for displaying a binding path (A). Thermodynamically defined subsets of binding events in the binding fountain can be obtained by looking at events in the isoenergetic or isoentropic rim (B).
Figure 3
Top view and properties of the binding fountain.
Sequential binding of a given antibody appears as a path with more or less rugged track (A). The frequency of interactions decreases by power law decay as we approach high energy binding with high entropic penalty (B). The levels of contact accounting for the entropic penalty increase by improved fit with stronger binding forces and by increased buried interface area (C). Conformations of binding surfaces share common origin with identical structural motifs closer to the "source" of the fountain, the region of low energy interactions (D).
Figure 4
Projections of the binding fountain.
An antibody entering the binding landscape engages in serial interactions with increasing energy, taking the molecule down a binding path (A). At an imaginary equilibrium natural antibodies (blue beads) and affinity matured thymus dependent antibodies (red beads) fill the holes of binding, arranged according to their conformation, entropic penalty and free energy level (B). The distance between any two binding events can be expressed as ΔΔ G, which represents the cross-reactivity of the two antibodies concerned. We can further project these events into an interaction space where a network is formed based on distance and binding capacity (C). Figure 5 | v3-fos-license |
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} | pes2o/s2orc | The Orai1 Store-operated Calcium Channel Functions as a Hexamer*♦
Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A “pore-inactive” subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative “trimer-of-dimers” channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.
The members of the Orai family of ion channels are ubiquitously expressed among cell types and mediate "store-operated" Ca 2ϩ entry signals, crucial in the control of many responses including gene expression, cell growth, secretory events, and cell motility (1)(2)(3). Orai channels are highly Ca 2ϩselective plasma membrane (PM) 4 channels activated through an elaborate intermembrane coupling mechanism by the Ca 2ϩsensing STIM proteins of the endoplasmic reticulum (ER) (1)(2)(3)(4)(5)(6). Alterations in the function of Orai channels and STIM proteins are implicated in a large number of immunological, muscular, and inflammatory disease states (1,(7)(8)(9). STIM1 undergoes a complex conformational rearrangement in the ER membrane in response to depletion of ER luminal Ca 2ϩ , and then translocates into discrete ER-PM junctions where it attaches to the PM surface and is able to directly tether and activate PM Orai1 channels (1, 2, 4 -6). The structural properties of Orai channels and STIM proteins and details of the molecular coupling they undergo are key to understanding their physiological activation, pharmacological modification, and pathophysiological role in disease states. Better understanding of the molecular coupling interface between STIM1 and Orai1 continues to emerge (10 -12), and significant advances are being made in discerning the mechanism by which Orai1 channels are gated by STIM1 (13)(14)(15). Recent studies have also provided a better understanding of the arrangement, dynamics, and stoichiometry of STIM proteins and Orai channels during their activation and coupling (16 -21).
Despite the many advances in identifying the molecular mechanisms of Orai channel function, considerable uncertainty still exists in some of the fundamental basic structural properties of Orai channels and how they become activated by STIM proteins. In particular, the multimeric assembly of the Orai1 channel, the most commonly expressed of the three-member mammalian Orai channel family, has remained a contentious issue (1,2,5,6,22,23). Recent crystallographic evidence reveals that Drosophila Orai has a hexameric subunit structure, a result reinforced by cross-linking and chromatographic evidence (22). The four transmembrane domains of Orai channels are exceedingly conserved from Drosophila to human, and the pore-lining residues revealed from the crystal structure coincide closely with residues predicted to lie in the pore from elegant structure-function studies (24 -27) that preceded the crystallization results. However, despite this congruity of structural understanding, several earlier studies revealed a fundamentally different, tetrameric subunit structure for Orai1. Thus, Shuttleworth and colleagues (28) compared the function of concatenated Orai1 channels (dimers, trimers, and tetramers) and concluded that the functional unit of the Orai1 channel is a tetramer. Shortly after, a number of other studies using a variety of approaches, including recovery after photobleaching, cross-linking, and electron microscopy, reported a similar tetrameric subunit structure for Orai channels (29 -33). Reports have also suggested that Orai channels may exist as dimers, which could associate into tetramers (29,33) or possibly hexamers (34). A seemingly more definitive study from Thompson and Shuttleworth (23) directly compared the functional channel properties of tetrameric and hexameric concatemers of Orai1. It was reported that only the Orai1 tetramer gave rise to the Ca 2ϩ -selective, inwardly rectifying, Ca 2ϩ releaseactivated Ca 2ϩ current (I CRAC ), the hallmark of authentic Orai1 channel function. In contrast, the hexameric concatemer gave an essentially non-selective cation conductance that was distinct from I CRAC , leading the authors to conclude that the hexameric Orai1 channel fails to replicate the function of endogenous CRAC channels (23).
Given the controversy in defining the functional Orai1 channel structure, we undertook a systematic investigation of the subunit configuration of the Orai1 channel. We generated a series of tdTomato-tagged concatenated Orai1 channel constructs containing from two to six Orai1 subunits. We expressed these concatemers in CRISPR-derived HEK cells in which endogenous Orai1 expression was eliminated. We confirmed that all of the Orai1 concatemers were PM-expressed and retained their concatemeric structure at the PM. Unexpectedly, the dimeric, trimeric, tetrameric, pentameric, and hexameric Orai1 constructs all mediated substantial store-operated Ca 2ϩ entry. Still more surprisingly, each one of the different concatemers mediated current with inward rectification and reversal potentials virtually identical to those observed with expressed Orai1 monomers. Replacement of specific subunits within each of the concatemers with Orai1 E106A "poredead" subunits revealed that the functional channels are predominantly formed from insertion of just the N-terminal pair of Orai1 subunits into the channel structure. The hexameric concatemer appears distinct from shorter concatemers in being able to at least partially form functional channels by itself. Our results argue strongly against a tetrameric configuration for Orai1 channels and provide evidence consistent with the conclusion that the functional Orai1 channel is hexameric.
Design and Expression of Orai1 Concatemers in a Null
Background-The recent controversy in understanding the molecular composition of Orai1 channels (22,23) led us to examine the function of concatenated multimers of Orai1. To ensure that there was no interference from endogenous Orai1 channels, we expressed the Orai1 concatemers in HEK cells in which the expression of endogenous Orai1 channels was eliminated through CRISPR/Cas9 gene editing. As shown in supplemental Fig. S1A, endogenous Orai1 was entirely absent in these cells. The Orai1-deleted HEK cells (HEK-O1 ko ) were transfected to stably express STIM1-YFP (HEK-O1 ko S1 ϩ cells) (supplemental Fig. S1, B and C). Using fura-2-loaded HEK-O1 ko S1 ϩ cells, the depletion of Ca 2ϩ stores with 2.5 M ionomycin resulted in no detectable Ca 2ϩ entry in Ca 2ϩ add-back experiments (supplemental Fig. S1D). The lack of any storeoperated Ca 2ϩ signals in these STIM1-overexpressing storedepleted cells reveals that there is no detectable Ca 2ϩ entry mediated by Orai1, Orai2, or Orai3 in the cells.
We made a series of Orai1 concatemers comprising two, three, four, five, or six subunits of Orai1 joined together with intervening 36-amino acid linker sequences (supplemental Fig. S2A). Each concatemer was tagged with the high fluorescence intensity tdTomato protein at the C terminus. Using the HEK-O1 ko S1 ϩ cells, we compared the plasma membrane expression and function of each of these Orai1 concatemers as well as tdTomatotagged Orai1 monomer. Deconvolved z axis images revealed that each tagged Orai1 construct was expressed at the PM (Fig. 1A). We verified this by imaging cells 10 min after plating and prior to cells spreading, which provides enhanced PM visibility (supplemental Fig. S2B). It was important to assess whether the PM-expressed surface Orai1 concatemeric proteins were authentic in size or had undergone alteration or degradation. Cells expressing Orai1 monomer or each of the Orai1 concatemers were biotinylated, and then surface proteins were isolated with NeutrAvidin resin, and following elution, assessed by Western analysis with an Orai1 antibody as shown in Fig. 1B. Each construct was observed as a discrete protein with molecular mass corresponding closely to the calculated size of the tdTomato-linked concatemers (see the legend for Fig. 1B). We also examined the proportion of expressed Orai1 concatemeric protein in the non-biotinylated state, which appeared in the NeutrAvidin resin flow-through, corresponding to the non-PM-expressed concatemer. As shown in Fig. 1C for the Orai1 hexamer, considerably more protein (ϳ70% of total) is expressed on the cell surface when compared with that within the cell.
Orai1 Concatemers Function Identically Regardless of Subunit Length-Having established the authentic cell surface expression of each Orai1 concatemer, we examined their function. Surprisingly, in each case (dimer, trimer, tetramer, pentamer, and hexamer), expression of the tagged Orai1 constructs resulted in large and comparable store-operated Ca 2ϩ entry, which matched almost exactly the rate and extent of Ca 2ϩ entry observed with the Orai1 monomer (Fig. 2, A and B). Non-transfected cells in each case showed no Ca 2ϩ entry. Considering previous results (23) in which only the Orai1 channel concate-meric tetramer was reported to give rise to authentic CRAC channel function, we examined the current properties of each construct. As shown in Fig. 2C, the Orai1 monomer and each of the five concatemeric Orai1 constructs all gave rise to similar levels of authentic CRAC current with the same inwardly rectifying properties that define the operation of Orai1 channels (1). Indeed, from numerous recordings with each construct, the reversal potential for the inward current mediated by all five Orai1 concatemers lay within a narrow range (51-58 mV) ( Fig. 2D) with no significant difference from that observed with monomeric Orai1 (p Ͻ 0.05).
In a previous study, dimeric and trimeric concatemers of Orai1 were observed to give rise to CRAC current, albeit smaller than the CRAC current observed with tetramer expres-sion (28). It was concluded that the dimers and trimers could give rise to current through incorporation of monomers of endogenous Orai1 channel subunits to form tetramers. However, in the present case, we undertook all our experiments in the HEK-O1 ko S1 ϩ cells in which endogenous Orai1 was eliminated, and there was no functional detection of any other Orai channel activity. Thus, the currents observed are attributable to the expressed constructs alone and not any combinations with endogenous Orai1 channels. The more recent report from Thompson and Shuttleworth (23) indicated that a hexameric Orai1 concatemer gave a current-voltage relationship very different from authentic CRAC channels. In that report, the hexameric construct was observed to have less inward rectification and prominent outward currents at reversal potentials of ϩ20 mV or above. In contrast, the tetrameric Orai1 construct in the same study gave rise to the strong inwardly rectifying current properties of an authentic CRAC current, very different from the properties of the hexamer. As shown in Fig. 2, C and D, we observe almost identical authentic CRAC current properties for both tetrameric and hexameric constructs, a result that is very different from the previous study (23). One possible difference could be the linker segments used: the earlier study used a 6-amino acid linker, whereas ours is 36 amino acids and is the same as that used to successfully attach STIM1 fragments to the Orai1 channel (16). It is possible that the longer linker could permit a more authentic configuration to be achieved in the multimers. However, in the earlier study (23), why there should have been a differential detrimental effect of a short linker only on the hexamer is difficult to reconcile. Also, in the previous studies and our own studies, the linkers are placed immediately upstream of the Orai1 N terminus of each conjoined Orai1 subunit. This N-terminal segment has ϳ70 amino acids that are redundant in the function of Orai1 channels (35,36), and secondary structure analysis reveals little predicted secondary structure. These flexible 70 amino acids may therefore assist in linking the Orai1 units, making it still less clear why the 6versus the 36-amino acid linker would selectively affect the function of Orai1 concatemers.
E106A Pore-dead Subunit Substitution in Orai1 Dimers and Tetramers-Next we sought to understand what could possibly allow all the concatemeric Orai1 constructs to function almost identically, and what we can determine about the nature of the functional Orai1 channel. The crystal structure of Drosophila Orai reveals it to be hexameric (22), and from its sequence similarity in critical transmembrane regions, the human Orai1 likely has a similar structure. We undertook functional studies on Orai1 concatemers in which we introduced one or more Orai1 residues that contained the E106A mutation. The Glu-106 residue is the crucial ion selectivity filter on the external surface of the channel pore (1), and its mutation to alanine completely prevents ion conductance (37). Initially, we substituted mutated Orai1 E106A subunits within the Orai1 dimer (Fig. 3A). Not surprisingly, mutation of either the first or the second subunit in the dimer to E106A (these constructs are termed "XO" or "OX," respectively) or mutation of both subunits in the dimer (XX) in each case almost completely prevented the function of the Orai1 dimer. Orai1 concatemers localize to the plasma membrane and exist in there with molecular masses corresponding to those predicted by their sequence. A, deconvolved fluorescence images of HEK-O1 ko S1 ϩ cells expressing tdTomato-tagged Orai1 monomer, dimer, trimer, tetramer, pentamer, or hexamer constructs revealing their plasma membrane localization. B, Western blot of surface-isolated proteins isolated from the HEK-O1 ko S1 ϩ cells expressing tdTomato-tagged Orai1 monomers, dimers, trimers, tetramers, pentamers, or hexamers shown in A. Cells were biotinylated, and surface proteins were isolated with NeutrAvidin resin, eluted with DTT-containing sample buffer, and assessed by Western analysis with Orai1 antibody. The calculated molecular masses for tdTomato-linked Orai1 concatemers are: 91.2 kDa (monomer); 127.1 kDa (dimer); 163 kDa (trimer); 198.9 kDa (tetramer); 234.8 kDa (pentamer); 270.7 kDa (hexamer). C, comparison of the surface (PM) expression of Orai1 hexamer with that expressed elsewhere in the cell. Western blotting analysis used an Orai1 antibody to detect surfacebiotinylated (left) and non-biotinylated (right) Orai1 protein derived from HEK-O1 ko S1 ϩ cells expressing tdTomato-tagged Orai1 hexamer. Following biotinylation of cells, lysates were applied to NeutrAvidin resin. The flowthrough lane is the fraction that was not retained on NeutrAvidin resin. The surface protein lane is the fraction eluted with sample buffer/DTT after binding to NeutrAvidin resin. Details are given under "Experimental Procedures." We also introduced E106A subunits into the Orai1 tetramer. Unexpectedly, the introduction of two E106A-mutated residues into the Orai1 tetramer caused entirely opposite results depending on the position of the mutated units. If the two N-terminal Orai1 residues were mutated to E106A (XXOO), the resulting construct was devoid of any Ca 2ϩ entry ( Fig. 3B) or CRAC current (Fig. 3C). In contrast, if the two C-terminal Orai1 residues were mutated (OOXX), then the construct retained full Ca 2ϩ entry and normal CRAC current activity (Fig. 3, B and C). We considered whether the tdTomato tag at the C terminus would somehow influence the ability of these Orai1 constructs to assemble into functional channels. To test such possible directionality, we constructed similar Orai1 concatemeric tetramers that were N-terminally tagged with tdTomato. As shown in Fig. 3D, these constructs gave virtually identical results, the XXOO constructs having no function and the OOXX constructs mediating full Ca 2ϩ entry indistinguishable from the OOOO construct. An earlier report (38) had examined the effects of substituting Orai1 concatenated tetramers with subunits containing the pore-inactive R91W mutation found in patients with severe combined immunodeficiency disease. The results were shown only for an "XXOO" version of the substituted concatemer, and revealed that it had essentially negligible function in agreement with our results. However, in that study, the "OOXX" version of the concatemer was not examined.
Substitution with E106A in the Orai1 Hexamer-Because we know that E106A has a powerful and dominant inhibitory effect on Orai1 channel function, we interpreted these results to suggest that the two distal (C-terminal) residues of the tetramer might not be participating in formation of the functional channel. We also considered that, if the real functional Orai1 channel is a hexamer, the substitution of E106A units in the hexameric concatemer might have different functional consequences than in the tetrameric concatemer. We therefore compared the effects of substituting a single E106A unit in the first two positions of the concatemeric Orai1 hexamer as opposed to the Orai1 tetramer. As shown in Fig. 4A, a single E106A unit in the tetramer, regardless of whether in the first or second position (XOOO or OXOO), almost completely blocked store-operated Ca 2ϩ entry (SOCE). In contrast, substitution of E106A in either of the first two positions of the hexamer gave constructs with much more activity (Fig. 4, B and C). Substituted at the first position (XOOOOO), SOCE was observed to be ϳ15% of the wild-type hexameric channel. Substituted at the second position (OXOOOO), the channel was ϳ35% as functional as the wild-type hexamer (OOOOOO). This indicates that the hexamer is behaving quite differently from the tetramer. A comparison of the relative Ca 2ϩ entry for each of the first or second E106A-substituted dimers, tetramers, and hexamers is shown in Fig. 4C, from which it is clear that the hexamer is a special case. FIGURE 2. Orai1 dimeric, trimeric, tetrameric, pentameric, and hexameric concatemers mediate store-operated Ca 2؉ entry and CRAC current indistinguishable from that mediated by Orai1 monomer. tdTomato-tagged Orai1 concatemer constructs were transiently expressed in HEK-O1 ko S1 ϩ cells. A, fura-2 ratiometric Ca 2ϩ add-back measurements for cells transfected with the concatemers shown. Ca 2ϩ stores were released upon the addition of 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). Traces (means Ϯ S.E.) are representative of three independent experiments. B, summary scatter plots with means Ϯ S.D. for peak Ca 2ϩ entry of all individual cells recorded in three independent experiments with the Orai1 concatemer constructs shown in A. C, representative I/V plots from whole-cell recordings from HEK-O1 ko S1 ϩ cells transiently expressing each of the tdTomato-tagged concatenated Orai1 constructs shown in A. D, summary statistics of the reversal potential measurements for each Orai1 concatemer construct described in C. Scatter plots are means Ϯ S.D. for all the cells recorded in three independent experiments. DECEMBER 9, 2016 • VOLUME 291 • NUMBER 50
Orai1 Functions as a Hexamer
In further experiments, we examined a series of hexamers in which we substituted a single E106A unit at the third (OOXOOO), fourth (OOOXOO), fifth (OOOOXO), or sixth (OOOOOX) positions in the hexamer. The results shown in Fig. 5A reveal that each of these concatemers (especially with X at the fourth, fifth, and sixth positions) had substantial Ca 2ϩ entry. A summary of results for all the single X mutations in the Orai1 hexameric concatemer is shown in Fig. 5A (right). These results indicate that the position of the E106A unit does not make that much of a difference, with the exception of the first place (XOOOOO), which shows rather little function (Fig. 4, B and C). This is important information indicating that all the residues in the expressed Orai1 hexameric concatemer can contribute to its channel function. We continued investigation of the hexamer by substituting two adjacent E106A units at three different positions (Fig. 5B). When the E106A units were at the first and second position in the hexamer (XXOOOO), there was almost no function. However, when the E106A units were at the third and fourth positions (OOXXOO) or at the fifth and sixth positions (OOOOXX) in the hexamer, there was again substantial Ca 2ϩ entry function.
Co-expression of Orai1 E106A Monomers with Orai1 Tetramers and Hexamers-In an earlier report, the Orai1 tetramer was reported to be uniquely resistant to the dominant negative effects of co-expressing the pore-dead Orai1 monomer, E106Q (28). Thus, it was argued that the Orai1 tetramer was a structurally intact channel that could resist Orai1 monomer insertion, unlike other concatemers (for example, the Orai1 trimer) that were blocked as a result of Orai1 E106Q monomer incorporation. In contrast, we observed that the Orai1 E106A monomer co-expressed with the Orai1 tetrameric concatemer substantially blocked the function of the latter, and also had the same inhibitory effects on the Orai1 hexamer (Fig. 6, A and B). To be sure that this effect did not result from competition of the E106A monomer for STIM1 binding, we introduced the L273D mutation (which completely eliminates STIM1 binding (16, 39)) into the E106A monomer. This Orai1 E106A/L273D monomer still substantially blocked the func- . Orai1 pore-inactive E106A subunit substitution in concatenated Orai dimers and tetramers. A, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 dimer constructs shown. Ca 2ϩ stores were released upon the addition of 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). Traces are for wild-type Orai1 dimer (OO, red), dimer with Orai1 E106A substitution at the first (N-terminal) position (XO, green), second (C-terminal) position (OX, blue), E106A substitution at both positions (XX, orange), or untransfected control cells (black). B, fura-2 ratiometric Ca 2ϩ responses as in A with cells expressing similar levels of tdTomato-tagged Orai1 tetrameric concatemers, including the all-wild-type Orai1 tetramer (OOOO, red), Orai1 tetramer with the first subunit pair E106A-mutated (XXOO, blue), Orai1 tetramer with the second subunit pair E106A-mutated (OOXX, green), or untransfected control cells (black). C, representative I/V plots from whole-cell recordings from HEK-O1 ko S1 ϩ cells transiently expressing each of the tdTomato-tagged concatenated Orai1 tetramer constructs shown in B. Summary scatter plot (means Ϯ S.D.) of reversal potential measurements taken from multiple I/V curves for the OOOO and OOXX constructs, as shown. Current for the XXOO construct was essentially zero. D, fura-2 ratiometric Ca 2ϩ responses as in B with cells expressing similar levels of tetrameric concatemers, but instead, all tagged with tdTomato at the N terminus. Traces are for the all-wild-type Orai1 tetramer (OOOO, red), Orai1 tetramer with the first subunit pair E106A-mutated (XXOO, blue), Orai1 tetramer with the second subunit pair E106A-mutated (OOXX, green), or untransfected control cells (black). For each of the results shown in A, B, and D, traces (means Ϯ S.E.) are representative of three independent experiments. In each case, summary scatter plots with means Ϯ S.D. of the percentage of peak Ca 2ϩ entry relative to wild type peak Ca 2ϩ entry are shown for all the cells derived from three independent experiments. tion of both the tetramer and the hexamer (Fig. 6, C and D). Thus, neither the tetramer nor the hexamer has any special exclusionary properties that lessen sensitivity to insertion and blockade by the monomer, a result further militating against the conclusion that the tetramer is the functional Orai1 channel configuration.
A Model for the Functional Assembly of Orai1 Channel Concatemers-The picture emerging from our concatemer studies shows a substantial difference between the operation of the dimer, tetramer, and hexamer. For dimers, both residues are essential and equal in their role in forming channels. For the tetramer, there is a complete distinction in the contribution of the first two (N-terminal) as opposed to the second two (C-terminal) subunits. In the hexamer, although all of the subunits can contribute to channel function, they do not contribute equally, and the double "XX" results reveal that the first two (N-terminal) subunits are more critical than the subsequent four residues. Based on the tetramer data, we conclude that channels are being formed by the concatemer in which some of the Orai1 subunits remain external from the functional channel core. Based on the strong crystallographic evidence that the channel is a hexamer (22), we postulate that the probable functional configurations of the dimers, tetramers, and hexamers are as shown in Fig. 7.
With the dimers, we assume that three dimers can assemble into a hexamer as a "trimer-of-dimers" (Fig. 7A). If both subunits in the dimer are E106A, there is no possibility of channel function. If one of the units in the dimer is E106A, there would be three E106A units in the hexamer, and this would likely also result in a channel with very little function. Indeed, the results with OX and XO (Fig. 3A) reveal very small Ca 2ϩ entry, but slightly more than observed with XX, which cannot form any functional channel protein. The lack of function of the XO and OX dimers indicates that functional channels are not formed from the assembly of six dimers each contributing just one functional monomer to the hexameric assembly. Thus, we conclude that the Orai channel may be assembled from dimers, in agreement with other studies (29,33,34) as well as with the crystallographic structure that reveals that Orai channels comprise a trimer-of-dimers (22).
With concatemeric tetramers, substitution with E106A at the first two N-terminal positions (XXOO) results in virtually no Ca 2ϩ entry (Fig. 3B). However, substituted at the third and fourth positions (OOXX), the channel has the same function as wild-type OOOO. Clearly, these two C-terminal residues of OOXX are not contributing to the hexamer and are likely extending out of the hexamer as depicted in Fig. 7B. Thus, we conclude that the N-terminal pair of residues in the tetramer is fed into a hexameric structure as a trimer-of-dimers, whereas the C-terminal pair remains external to the channel. Remarkably, the pair of subunits feeding into the hexamer is not altered by the position of the tdTomato tag. Thus, when we take this large fluorescent tag and place it on the N terminus instead of the C terminus of the Orai1 tetramer, the functional results with the tetramers are exactly the same (Fig. 3D). Hence, the dimers of Orai1 still feed into the hexamer from the N-terminal end, despite the presence of the bulky tdTomato residue immediately adjacent to the N terminus. This is not so unexpected because tdTomato-tagged Orai1 monomers themselves can form functional channels (Fig. 2) with tags present on each Orai1 unit in the hexameric channel.
If we did not have the hindsight of the hexameric channel crystal structure (22), would our results with the dimers and tetramers be consistent with the previously proposed Orai1 tetramer model (23,28)? The dimer data would be explainable because if the dimers were simply combining to form tetrameric channels, the OX and XO constructs would have no function. However, our results with the tetramers reveal that they are clearly not forming simple tetrameric ring structures as concluded in the earlier studies (23,28). Thus, there would be no explanation for the completely opposite effects of OOXX as opposed to XXOO (Fig. 3, B-D) under the tetramer theory. Also, the XOOO and OXOO tetramers might be expected to FIGURE 4. The Orai1 tetrameric and hexameric concatemers differ in the effects of substituting Orai1 pore-inactive E106A subunits at the first or second positions. A, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar fluorescence levels of the C-terminal tdTomato-tagged Orai1 tetramers shown. Ca 2ϩ stores were released upon the addition of 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). Left, traces are for wild-type Orai1 tetramer (OOOO; red) or Orai1 tetramer substituted with E106A at the first position (XOOO, orange). Middle, traces are for wild-type Orai1 tetramer (OOOO, red) or Orai1 tetramer substituted with E106A at the second position (OXOO, purple). Untransfected control cells are shown for each experiment (black). Traces (means Ϯ S.E.) are representative of three independent experiments. Right, summary scatter plots of peak Ca 2ϩ entry relative to wild type; results are means Ϯ S.D. of all cells derived from three independent experiments. B, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca 2ϩ measurements were as in A. Left, traces are for wild-type Orai hexamer (OOOOOO, red) or Orai1 hexamer substituted with E106A at the first position (XOOOOO, turquoise). Right, traces are for wild-type Orai1 hexamer (OOOOOO, red) or Orai1 hexamer substituted with E106A at the second position (OXOOOO, brown). Untransfected control cells are shown for each experiment (black). Traces (means Ϯ S.E.) are representative of three independent experiments. C, summary scatter plots of peak Ca 2ϩ entry relative to wild type; results are means Ϯ S.D. of all cells from three independent experiments. The summary results for the XO and OX dimers from Fig. 3A are included for comparison. DECEMBER 9, 2016 • VOLUME 291 • NUMBER 50 have some function in a ring tetramer because only one out of the four subunits is deficient. However, these constructs are almost devoid of function (Fig. 4A).
Orai1 Functions as a Hexamer
Our results with the hexameric concatemers make a strong argument that the functional Orai1 channel is a hexamer, supporting the crystallographic identification of a hexameric channel structure. Thus, in the hexamers, substitution with a single E106A at any of the positions in the hexamer reduces function. However, the hexamer can still have good channel function, which would be expected with a single E106A substitution. Therefore, we conclude that the hexamer can indeed form a single ring that functions as a channel as shown in Fig. 7C. However, it seems that this is not the only functional assembly, and that the concatemeric hexamer can also form a functional trimer-of-dimers species equivalent to that formed by the tetramer, but now with four Orai1 units extending out from the hexameric core (Fig. 7C). This conclusion is strongly supported by the function of the double E106A-substituted hexamers shown in Fig. 5B. Thus, E106A subunits in the first two positions (XXOOOO) result in almost no channel function, whereas a pair of E106A units in the third and fourth, or fifth and sixth positions, results in significant channel function. This conclusion is reinforced by the observation that single E106A substitutions at different positions in the hexamer are not equal, those at the N terminus having more effect than those at the C terminus (Fig. 5A, right). If all the hexamer were in the ring configuration, there would be no positional difference observed with single E106A substitutions. If all the hexamer were in the trimer-of-dimers configuration, the two XOOOOO and OXOOOO constructs would have little activity. Thus, our hexameric concatemer is likely forming a combination of both configurations. Importantly, the hexamer is a special case and is able to tolerate a single X at the first (XOOOOO) or second position (OXOOOO) because at least some fraction of the hexamer can form as a hexameric ring. This sets it apart from the tetramer, which is unable to form a channel with a single X in either of the first two positions (XOOO, OXOO), because in contrast to the hexamer, the tetramer cannot form a complete ring assembly by itself.
The observation that Orai1 hexamers can function with one or more pore-inactive subunits has relevance to the expression of pore-inactive mutations (for example, R91W) in severe combined immune deficiency (SCID) patients. Ca 2ϩ entry responses in T cells derived from heterozygous disease carriers were relatively normal although diminished under conditions of lowered external Ca 2ϩ (40). Whether structures excluding pore-inactive subunits may result in the relatively normal behavior of Orai1 channels in the heterozygous case is an interesting possibility.
The Selectivity Filter Glu-106 Appears Critical in Orai1 Channel Assembly-Interestingly, increasing the number of E106A units beyond two provided some further perspective on FIGURE 5. Substitution of pore-inactive E106A subunits in the Orai1 hexamer reveals that all the subunits in the hexamer contribute to channel function. A, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar fluorescence levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca 2ϩ stores were released upon the addition of 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). In each case, traces for wild-type Orai1 hexamer (OOOOOO, red) are compared with traces for Orai1 hexamer substituted with E106A at the third (OOXOOO, orange), fourth (OOOXOO, gray), fifth (OOOOXO, blue), or sixth position (OOOOOX, purple). Untransfected control cells are also shown (black). B, Ca 2ϩ add-back measurements were as in A using hexamers substituted with pairs of E106A residues. In each case, traces for wild-type Orai1 hexamer (OOOOOO, red) are compared with traces for Orai1 hexamer substituted with two E106A subunits at the first and second (XXOOOO, purple), third and fourth (OOXXOO, green), or fifth and sixth position (OOOOXX, blue). Untransfected control cells are also shown (black). Traces (means Ϯ S.E.) are representative of three independent experiments. Note that for the OOOOXO data (A, third panel), the wild-type trace (OOOOOO) is the same as that shown for the OXOOOO data (Fig. 4B, right panel) because the data were obtained in the same experiment. In each case, summary scatter plots of peak Ca 2ϩ entry relative to wild type are shown; results are means Ϯ S.D. of all cells from three independent experiments.
Orai1 Functions as a Hexamer
the function and assembly of the Orai1 channel. E106A units in the first three positions of the hexamer (XXXOOO) resulted in a nonfunctional channel (Fig. 8, A and C). Similarly, E106A substituted at the second, fourth, and sixth positions (OXOXOX) resulted in no function. These results would be expected, because regardless of the two alternative functional hexameric structures shown in Fig. 7C, these concatemer constructs would result in at least three E106A subunits in the channel and would therefore have little function. When we expressed the hexamer with three E106A subunits in the fourth, fifth, and sixth positions (OOOXXX), we obtained small but significant Ca 2ϩ entry (Fig. 8, D and F). This would again be expected because some functional channel likely arises through the trimer-of-dimers structure shown in Fig. 8C, whereas any ring structure would be nonfunctional by virtue of the three E106A units. Quite unexpectedly, introducing a fourth E106A residue into this hexamer (OOXXXX) resulted in high Ca 2ϩ entry, almost identical to that of the complete wild-type hexamer (OOOOOO) (Fig. 8, E and F). Thus, there is an extraor-dinary recovery of function by simply replacing the third subunit of the OOOXXX hexamer with E106A. This result appears counter-intuitive because the addition of a further non-functional channel subunit would be expected to further reduce rather than dramatically increase channel function.
This unexpected result has a most interesting possible rationale and may provide information about how the Orai1 channel assembles. We interpret the result to indicate that the OOXXXX construct has been forced to exist exclusively in the trimer-of-dimers configuration. Because the Glu-106 residue is itself the crucial Ca 2ϩ binding selectivity filter of the Orai1 channel pore, we postulate that by binding Ca 2ϩ , this residue may also function to instigate the hexameric assembly of the Orai1 channel around a central Ca 2ϩ ion. We would assume that the OOOOOO hexamer would favor formation of the ring structure, although we do not know the proportion of ring versus trimer-of-dimers species present with that construct. As more E106A units are added to the C terminus, the channel not only becomes less conductive as a hexameric ring, but may also FIGURE 6. The function of the Orai1 tetramer or Orai1 hexamer is substantially reduced by co-expression of pore-inactive Orai1 E106A monomer, and this effect is independent of any STIM1 binding of the monomer. A, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 tetramer (left) or hexamer (right), either with (blue traces) or without (red traces) co-expression of Orai1 E106A monomer. Ca 2ϩ stores were released with 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). In each case, traces (means Ϯ S.E.) are representative of all cells in two independent experiments. B, summary scatter plots of peak Ca 2ϩ entry in the presence of E106A monomer when compared with Ca 2ϩ entry without monomer; results are means Ϯ S.D. of three independent experiments represented in A. C, exactly the same experimental design as in A except that the monomer used is the double mutant, Orai1 E106A/L273D, which is devoid of any ability to bind STIM1. Traces (means Ϯ S.E.) are representative of three independent experiments. D, summary scatter plots of peak Ca 2ϩ entry in the presence of E106A monomer when compared with Ca 2ϩ entry without monomer; results are means Ϯ S.D. of all cells in two independent experiments represented in C. Note that the levels of expression of tdTomato-tagged Orai1 tetramer or hexamer were not affected by Orai1 E106A monomer expression, and in each experiment, the level of tdTomato fluorescence, with or without monomer expression, was the same. DECEMBER 9, 2016 • VOLUME 291 • NUMBER 50 become less susceptible to forming a ring nucleated by a Ca 2ϩ ion. By default, this would favor assembly of trimer-of-dimers species. With OOOOXX or OOOXXX, there may still be sufficient driving force for some ring formation, but such rings are with little or no channel function. Thus, three or four N-terminal O's might still be effective in preventing the trimer-of-dimer assembly as a result of promoting ring formation. However, with the OOXXXX structure, there is now no driving force for ring formation, and the assembly of trimer-of-dimers is strongly favored.
Orai1 Functions as a Hexamer
Concluding Comments-Our results provide strong support for the hexameric structure of the Orai1 channel. Moreover, our studies contradict previous studies that were interpreted to favor the tetramer model. First, concatemeric hexamers and tetramers both give rise to authentic CRAC currents, identical in properties to those mediated by the expression of Orai1 channels in native cells (Fig. 2). This directly refutes the previous evidence (23) that the tetramer is functionally distinct from the hexamer in mediating CRAC channel activity. Second, the function of dimers, trimers, and other multimers of Orai1 does not require the presence of endogenous Orai1 subunits to reconstitute functional CRAC channel activity as suggested earlier (28); thus, we observe the function of all Orai1 multimers in cells devoid of endogenous Orai1. Third, our experiments disprove the earlier report that the Orai1 tetramer is uniquely resistant to the dominant negative effects of co-expressing the pore-dead Orai1 monomer, E106Q (28).
Overall, our results provide functional data for the Orai1 channel that are highly consistent with the compelling structural data (22) from Drosophila Orai, indicating that the Orai1 channel is a hexamer. The crystal structure reveals that Drosophila Orai exists as a trimer-of-dimers, with each pair held together by hydrophobic interactions between the C termini of each of the two Orai subunits in the dimer. Such an assembly is consistent with other reports that indicate Orai channels are assembled from dimers (29,33). Indeed, recent data indicate that PM dimers predominate in the resting state (90%) and can assemble into multimers up to hexamers, upon store depletion (34). This lends support to the evidence presented here that several combinations of concatemers may form hexamers through the assembly of dimers. When this happens, it seems that the dimers are always donated from the N terminus of concatemers. The presence of the E106A mutation may not only prevent the function of channels formed in this way, but it may also actively prevent the assembly of channels. This explains why the N-terminal Orai1 subunit in the concatemeric hexamer is the most susceptible to mutation (Figs. 4 and 5). We speculate that Ca 2ϩ binding to Glu-106 in this subunit may mediate a "seeding" role in Orai1 channel assembly; thereafter, the bound Ca 2ϩ functions as a template for channel assembly as described above. The strong inhibitory action we observe of E106A monomer co-expression with tetramers or hexamers (Fig. 6) may therefore reflect its dominant role in preventing channel assembly as well as channel function. Clearly, investigation of the subunit assembly of Orai1 channels will be an important area for future investigation.
Experimental Procedures
DNA Constructs-The human Orai1 with 36-amino acid linker sequence was derived by PCR from the Orai1 S-S constructed previously (16). The DNA sequence was inserted into the pEGFP-N1 vector with the BamHI and XhoI restriction sites. The original EGFP tag was deleted using the BamHI and NotI restriction sites, and the tdTomato tag from ptdTomato-N1 with the same restriction sites was inserted into the construct to give ptdTomato-N1-Orai1. For the concatenated Orai1 dimer, the ptdTomato-N1-Orai1 was digested by BamHI and XhoI to obtain the Orai1-linker fragment, and this fragment was inserted into the same plasmid digested by BamHI and SalI. The trimer, tetramer, pentamer, and hexameric concatemers were constructed by repeating these digestion and ligation steps. Orai1 E106A mutations were derived using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent; 210518).
Generating Human ORAI1 Knock-out Cell Lines Using the CRISPR-Cas9 Nickase System-Orai1 sequence-specific guide RNAs were inserted into the lentiCRISPR V2 vector (Addgene 52961) with the BsmBI restriction site to create a gRNA-Cas9encoding plasmid. HEK cells were transfected with the gRNA-Cas9 plasmid using the cell line Nucleofector Kit (Lonza, VCA-1003) and the Amaxa Biosystems Nucleofector protocol (Lonza) according to the manufacturer's protocol. 48 h after transfection, cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin containing puromycin (2 g/ml) (Gemini Bio Products, West Sacramento, CA) in 5% CO 2 at 37°C. 6 days after puromycin selection, cells were collected and seeded at one cell per well into 96-well plates. Disruption of the ORAI1 gene in individual colonies was detected using the Guide-it Mutation Detection Kit (Clontech Laboratories, 631443) and confirmed by sequencing, as well as Western blotting and functional responses FIGURE 7. Model of the possible assembly of concatenated Orai1 constructs to form functional hexameric channels. The model depicts the ability of concatenated Orai1 dimers (A) or tetramers (B) to form functional hexameric channels through the insertion of three N-terminal dimers into a hexameric trimer-of-dimers complex. The concatenated Orai1 hexamer is distinct from the dimer and tetramer in being able to form a hexameric ring (C) in which all six of the subunits contribute to the function of the channel. The data suggest that the hexamer can additionally form a trimer-of-dimers complex similar to that formed by the tetramer. Details of the evidence and logic favoring formation of these functional assemblies are given under "Results and Discussion." 36-aa linker, 36-amino acid linker.
Cell Culture and Transfection-The stable HEK-O1 ko S1 ϩ cell line expressing STIM1-YFP was derived from HEK-O1 ko cells using the transfection and selection methods described previously (41,42). Cells were cultured in DMEM (Corning Cellgro) supplemented with 10% fetal bovine serum, penicillin, and streptomycin containing puromycin (2 g/ml) (Gemini Bio Products) at 37°C with 5% CO 2 . All transfections were performed by electroporation at 180 V, 25 ms in 4-mm cuvettes (Molecular Bio-Products) using the Bio-Rad Gene Pulser Xcell system in OPTI-MEM medium as described previously (43). Experiments were all performed 18 -24 h after transfection.
Cytosolic Ca 2ϩ Measurements-Cytosolic Ca 2ϩ levels were measured as described earlier (44) by ratiometric imaging using fura-2 (Molecular Probes). Cells were used 18 -24 h after transfection, and then incubated with 2 M fura-2 in buffer containing: 107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl 2 , 1 mM CaCl 2 , 11.5 mM glucose, 0.1% BSA, 20 mM HEPES, pH 7.2, for 30 min at room temperature, followed by treatment with fura-2-free solution for another 30 mins. Fluorescence ratio imaging was measured utilizing the Leica DMI6000B fluorescence microscope and Hamamatsu camera ORCA-Flash 4 controlled by SlideBook 6.0 software (Intelligent Imaging Innovations; Denver, CO) as described previously (45). Consecutive excitation at 340 nm (F 340 ) and 380 nm (F 380 ) was undertaken every 2 s, and emission fluorescence was collected at 505 nm. Intracellular Ca 2ϩ levels are shown as F 340 /F 380 ratios obtained from groups of 8 -25 single cells on coverslips. For the Orai1 concatemer experiments, data are shown for cells expressing a narrow range of tdTomato fluorescence to maximize the consistency of responses between cells. For each concatemer experiment, we simultaneously examined each set of mutants with the corresponding wild-type concatemer. The expression levels for wildtype and mutant concatemers were within a similar narrow FIGURE 8. The position and number of pore-inactive E106A subunits substituted in the Orai1 hexamer reveal profound differences in channel function likely reflecting different combinations of assembly as described under "Results and Discussion." A, fura-2 ratiometric Ca 2ϩ add-back measurements for HEK-O1 ko S1 ϩ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca 2ϩ stores were released with 2.5 M ionomycin in Ca 2ϩ -free medium followed by the addition of 1 mM Ca 2ϩ (arrows). The trace for wild-type Orai1 hexamer (OOOOOO, red) is compared with that for Orai1 hexamer substituted with E106A at the first three positions (XXXOOO, orange). B, Ca 2ϩ add-back measurements as in A using Orai1 hexamer substituted with three E106A residues in the second, fourth, and sixth positions (OXOXOX, olive). Traces (means Ϯ S.E.) are representative of three independent experiments. C, summary scatter plots of peak Ca 2ϩ entry when compared with wild type; results are means Ϯ S.D. of all cells in three independent experiments represented in A or B. D, Ca 2ϩ add-back measurements as in A using Orai1 hexamer substituted with three E106A residues in the last three positions (OOOXXX, cyan). E, Ca 2ϩ add-back measurements as in A using Orai1 hexamer substituted with four E106A residues in the last four positions (OOXXXX; green). Traces (means Ϯ S.E.) are representative of three independent experiments. F, summary scatter plots of peak Ca 2ϩ entry when compared with wild type; results are means Ϯ S.D. of all cells in three independent experiments represented in D or E. range of fluorescence intensity. All Ca 2ϩ imaging experiments were performed at room temperature, and representative traces of at least three independent repeats are shown. Scatter plots with means Ϯ S.D. are shown for all the cells recorded.
Deconvolved Fluorescence Image Analysis-For the imaging of cells, we used the inverted Leica DMI6000B automated fluorescence microscope and Hamamatsu ORCA-Flash 4 camera controlled by SlideBook software to collect and analyze high resolution fluorescence images. For the PM localization studies of tdTomato-tagged Orai1 concatemers, stacks of 10 -20 3D Z-axis image planes close to the cell-glass interface were collected at 0.35-m steps. The no-neighbor deconvolution function of the SlideBook 6.0 software was used to analyze images and derive enhanced deconvolved images with minimized fluorescence contamination from out-of-focus planes. The tdTomato-tagged Orai1 concatemer images shown were typical of at least three independent analyses.
Electrophysiological Measurements-Whole-cell patch clamp recording was performed on HEK-O1 ko S1 ϩ cells transiently transfected with tdTomato Orai1 concatemer constructs, or not transfected, as described (11). To passively deplete ER Ca 2ϩ stores, the pipette solution contained: 135 mM cesium (aspirated), 10 mM HEPES, 8 mM MgCl 2 , and 10 mM BAPTA (pH 7.2 with CsOH). The bath solution contained: 130 mM NaCl, 4.5 mM KCl, 5 mM HEPES, 10 mM dextrose, 10 mM tetraethylammonium chloride (TEA-Cl), and 20 mM CaCl 2 (pH 7.2 with NaOH). Currents were recorded in the standard whole-cell configuration using the EPC-10 amplifier (HEKA). Glass electrodes with a typical resistance of 2-4 megaohms were pulled using a P-97 pipette puller (Sutter Instruments). A 50-ms step to Ϫ100 mV from a holding potential of 0 mV, followed by a 50-ms ramp from Ϫ100 to 100 mV, was delivered every 2 s. Currents were filtered at 3.0 kHz and sampled at 20 kHz. A ϩ10-mV junction potential compensation was applied to correct the liquid junction potential between the bath and pipette solutions. The current measure at Ϫ100 mV was used in I-V curves. All data were acquired with PatchMaster and analyzed using FitMaster and GraphPad Prism 6.
Western Blotting and Biotinylation Analyses-Cells were washed with ice-cold PBS and lysed on ice using lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 1ϫ protease inhibitor cocktail) for 30 min, followed by centrifugation at 14,000 ϫ g for 10 min at 4°C. Supernatants were collected, and protein was quantified using Bio-Rad DC kits. Proteins were resolved on 4 -15% NuPAGE Bis-Tris precast gels and transferred to Bio-Rad Immuno-Blot PVDF membranes. After blocking in 5% nonfat milk for 1 h at room temperature, the membrane was incubated with primary antibody at 4°C overnight. Membranes were washed three times in PBST (phosphate-buffered saline supplemented with 0.1% Tween-20) and incubated with secondary antibody for 1 h at room temperature. Subsequently, membranes were washed three times with PBST. Peroxidase activity was examined with Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific), and fluorescence was collected using the FluorChem M imager (ProteinSimple). Cell surface proteins were isolated after biotinylation using the Pierce Cell Surface Protein Isolation kit (Thermo Scientific, 89881). HEK-O1 ko S1 ϩ cells were transfected with ptdTomato-N1-Orai1 or each of the concatemers described (dimers through hexamers) for 24 h followed by washing cells twice with ice-cold PBS. Cells were incubated with 0.25 mg/ml Sulfo-NHS-SS-Biotin for 30 mins at 4°C. Thereafter, quenching solution (Thermo Scientific, 1859386) was added to stop the reaction. Cells were collected and rinsed with TBS. Cells were then lysed in 200 l of Lysis Buffer (Thermo Scientific, 1859387) on ice for 30 min and centrifuged at 10,000 ϫ g for 10 mins at 4°C. The supernatant containing biotinylated proteins was incubated with Immobilized Neutr-Avidin Gel (Thermo Scientific, 1859388) in the kit columns at room temperature for 1 h on a rocking platform. After centrifuging at 1,000 ϫ g for 2 min, flow-through from the columns was collected and applied to gels to assess the level of nonbiotinylated Orai1 concatemeric protein, that is, the levels of concatemer that did not reach the PM. Columns were washed three times with Wash Buffer (Thermo Scientific, 1859389), and the biotinylated protein was eluted with 200 l of SDS-PAGE Sample Buffer with 50 mM DTT and resolved on 4 -12% NuPAGE Bis-Tris precast gels (Life Technologies) as described above. | v3-fos-license |
2018-06-30T00:51:45.507Z | 2018-06-01T00:00:00.000 | 48364769 | {
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} | pes2o/s2orc | Exosomes Derived from Human Induced Pluripotent Stem Cells Ameliorate the Aging of Skin Fibroblasts
Stem cells and their paracrine factors have emerged as a resource for regenerative medicine. Many studies have shown the beneficial effects of paracrine factors secreted from adult stem cells, such as exosomes, on skin aging. However, to date, few reports have demonstrated the use of exosomes derived from human pluripotent stem cells for the treatment of skin aging. In this study, we collected exosomes from the conditioned medium of human induced pluripotent stem cells (iPSCs) and investigated the effect on aged human dermal fibroblasts (HDFs). Cell proliferation and viability were determined by an MTT assay and cell migration capacity was shown by a scratch wound assay and a transwell migration assay. To induce photoaging and natural senescence, HDFs were irradiated by UVB (315 nm) and subcultured for over 30 passages, respectively. The expression level of certain mRNAs was evaluated by quantitative real-time PCR (qPCR). Senescence-associated-β-galactosidase (SA-β-Gal) activity was assessed as a marker of natural senescence. As a result, we found that exosomes derived from human iPSCs (iPSCs-Exo) stimulated the proliferation and migration of HDFs under normal conditions. Pretreatment with iPSCs-Exo inhibited the damages of HDFs and overexpression of matrix-degrading enzymes (MMP-1/3) caused by UVB irradiation. The iPSCs-Exo also increased the expression level of collagen type I in the photo-aged HDFs. In addition, we demonstrated that iPSCs-Exo significantly reduced the expression level of SA-β-Gal and MMP-1/3 and restored the collagen type I expression in senescent HDFs. Taken together, it is anticipated that these results suggest a therapeutic potential of iPSCs-Exo for the treatment of skin aging.
Introduction
Skin undergoes physiological changes as a consequence of the aging process. There are two basic types of skin aging, i.e., intrinsic and extrinsic aging. Intrinsic aging is genetically determined, which indicates that it occurs inevitably as time passes. Many studies have suggested epigenetic changes and post-translational mechanisms are more important pathways of intrinsic aging rather than genetic influence. On the other hand, extrinsic aging occurs by external factors such as smoking, air pollution, and unbalanced nutrition. Among them, UV exposure is the most important cause of extrinsic aging. Therefore, the skin damages induced by UV exposure is called "photoaging" [1,2]. Photoaging is characterized by irregular pigmentation, dryness, sallowness, roughness, premalignant implicated in cell-to-cell communication and the progression of several diseases such as cancer [30,31].
Recently, many studies have demonstrated the therapeutic applications of exosomes derived stem cells to ameliorate various injuries, including cardiovascular, renal, and lung injuries [32,33].
We previously demonstrated the stimulatory effects of human induced pluripotent stem cell-conditioned medium (iPSC-CM) on the proliferation and migration of dermal fibroblasts [34]. Herein, we hypothesized that the iPSCs-CM contained exosomes and the human induced pluripotent stem cells-derived exosomes (iPSC-Exo) played a key role in these effects of iPSC-CM. To address this hypothesis, we isolated exosomes from iPSC-CM and examined their effects on several cellular responses associated with skin aging, as well as the proliferation and migration in HDFs. To induce photoaging and natural senescence, HDFs were irradiated by UVB or subcultured and serially passaged, respectively. Then, we explored the protective effect of iPSC-Exo on UVB irradiation-induced cellular damage. Furthermore, we examined the effect of iPSC-Exo on the expression of MMP-1, MMP-3, and collagen type I, which are important in the construction of ECM, in UVB-induced and naturally senescent HDFs, respectively.
Characterization of Exosomes Derived from Human iPSCs
Exosomes secreted from human iPSCs were isolated using ExoQuick-TC TM solution (System Biosciences, Palo Alto, CA, USA) as described in the experimental section and characterized by size and morphology. We utilized nanoparticle tracking analysis (NTA) to evaluate exosome numbers and their size profiles. The NTA results showed that the size distribution of iPSC-Exo had a major peak at~100 nm and the mean diameter was 85.8 nm, although a small number of larger vesicles was included ( Figure 1A). This result indicated that most of the extracellular vesicles used in this study were exosomes, because the sizes of other microvesicles or apoptotic bodies are relatively larger than exosomes. Transmission electron microscopy (TEM) images indicated a similar tendency as the NTA result, showing that iPSC-Exo had a spherical membrane structure ( Figure 1B). The sizes of exosomes shown in the TEM image were smaller than that from the NTA result. It is believed that this is due to condensation of the iPSC-Exo during the drying step for TEM analysis [35]. In addition, dynamic light scattering (DLS) results showed that the average zeta potential of the isolated iPSC-Exo was −15.6 mV ( Figure 1C). These results suggest that the physical characteristics of iPSC-Exo used in this study were similar to those of exosomes derived from other cell lines [36,37].
Proliferation and Migration of HDFs Were Accelerated by iPSC-Exo
We previously demonstrated that the iPSC-CM could promote proliferation and migration of HDFs. As the iPSC-CM was supposed to contain exosomes, we examined the effect of iPSC-Exo on proliferation and migration of HDFs. The mitogenic effect was determined by the MTT assay with different doses of iPSC-Exo, ranging from 3 to 315 × 10 8 particles/mL. The proliferation of HDFs was increased by iPSC-Exo in a dose-dependent manner up to 50 × 10 8 particles/mL and maintained with a higher concentration ( Figure 2). In our previous study, some toxicity had been shown at high concentrations of iPSC-CM [34]. However, no toxic effect was observed when iPSC-Exo was treated even with higher concentrations. Although the mitogenic effect was saturated up to 50 × 10 8 particles/mL, we used iPSC-Exo with 20 × 10 8 particles/mL in further experiments because the differences were not significant. To investigate the effect of iPSC-Exo on migration of HDFs, we performed scratch wound assays. The stimulatory effect on cell migration was determined by evaluating the closure of the scratched area in this assay. As shown in Figure 3A, the rates of gap-filling in HDFs treated with iPSC-Exo were significantly higher than the control group, indicating that iPSC-Exo promoted the migration of HDFs. The migration-promoting effect was also demonstrated in a transwell migration assay. When the cells were seeded on the upper side of a porous membrane, they migrated to the opposite side across the membrane. HDFs that migrated toward the bottom side of the membrane were stained by crystal violet, and the intracellular crystal violet was quantitated by measuring the absorbance at 560 nm after dissolving it with an acetic acid solution. Bright field images showed that the number of migrated cells was significantly increased by the treatment of iPSC-Exo ( Figure 3B). The result of the quantitative analysis also demonstrated the stimulatory effect of iPSC-Exo on the migration of HDFs ( Figure 3C). Transwell membrane with the stained HDFs was cut out and immersed in a 50% acetic acid solution to dissolve the crystal violet. The amount of stained crystal violet was determined as absorbance at 560 nm. *** p < 0.001 compared to the iPSC-Exo-untreated group. Error bars indicate standard deviations of triplicate samples in a single representative experiment.
Effect of iPSC-Exo on UVB-Induced HDFs Damages
To investigate whether iPSC-Exo can rescue HDFs from the damage induced by UVB irradiation, cells were treated with 20 × 10 8 particles/mL of iPSC-Exo prior to UVB irradiation. Since it will take some time for iPSC-Exo to pass through the plasma membrane and mediate cellular signaling pathways caused by UVB irradiation, it was considered that the treatment of iPSC-Exo after UVB irradiation would not show significant recuperative effects. When the cells were irradiated by 40 mJ/cm 2 UVB, the difference was not statistically significant (0.05 < p < 0.1) 24 h after UVB irradiation, although the iPSC-Exo-treated group showed slightly higher viabilities. However, when the cells were irradiated by 80 mJ/cm 2 UVB, the viabilities of the untreated group were significantly reduced to 65.6% ± 8.6%, while the iPSC-Exo-treated group showed viabilities of 88.4% ± 2.1% ( Figure 4). After 48 h of UVB irradiation, the effect of iPSC-Exo on UVB-induced cell damage was more clearly demonstrated when the cells were irradiated, with 40 mJ/cm 2 as well as 80 mJ/cm 2 UVB. These results indicate that iPSC-Exo can protect human skin cells from UV-induced photoaging.
Effect of iPSC-Exo on the Expression of MMP-1, MMP-3, and Collagen Type I at mRNA Level
Collagen type I predominates in the dermis and is responsible for the tensile strength of the skin tissue. MMPs are a family of the structurally related matrix-degrading enzymes. It has been reported that UV irradiation inhibits collagen type I synthesis and induces the expression of MMPs, which eventually results in premature skin aging [10,38,39]. As UVB irradiation has been reported to alter the expression of a wide range of genes through the regulation of several transcription factors [39,40], we investigated the effect of iPSC-Exo on mRNA expression of genes related to photoaging. After UVB irradiation of HDFs, the levels of MMP-1, MMP-3, and collagen type I mRNA were evaluated using quantitative real-time PCR (qPCR). UVB irradiation (80 mJ/cm 2 ) increased the mRNA expression of MMP-1 and MMP-3 by 7.6 ± 0.7 and 7.3 ± 0.3 times, respectively. On the other hand, the mRNA expression of collagen type I was reduced by 68.3% ± 0.5% by UVB irradiation. However, it was clearly demonstrated that iPSC-Exo treatment significantly increased transcript levels of collagen type I, which is a crucial component of the skin dermis ( Figure 5A). On the contrary, the expression levels of matrix-degrading enzymes, MMP-1 and MMP-3, were reduced in the HDFs treated with iPSC-Exo compared to the untreated group ( Figure 5B,C). These results indicate that iPSC-Exo restored the mRNA expression disturbed by UV irradiation in the dermal fibroblasts.
iPSC-Exo Reversed Senescence-Related Gene Expressions in HDFs
We examined whether iPSC-Exo could restore alterations of gene expression even in naturally senescent HDFs. SA-β-Gal is a typical biomarker expressed in senescent fibroblasts. It has been demonstrated that the expression level of SA-β-Gal increases with age in epidermal keratinocytes and dermal fibroblasts of human skin biopsies [41]. Many studies have examined the expression of SA-β-Gal by staining senescent cells with a substrate at pH 6.0 [42,43]. Figure 6A shows that the expression of SA-β-Gal was higher in senescent HDFs (passage number 31) compared to young HDFs (passage number 5). However, the treatment of iPSC-Exo significantly reduced the number of SA-β-Gal-positive cells. For quantitative analysis, the cells were classified into three groups: unstained, weakly stained, and strongly stained, according to the expression of SA-β-Gal, and then the number of cells in each group was counted from three different images. As a result, it was clearly demonstrated that the proportion of SA-β-Gal-positive cells was reduced in the cells treated with iPSC-Exo compared to the untreated senescent cells ( Figure 6B). Next, we investigated the effect of iPSC-Exo on gene expression associated with ECM construction in senescent HDFs. As in the case of photoaging, the mRNA levels of MMP-1 and MMP-3 were increased, and iPSC-Exo significantly reduced the expression of these matrix-degrading enzymes. On the contrary, collagen type I mRNA was reduced in senescent HDFs, and it was completely recovered through treatment of iPSC-Exo (Figure 7).
Discussion
Several factors are involved in skin aging, such as extrinsic factors, e.g., UV light and air pollution, as well as intrinsic factors, such as natural senescence. Tissue integrity is disrupted in the aging skin, resulting in the reduction of elasticity and the formation of wrinkles [44]. Many studies have demonstrated that stem cells and their paracrine factors are potential therapeutics for the treatment of several diseases, and that exosomes play an important role in their functions [19,45,46]. In recent years, there have been advances in exploring the role of exosomes secreted from stem cells in tissue regeneration. Many cytokines, RNAs, and some proteins that can induce repair following cellular injury, are located within the lipid-bilayer membranes of exosomes [28]. For this reason, exosomes are believed to have many advantages, such as reliably preserving the useful components, and delivering them into the cells in the injured tissues. Zhang et al. mentioned that exosomes of human iPSC-derived MSCs facilitate cutaneous wound healing by inducing collagen synthesis and angiogenesis [47]. Hu et al. demonstrated through in vivo tracking experiments that exosomes derived human adipose MSCs accelerate cutaneous wound healing [37]. Furthermore, Nong et al. reported the hepatoprotective effect of exosomes originating from human iPSC-derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury [48]. On the other hand, some studies have revealed the therapeutic effects of human pluripotent stem cells-derived exosomes. Khan et al. reported the effect of mouse embryonic stem cell-derived exosomes for cardiac regeneration in ischemic myocardium [49]. Park and colleagues generated exosome-like nanovesicles from human embryonic stem cells through devices that physically impacts the cells. They demonstrated that the generated nanovesicles were able to enter mouse skin fibroblasts and promote cell proliferation [50]. However, according to our finding, therapeutic effects of exosomes derived from human pluripotent stem cells on the aging of skin cells as well as an in vivo study have not been reported.
We purified exosomes from the medium conditioned by human iPSCs and characterized iPSC-Exo as having spherical morphologies with approximate diameters ranging from 30 to 120 nm; these features were like the exosomes derived from other cell lines (Figure 1). In the previous report, when the iPSC-CM was treated to HDFs, it has been demonstrated that the cell proliferation was increased in a dose-dependent manner, up to 75% iPSC-CM (iPSC-CM:fresh basal medium = 3:1 mixture) but was reduced at 100%. It was supposed to be due to accumulation of toxic metabolites such as lactic acid and the depletion of nutrients in the iPSC-CM. For this reason, it was shown in the result of the MTT assay that the maximum increase in HDFs proliferation was limited, up to about 40% [34]. On the other hand, when iPSC-Exo was applied, the cell proliferation has increased by up to 80% without any significant cytotoxicity ( Figure 2). Furthermore, the treatment of iPSC-Exo promoted the migration of HDFs similar with iPSC-CM. Although the rate of gap-filling may be affected by the proliferation as well as the migration in scratch wound assay ( Figure 3A), the stimulatory effect of iPSC-Exo on the migration of HDFs was also clearly demonstrated in transwell migration assay ( Figure 3B,C). These results indicate that iPSC-Exo contains key factors to induce the proliferation and migration of HDFs and is more suitable for clinical application than iPSC-CM because of its lower toxicity. There may be concern that nonexosomal proteins were contained in the isolated iPSC-Exo samples. However, it is likely that only a small fraction of the total nonexosomal proteins contained in the iPSC-CM has been precipitated with iPSC-Exo, and consequently, the concentration of the nonexosomal proteins was significantly lower when HDFs were treated with the isolated iPSC-Exo samples. It is also possible that some cytokines and growth factors among nonexosomal proteins were inactivated during the precipitation because they are known to be very labile (e.g., basic fibroblast growth factor). Taken together, it is unlikely that nonexosomal proteins exerted significant effects on the results shown in our study. However, a recent study revealed that ultracentrifugation method provided higher purity than ExoQuick TM in exosome purification, although nonexosomal proteins such as serum albumin and apolipoprotein E were not completely removed even in this method [51]. They also suggested that a density gradient centrifugation is superior to protocols based on ultracentrifugation and precipitation in terms of purity, despite disadvantages such as high labor intensity. For further applications, we believe that it is necessary to develop an optimized iPSC-Exo purification protocol providing higher purity and exosome yield.
The present study also demonstrated the beneficial effects of iPSC-Exo on damage and alterations of gene expression induced by not only UVB irradiation but also natural senescence. UV in sunlight, consisting of UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), is a major environmental factor in causing skin photoaging [38,52]. UVB results in cellular damage by inducing DNA mutations as well as indirectly via oxidative stress [53]. In addition, exposure of dermal fibroblasts to UVB activates cell surface receptors, which subsequently stimulate MAPK signaling pathways. MAPK signal transduction is associated with the transcription factor activator protein-1 (AP-1), which reduces the synthesis of collagen type 1 and enhances the expression of MMPs [3,38,39]. When iPSC-Exo was applied to HDFs, we found that cells were protected from damage induced by UVB irradiation. Since we have observed that iPSC-Exo promoted the proliferation of HDFs even without UV irradiation (Figure 2), the viability of each cell group was normalized by that of the UVB-unexposed group. Nevertheless, the protective effect of iPSC-Exo on UV-induced damage was clearly demonstrated ( Figure 4). Meanwhile, we also found that the pretreatment of iPSC-Exo inhibited the upregulation of MMP-1 and MMP-3 and downregulated collagen type I mRNA expression induced by UVB irradiation ( Figure 5). Next, the effects of iPSC-Exo on multiple genotypic changes involved in natural senescence were further demonstrated. The expression of SA-β-Gal, a typical senescence-associated marker [41], was significantly decreased by iPSC-Exo ( Figure 6). Then, we found that iPSC-Exo restored altered expression of MMP-1, MMP-3, and collagen type I in senescent HDFs, as shown in the case of photoaging (Figure 7). These results suggest that iPSC-Exo contains useful factors to mediate the rebalancing process of the matrix in the aging skin and they can successfully be delivered into dermal fibroblasts. It is believed that those factors modulate the expression of aging-related genes and promote the reconstitution of dermal matrix by increasing the content of structural proteins such as collagen type I in aged skin.
Several studies have reported proteomic analysis of extracellular vesicles derived from various stem cells. Kim et al. profiled the proteome of microvesicles from MSCs (MSC-MVs) using mass spectrometry-based approach and identified 730 MV proteins. They identified potential MV protein candidates that can be implied in the therapeutic effect of MSC-MVs on damaged tissues via the integration of the MSC-MV proteome with the transcriptome of MSCs and the proteome of MSC-conditioned medium [54]. Anderson et al. identified 1927 proteins in MSCs-derived exosomes using high resolution isoelectric focusing liquid coupled chromatography tandem mass spectrometry (HiRIEF LC-MS/MS) and these proteins include several putative paracrine factors modulating angiogenesis [55]. Meanwhile, several proteomic analyses have identified numerous proteins, including cytokines, signaling molecules, chaperones, and plasma membrane proteins, from human pluripotent stem cell [56][57][58][59]. Similar to MSC-derived exosomes, iPSC-Exo is believed to contain many kinds of those human iPSC-derived proteins. iPSC-Exo may also contain various mRNAs, miRNAs, and lncRNAs, which can affect cellular physiologies. For the reason, the beneficial effects of iPSC-Exo on skin fibroblasts are presumably due to the complex action of various components such as cytokines, membrane proteins, and RNAs.
In conclusion, our results have revealed the beneficial effects of human iPSC-Exo on human skin damaged by photoaging and on natural senescence. To the best of our knowledge, this is the first study to investigate the effect of human pluripotent stem cell-derived exosomes on the treatment of aging skin. Exosomes are considered to have several advantages in that they can stably preserve useful components to prevent skin aging and efficiently deliver them to the skin tissues. Because many complicated processes are involved in skin aging, further study should be required to demonstrate the therapeutic potential of iPSC-Exo. However, the present study is expected to serve as a technical insight toward the clinical application of exosomes derived from human induced pluripotent stem cells for the treatment of skin aging.
Cell Culture
In this study, we used a human iPSC line obtained from The National Center for Stem Cell and Regenerative Medicine in Korea. As described in our previous study [34], this cell line was generated from human dermal fibroblasts by introducing Oct4, Sox2, cMyc, and Klf4 using Sendai Virus. When the confluency was reached at 80%-90%, the iPSCs were dissociated by treatment with 0.5 mM EDTA and plated onto a truncated human vitronectin-coated culture dish. The cells were cultured in Essential 8 medium (Thermo Fisher Scientific, Waltham, MA, USA) and passaged every 4 to 5 days. In addition, human dermal fibroblasts (HDFs) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. To induce replicative senescence, HDFs were cultured for a long period of time with repeated passaging. When the confluency was reached at 70%-80%, HDFs were plated onto new dish at 2 × 10 4 cells/cm 2 and cultured until getting 70%-80% confluent again. All cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 .
Exosome Isolation
Culture medium was replaced with fresh Essential 8 medium daily in human iPSCs culture. The spent medium was collected daily from day 2 to the end of culture and filtered through 0.45 µm syringe filter. Exosomes were isolated from the conditioned medium, using ExoQuick-TC TM (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's instructions. Briefly, the conditioned medium was incubated with the exosome precipitation solution at 4 • C for 12 h and subsequently centrifuged at 1500× g for 30 min. After discarding the supernatant, the pellet was resuspended in phosphate buffered saline (PBS) and the collected iPSC-Exo was stored at −80 • C until use.
Characterization of iPSC-Exo
Size distribution and concentration of iPSC-Exo were analyzed by nanoparticle tracking analysis (NTA) using NanoSight NS300 (Malvern Panalytical, Malvern, UK). The solution containing iPSC-Exo was injected into the laser chamber using a 1 mL syringe and three 30 sec recordings were performed. The mode, mean size, and concentration of iPSC-Exo were determined by NTA 3.0 software. Camera level, threshold, and focus were kept under the same conditions in all experiments. Morphology of iPSC-Exo was visualized by transmission electron microscopy (TEM) using a JEM-1010 electron microscope (JEOL, Tokyo, Japan). First, iPSC-Exo was absorbed in a formvar/carbon-coated grid for 10 min and fixed with 2% paraformaldehyde. After negative staining with 2% uranyl acetate for 10 min, iPSC-Exo was observed by TEM operated at 60 kV. Zeta potential was measured using a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK).
MTT Assay
HDFs were plated at 2 × 10 4 cells/cm 2 in 96-well culture plates and incubated in growth medium for 24 h. For the serum starvation, the medium was then replaced with DMEM supplemented with 0.5% FBS and cells were cultured for a further 24 h. Following treatment of iPSC-Exo in serum-free DMEM/F12 for 24 h, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (Sigma Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. After removal of the supernatant, dimethyl sulfoxide was added to dissolve formazan and subsequently the absorbance at 560 nm was determined using a microplate reader.
Migration Assay
The effect of iPSC-Exo on the migration of HDFs was determined by scratch wound assay and transwell migration assay as described in the previous study [34]. In scratch wound assay, briefly, a scratch was created by scoring 70%-80% confluent monolayer of serum-starved HDFs with a sterile pipette tip. After incubation with 20 × 10 8 particles/mL iPSC-Exo in serum-free DMEM/F12 for 24 h or 48 h, migration of HDFs into the scratched area was monitored under an optical microscope. For the transwell migration assay, HDFs were plated onto upper chambers of transwell plates (Corning, Lowell, MA, USA) containing serum-containing growth medium and cultured for 24 h. Following replacement of the medium with serum-free DMEM/F12 containing iPSC-Exo, cells were incubated for a further 24 h to induce migration toward the opposite side of the transwell membrane. After removal of cells remaining on the top of the membrane by a cotton swab, the cells at the bottom of the membrane were fixed with 4% paraformaldehyde and then stained by 0.5% crystal violet (Sigma Aldrich, St. Louis, MO, USA) to visualize the migrated cells. The staining intensity was determined as absorbance at 560 nm after dissolving the crystal violet using 50% acetic acid.
UVB Irradiation
HDFs were plated in 96-or 24-well plates, depending on the assay, and cultured for 24 h. Then, cells were treated with 20 × 10 8 particles/mL iPSC-Exo in serum-free DMEM/F12 for 24 h. After washing three times, 50 µL and 200 µL of PBS were added to 96-and 24-well plates, respectively. Cells were irradiated with UVB (315 nm) generated by a Bio-Sun illuminator (Vilber Lourmat, Eberhardzell, Germany). The light intensity was estimated according to the distance between the UV illuminator and the each well plate. After the UV irradiation, PBS was replaced with DMEM/F12 and the cells were further incubated for 24 h or 48 h prior to specific assays. For MTT assay, cells were divided into iPSC-Exo-untreated (Control) and treated group (iPSC-Exo). Each group was further divided into following three groups: UVB-unexposed, 40 mJ/cm 2 UVB-exposed, and 80 mJ/cm 2 UVB-exposed group.
SA-β-Gal Staining
Senescent HDFs were plated in 96-well plates and cultured for 24 h. Following 20 × 10 8 particles/mL iPSC-Exo treatment for 24 h, the cells were further cultured for 48 h in serum-free DMEM/F12. The cells were then fixed with 4% paraformaldehyde and SA-β-Gal was stained using senescent cells histochemical staining kit (Sigma Aldrich, St. Louis, MO, USA). Three images per each well were collected, and the SA-β-Gal-stained cells were counted.
RNA Isolation and Real-Time RT-PCR
Total RNA was extracted from HDFs using a GeneAll Ribospin TM total RNA purification kit (GeneAll Biotechnology Co. Ltd., Seoul, Korea) according to the manufacturer's instructions. Purified total RNA was reverse-transcribed to cDNA using TOPscript TM RT DryMIX (Enzynomics Co. Ltd., Daejeon, Korea) with a dT 18 plus primer. Subsequently, a PCR step was performed from the cDNA samples using specific primers and TOPreal TM qPCR 2X PreMIX (SYBR Green with high ROX, Enzynomics Co. Ltd. Daejeon, Korea) in triplicate on the Eco Real-Time PCR System (Illumina, San Diego, CA, USA). The primers used in this study are summarized in Table 1. The standard cycle conditions were as follows: 95 • C for 1 min, 40 cycles of denaturation at 95 • C for 15 s, and annealing-extension at 60 • C for 30 s. The expression level of specific RNAs was normalized by actin as an endogenous control.
Statistical Analysis
All results were expressed as mean ± standard deviation and were statistically analyzed by a Student's t-test. Statistical significance was indicated at p < 0.05. | v3-fos-license |
2019-04-16T13:22:02.589Z | 2019-04-29T00:00:00.000 | 115202724 | {
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} | pes2o/s2orc | CFTR interacts with Hsp90 and regulates the phosphorylation of AKT and ERK1/2 in colorectal cancer cells
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF cells and tissues exhibit various mitochondrial abnormalities. However, the underlying molecular mechanisms remain elusive. Here, we examined the mechanisms through which CFTR regulates Bcl‐2 family proteins, which in turn regulate permeabilization of the mitochondrial outer membrane. Notably, inhibition of CFTR activated Bax and Bad, but inhibited Bcl‐2. Moreover, degradation of phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) and AKT increased significantly in CFTR‐knockdown cells. Dysfunction of CFTR decreased heat‐shock protein 90 (Hsp90) mRNA levels, and CFTR was found to interact with Hsp90. Inhibition of Hsp90 by SNX‐2112 induced the degradation of phosphorylated AKT and ERK1/2 in Caco2 and HRT18 cells. These findings may help provide insights into the physiological role of CFTR in CF‐related diseases.
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF cells and tissues exhibit various mitochondrial abnormalities. However, the underlying molecular mechanisms remain elusive. Here, we examined the mechanisms through which CFTR regulates Bcl-2 family proteins, which in turn regulate permeabilization of the mitochondrial outer membrane. Notably, inhibition of CFTR activated Bax and Bad, but inhibited Bcl-2. Moreover, degradation of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT increased significantly in CFTR-knockdown cells. Dysfunction of CFTR decreased heat-shock protein 90 (Hsp90) mRNA levels, and CFTR was found to interact with Hsp90. Inhibition of Hsp90 by SNX-2112 induced the degradation of phosphorylated AKT and ERK1/2 in Caco2 and HRT18 cells. These findings may help provide insights into the physiological role of CFTR in CF-related diseases.
Cystic fibrosis (CF) is an autosomal recessive genetic disease that is most common in Caucasians that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. CFTR encodes a cAMP-regulated anion channel expressed at the apical membrane of epithelial cells in various organs [2]. The CFTR channel can transport chloride, bicarbonate, and glutathione [3,4]. CFTR interacts either directly or indirectly with some proteins by forming a macromolecular complex [5][6][7][8]. More than 2000 mutations have been identified in CF, and deletion of the phenylalanine at position 508 (DF508) is the most frequent mutation occurring in over 80% of patients with CF [9][10][11][12]. CFTR mutations affect the mucosal physiology of the respiratory, digestive, and reproductive tracts, leading to systemic diseases, including pancreatic insufficiency, focal biliary cirrhosis, infertility, intestinal obstruction, increased cancer risk, and chronic airway obstruction [13,14].
Interestingly, mitochondrial abnormalities have been found in CF cells and tissues [15][16][17]. For example, calcium uptake and oxygen consumption are altered in the mitochondria of patients with CF [18]. Additionally, fragmentation of mitochondria and a decrease in the mitochondrial membrane potential were observed in CF cells compared with control cells [19]. Mitochondrial proteins have also been shown to exhibit differences in the two-dimensional electrophoretic patterns in CF [20]. Moreover, reactive oxygen species and autophagy may be related to mitochondrial dysfunction in patients with CF [21][22][23]. However, the underlying molecular mechanisms remain elusive.
Thus, in this study, we aimed to evaluate the possible roles of CFTR in mitochondrial abnormalities. Our results provided important insights into the roles of CFTR in mitochondrial dysfunction via a mechanism involving heat-shock protein 90 (Hsp90)-mediated extracellular signal-regulated kinase (ERK) 1/2 and AKT, suggesting the involvement of CFTR in CF-related diseases.
Cell culture and lentiviral transduction
The human colon cancer cell lines Caco2 and HRT18 (American Type Culture Collection, Manassas, VA, USA) were cultured in MEM or RPMI-1640, respectively, supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin in an incubator at 37°C in an atmosphere containing 5% CO 2 .
Lentiviral transduction particles encoding short hairpin RNA (shRNA) against CFTR were purchased from Jima Inc. (Shanghai, China). The sequences (5 0 to 3 0 ) of the two shRNAs were GAAGTAGTGATGGAGAATGTA and TTGGAAAGGAGACTAACAAGT, respectively. These two shRNA duplexes were from two different regions of human CFTR mRNA. Cells were plated in 24-well cell culture plates, incubated with 5 lL lentivirus (1 9 10 9 TUÁmL À1 , 1 9 10 9 TUÁmL À1 ) for 24 or 48 h, and collected for analysis. Viral vectors containing noncoding shRNA were used as a control.
Western blotting
Cells were lysed in RIPA buffer with 1 : 100 protein inhibitor for 30 min on ice. The supernatants were collected as total protein after centrifugation at 15 000 g for 30 min. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide electrophoresis followed by western blotting. The protein bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA, USA) according to the manufacturer's instructions, and data were quantified using densitometry analysis. Experiments were repeated three times, and the bands were scanned and quantified.
Immunohistochemistry and fluorescent immunocytochemistry
The sections were incubated with phosphate-buffered saline (PBS) for 5-10 min and retrieval buffer (citrate buffer, pH 6.0) for 30 min. After washing in PBS, the sections were incubated with primary antibodies overnight at 4°C. The sections were then incubated with secondary antibodies for 60 min at room temperature. Finally, the sections were mounted with coverslips and visualized by microscopy.
RNA extraction and real-time quantitative polymerase chain reaction (RT/qPCR)
Total RNA was extracted using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and was reverse-transcribed using a PrimeScript RT Master Mix kit (TaKaRa, Dalian, China) for standard real-time PCR analysis. RT/qPCR was performed using a TB Green Premix EX Taq II detection system in a Roche LightCycler 96 qPCR machine (Roche Diagnostics, Mannheim, Germany). Glyceraldehyde 3phosphate dehydrogenase was used as a control. The genespecific primers are summarized in Table S1.
Co-immunoprecipitation (Co-IP)
CFTR was overexpressed in 293T cells, and cells were cultured for 48 h. Cell lysates were collected in IP Lysis/Wash Buffer with a Pierce Crosslink Magnetic IP/Co-IP Kit (cat. no.: 88805; Thermo Scientific, Rockford, IL, USA). Cell lysates were then incubated with Protein A/G Magnetic Beads bound to CFTR antibodies for 2 h at room temperature. The beads were washed with IP wash buffer and then eluted in elution buffer to collect the target protein. Co-IP was performed according to the instructions provided by the kit manufacturer.
Three-dimensional (3D) structure of the CFTR/ Hsp90 complex was built using PYMOL, version 2.0. The model contained the amino acids residues predicted as possible interacting sites [24,25].
Statistical analysis
Data were expressed as means AE standard deviations. Differences in measured variables between two groups were analyzed using Student's t-tests, and differences between more than two groups were analyzed by one-way analysis of variance. Results with P values of < 0.05 were considered statistically significant.
Knockdown of CFTR
Initially, we assessed the effects of shRNA against CFTR by western blotting. Caco2 and HRT18 cells were transfected with lentivirus for 48 h. Western blot analysis of CFTR protein was then performed. The results showed that CFTR was significantly downregulated (Fig. 1A). Moreover, in Caco2 cells, CFTR was not expressed at the apical membrane after knockdown (Fig. 1B).
Knockdown of CFTR induced mitochondrial dysfunction by regulating the phosphorylation of AKT and ERK1/2
To determine whether CFTR knockdown affected mitochondrial function in Caco2 and HRT18 cells, we performed western blot analysis of Bcl-2 family members, including Bcl-2, Bad, and Bax. The results showed that Bax and Bad were upregulated, and Bcl-2 was downregulated in CFTR-knockdown cells ( Fig. 2A). In addition, the mRNA levels of Bax and Bad were upregulated, whereas Bcl-2 mRNA was downregulated in CFTR-knockdown cells (Fig. 2B).
To further explore the molecular mechanisms involved in mitochondrial dysfunction induced by CFTR knockdown, we measured the phosphorylation of AKT and ERK1/2, which regulate Bcl-2 family proteins. Western blot analysis indicated that phospho-AKT and phospho-ERK1/2 levels were decreased in CFTR-knockdown cells (Fig. 2C). These findings suggested that knockdown of CFTR induced mitochondrial dysfunction in Caco2 and HRT18 cells by regulating the phosphorylation of AKT and ERK1/2.
CFTR interacted with Hsp90
AKT and ERK1/2 are regulated by Hsp90 [26]; indeed, inhibition of Hsp90 induces the degradation of phospho-AKT and phospho-ERK1/2 [27][28][29]. Therefore, we next investigated the protein levels of Hsp90 in CFTR-knockdown cells. The results showed that both Hsp90a and Hsp90b decreased significantly in CFTRknockdown Caco2 and HRT18 cells (Fig. 3A). To further investigate the relationships between CFTR and Hsp90, we performed Co-IP in CFTR-overexpressing 293T cells. The results showed that Hsp90 could be pulled down by CFTR (Fig. 3B). Although the 3D structures of both CFTR and Hsp90 were solved, the structure of the complex between these two proteins has not been determined experimentally yet. Therefore, we then built a model of the protein-protein interaction with PyMOL to predict the complex (Fig. 3C). These results indicated that degradation of phospho-ERK1/2 and phospho-AKT in CFTR-knockdown cells might be associated with decreased Hsp90 expression.
Discussion
CFTR acts as a chloride channel and can interact directly or indirectly with several proteins by forming a complex to regulate cell signaling processes [31]. Knockdown of CFTR suppresses the proliferation of ovarian cancer in vitro and in vivo [32]. In contrast, CFTR has also been reported to function as a tumor suppressor [33], and downregulation or mutation of CFTR activates proliferation, invasion, migration, and the epithelial-mesenchymal transition in breast cancer and prostate cancer [34][35][36]. Hyperproliferation has been observed in CF airways [37], and downregulation of CFTR has been reported in cancer tissues [33]. In addition, mitochondrial abnormalities have been reported in CF cells or tissues [38]. Differential expression of genes or proteins and alterations in calcium homeostasis, membrane potential, reactive oxygen species, and complex I activity have all been reported in mitochondria in the context of CFTR knockdown [39]. These mitochondrial effects in turn induce changes in the ratio of reduced/oxidized glutathione and trigger proliferation or apoptotic events.
Moreover, these alterations may influence the phenotype or clinical manifestations of CF [19,21,38,40]. However, the molecular mechanisms underlying mitochondrial dysfunction remain elusive.
In this study, we showed, for the first time, that disruption of the CFTR/Hsp90 interaction induced mitochondrial dysfunction via degradation of phospho-AKT and phospho-ERK1/2 in Caco2 and HRT18 cells. In CFTR-knockdown cells, Bax and Bad were activated, whereas Bcl-2 was inhibited, indicating the involvement of mitochondrial dysfunction. The mitochondria play important roles in apoptosis and proliferation [40]. Thus, further studies are needed to evaluate the roles of CFTR in mitochondrial dysfunction and subsequent cell proliferation and apoptosis.
AKT and ERK1/2 are crucial proteins that regulate the Bcl-2 family [41]. AKT upregulates Bcl-2 expression through cAMP-response element-binding protein [42]. Bad is a distant member of the Bcl-2 family and can be phosphorylated by AKT and ERK1/2 [43][44][45]. In this study, both phospho-ERK1/2 and phospho-AKT levels were significantly decreased in CFTRknockdown cells, suggesting that mitochondrial dysfunction induced by CFTR dysfunction may be regulated by the AKT and ERK1/2 pathways. AKT and ERK1/2 are the target proteins of Hsp90 [46], and inhibition of Hsp90 induces degradation of these proteins [47]. In this study, we found that Hsp90 was downregulated in CFTR-knockdown cells. Hsp90 interacted with CFTR. Hsp90 inhibitors induce the degradation of AKT and ERK1/2. Additional studies are needed to clarify the mechanisms mediating the interactions between CFTR and Hsp90. The regulation mechanisms of CFTR and Hsp90 on mitochondria, especially how CFTR and Hsp90 work together to maintain the function of mitochondria, will be investigated in the future work.
Taken together, our findings revealed a previously unidentified role of CFTR defects in mitochondrial abnormalities via the Hsp90-mediated degradation of phosphorylated ERK1/2 and AKT. These results provided novel insights into the physiological functions of CFTR and CF-related diseases. | v3-fos-license |
2018-04-03T00:16:02.534Z | 2013-11-11T00:00:00.000 | 1272317 | {
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} | pes2o/s2orc | Concomitant Effects of Ramadan Fasting and Time-Of-Day on Apolipoprotein AI, B, Lp-a and Homocysteine Responses during Aerobic Exercise in Tunisian Soccer Players
Objective To examine the time-of-day and Ramadan fasting (RF) effects on serum apolipoprotein-AI (Apo-AI) and B (Apo-B), lipoprotein particles-a (Lp-a), high-sensitive C-reactive-protein (hs-CRP), and homocysteine (Hcy) during the Yo-Yo intermittent recovery test (YYIRT). Design Performance and biochemical measures were completed at two times-of-day (07:00 and 17:00 h), 1-week before RF (BR), the second week of RF (SWR), and the fourth week of RF (ER). Setting For each session, subjects performed the YYIRT, and blood samples were taken before and 3-min after the test for biochemical measures. Participants Fifteen soccer players. Main Outcome Measures Total distance during the YYIRT, core temperature, body composition, dietary intakes, lipid (HDL-C, LDL-C, Apo-AI, B and Lp-a) and inflammatory (hs-CRP and Hcy) profiles. Results Performances during the YYIRT were higher in the evening than the morning BR (P < 0.05), but this fluctuation was not observed during RF. Moreover, LDL-C, ApoB, and Lp-a were stable throughout the daytime BR. However, during RF, they decreased at 17:00 h (P < 0.05). Likewise, HDL-C and Apo-AI increased after the exercise and were higher at 17:00 h BR (P < 0.001). Moreover, these parameters increased during RF (P < 0.01). Furthermore, Hcy and hs-CRP increased during the exercise (P < 0.01) with higher evening levels BR. During ER, the diurnal pattern of Hcy was inversed (P < 0.001). Conclusions This study concluded that caloric restriction induced by RF seems to ameliorate lipid and inflammatory markers of cardiovascular health during intermittent exercise performed in the evening.
Introduction
The Ramadan fast (RF) is observed by Muslims every year when they are required to abstain from food and fluid intake between dawn and dusk. It is well established that RF induces many beneficial effects on lipid metabolism in healthy subjects [1][2][3][4]. Indeed, it has been demonstrated that RF is accompanied with reduced serum low density lipoprotein (LDL-C), apolipoprotein B (Apo-B), a major protein component of LDL-C, total cholesterol (TC), triglycerides (TG), and increased apolipoprotein AI (Apo-AI) and high density lipoprotein (HDL-C) levels, with unchanged or reduced body mass despite, in some cases, increased caloric intake [1,3,5]. In this context, Apo B, as a major protein component of LDL-C, has been proposed to be a better indicator for coronary artery diseases than TC and TG [6]. Moreover, Apo-AI-containing lipoprotein particles (Lp-a) seem to be a more powerful discriminating marker for cardiovascular risk than Apo-AI and HDL-C [7]. However, several other studies have found a decline in HDL-C levels and an increase in LDL-C levels following Ramadan fasting [8,9].
In the study of Adlouni et al [3], Apo-AI increased in the day 8 and 29 of RF and remained elevated one month later in comparison with before RF. However, Apo-B levels were lower during the second and the last week of RF and remained low one month after RF. Moreover, it has been reported that RF positively influenced the inflammatory status by a decrease in the pro-inflammatory cytokine (ie, IL-6) concentrations associated with decreases in both high-sensitive C-reactive protein (hs-CRP) and the cardiovascular-risk marker (ie, homocysteine (Hcy)) [10]. Nevertheless, the studies cited above were conducted in healthy sedentary subjects. In highly trained subjects, very few studies investigated the influence of RF on lipid metabolism. Chaouachi et al [11] reported changes in some serum lipids and apolipoproteins (ie, increase in HDL-C, LDL-C, and Apo-AI) in elite judo athletes. Conversely, only free-fatty-acid and HDL-C levels have been shown to increase during RF in middle-distance runners [1,3,5] and physically active men [13]. Particularly, it has recently been identified that aerobic training during RF may increase muscle oxidative capacity [14] and enhance exercise-induced intramyocellular lipid degradation [15]. It seems that the effect of RF on serum lipid levels may be closely related to the feeding behavior or biochemical responses to starvation [3]. Concerning the RF effect on exercise, the few studies conducted in this context indicated an increase in plasma TG and HDL-C concentrations during submaximal exercise [16,17].
Otherwise, most clinical biochemical entities have been shown to exhibit circadian rhythms at rest [18] and during exercise [19,20] in soccer players. In this context, it has been indicated that fasting and postprandial lipid, lipoprotein, and apolipoprotein concentrations were affected by circadian factors [21,22]. Moreover, it has been established that a given nutrient ingested at an unusual time could induce different metabolic effects [23]. In fact, our previous findings indicated that the resting circadian pattern of lipid metabolism could influence post-exercise measurements [24]. Furthermore, we recently observed that various components of physical performances displayed diurnal variations (ie, higher evening values) which tend to disappear during RF [25]. Even if the circadian rhythm in core temperature is not necessarily the cause of the rhythm in muscle performance [26], previous research has suggested that the simultaneous increases in body temperature and muscular power are causally related. Moreover, it is well known that RF modifies the diurnal pattern of core temperature, which could affect the diurnal patterns of biochemical measures [27].
In view of the above considerations, the modified biochemical response during RF could be a consequence of various external factors. The objective of the present study was to examine the RF effects on daily fluctuations in serum lipoproteins, Apo-AI, Apo-B, Lp-a as well as hs-CRP and Hcy responses during a maximal aerobic exercise in soccer players. The selected markers are important indicators of cardiovascular health and post exercise measurements during RF. Therefore, it would be of interest to investigate the modified metabolic response to exercise. We hypothesize that RF influences the lipid profile and inflammatory status through the concomitant effects of many mechanisms (ie, diet, time-ofday, week of Ramadan).
Subjects
Fifteen male professional soccer players (age, 17.3 ± 0.3 years, body mass, 69.1 ± 4.2 kg, height, 179.7 ± 3.6 cm) volunteered to participate in this study. The participants were recruited on the basis of: (i) they trained in the evening at least 4 days per week for an average of 2 h daily in addition to the weekend match and (ii) they had a minimum of 5 years of training experience and were members of the same youth team competing in the first division of the Tunisian football league. The training program during RF consisted of high intensity short duration intermittent exercises. We interviewed all players and coaches in order to provide information concerning the number of years of football practice and hours of regular training per week. Moreover, goalkeepers and players who experienced injuries were excluded from the analysis. So, the volunteers were homogeneous in physical characteristics (ie, VO2max, strength, anaerobic power and capacity). Approval for the study was obtained from the club. Subjects abstained from food, drinks and sexual activity about 16 h/day during Ramadan. The last meal was taken at 1:00-2:00 h. The study was carried out in Sfax, Tunisia in 2010 when Ramadan started on the 11 th of August 2010 and ended on the 10 th of September 2010. The length of each fasting day was approximately 15-16 h. To ensure that participants were all of a "moderately morning type" (n = 5) or "neither type" (n = 10), they were selected based on their responses to the Horne and Östberg's selfassessment questionnaire [28]. The Horne and Östberg's questionnaire allowed selecting subjects of similar sleeping and daily habits which allows a better understanding of the modified diurnal pattern of biochemical responses. Subjects were nonsmokers and did not consume nutritional supplements, caffeine, drugs, or alcoholic beverages.
Experimental Design
The experimental design consisted of three testing periods: (i) one week before RF (BR), the second week of RF (SWR), and the fourth week of RF (ER). For example, the major change of HDL-C and LDL-C levels were observed during the first and the last weeks of RF [29], that's why we chose to evaluate our subjects in the second and the fourth weeks of Ramadan. At each phase, the subjects performed two test sessions (TS) at two times-of-day (ie, 07:00 and 17:00 h) with a recovery period ≥ 36 h in-between. These two time points were chosen because they correspond to the peak and trough of football-specific skills and measures of physical performance [30]. TS were completed in a counterbalanced design and each one started with measure of oral temperature with a digital clinical thermometer (Omron, Paris, France; accuracy ± 0.05°C) inserted sublingually for at least 3 min after a 10-min rest in a seated position. To ensure a reliable measure of temperature, subjects were instructed not to talk or drink during measurement. Then, the soccer players participated in an endurance specific test (the Yo-Yo intermittent recovery test level-1 (YYIRT)). Heart rate was recorded during the YYIRT using a Polar heart rate monitor (Polar Electro Oy, T61-coded, Finland).
Body composition was assessed using an electronic scale (Tanita, Tokyo, Japan) during each testing phase [19]. Throughout the experimental period, participants were required to maintain their habitual physical activity and to avoid strenuous physical efforts 24 h before each TS.
The Yo-Yo Intermittent Recovery Test Level-1 (YYIRT)
As described previously [24], the test consisted of 20-m shuttle runs performed at increasing velocities with 10 s of active recovery between runs until exhaustion. Moreover, the reliability of the YYIRT was established in a previous study [31]. The end of the test was considered when the participant twice failed to reach the front line in time or he felt unable to complete another shuttle at the dictated speed. The total distance covered during the YYIRT (including the last incomplete shuttle) was considered as the test score. All players were already familiar with the test as it was part of their usual fitness assessment program. We have chosen the YYIRT due to its specificity to soccer. Indeed, Castagna et al [31] reported a significant correlation (r = 0.65) between the total distance covered during the YYIRT and the total distance covered during a soccer match.
Dietary Records
A seven consecutive day dietary record was completed. Dietary records were done in the same weeks as TS. Subjects received a detailed verbal explanation and written instructions on data collection procedures. They were asked to be as accurate as possible in recording the amount and type of food and fluid consumed. Each individual's diet was calculated using the Bilnut 4 software package (SCDA Nutrisoft, Cerelles, France) and the food composition tables published by the Tunisian National Institute of Statistics in 1978 (Table 1). To additionally substantiate the effect of acute exercise on the estimation of Hcy, Folate and vitamin B12 intakes were estimated from the subjects' dietary intake using a selfadministrated quantitative food questionnaire [32].
Blood Sample Variables Analysis
Blood samples were obtained from a forearm vein after 5 min of seated rest and 3 min after the YYIRT. TC, TG, and HDL-C were estimated by standard enzymatic analysis using reagents, standards, and controls from Randox Laboratories Ltd. (Antrim, UK). The coefficients of variation (CVs) for these parameters were < 7%. LDL-C was calculated by the Friedewald formula for TG levels below 400 mg/dL [24]. The above measures were done as adapted for the autoanalyzer by Synchron CX systems (Beckman Instruments, Danville, California, USA).
Determination of Apo-AI, Apo-B, Lp-a, and hs-CRP were performed by rate nephelometry on an immunonephelometer analyzer (BN Prospec, Siemens Diagnostics) using mono-and polyclonal antibodies. The inter-and intra-assay CVs for these parameters were < 2.8% and < 4.7% respectively. Hcy was determined using immunoassay IMX Analyzer (Abbott Laboratories, Lake Bluff, Illinois, USA). The detection limit of the assay was 0.8 μmol·L -1 and the intra-and inter-assay imprecision CVs were 2.3% and 3.2% respectively. Plasma volume changes were estimated based on resting and postexercise haematocrit and hemoglobin using the equations of Dill and Costill [33]. Haematocrit was measured by microcentrifugation and hemoglobin was assessed by the cyanmethemoglobin method.
Statistical Analysis
All statistical tests were processed using STATISTICA Software (StatSoft, France). The Shapiro-Wilk W-test of normality revealed that the data were normally distributed. The data were analyzed using a two-way analysis of variance (ANOVA) [3 (Ramadan) × 2 (time-of-day)] for the YYIRT, oral temperature, and peak heart rate (HRpeak), and a three-way ANOVA [3 (Ramadan) × 2 (time-of-day) × 2 (blood samples)] with repeated measures for the biochemical measurements. When appropriate, significant differences between means were tested using the Tukey post-hoc test. Paired t-tests were used to compare the morning-evening differences between BR, SWR, and ER. A probability level of 0.05 was selected as the criterion for statistical significance.
Ethical Considerations
After receiving a description of the protocol and its benefits and possible risks, each volunteer (and parents/tutors for the minors) provided a written informed consent. The experimental design was approved by the Clinical Research Ethics Committee of the National Center of Medicine and Sciences in Sport of Tunis and met the ethical standards of the Declaration of Helsinki.
Body Composition and Dietary Intake
Body mass (BM), fat mass (FM), and lean mass data are shown in Table 1. Statistical analyses indicated a decrease in BM and FM by the ER with respect to BR. Nevertheless, lean mass was unaffected by RF. Likewise, there was no significant difference between SWR and ER.
Compared with BR, the estimated daily energy intake was substantially reduced by the ER. This change was associated with a decrease in fat and saturated fat intakes and an increase in polyunsaturated fat, while carbohydrates and protein intakes remained stable during RF. Moreover, Folate, Vitamin A, Vitamin B12, Vitamin C, and Vitamin E intakes were unchanged during RF. For all these parameters, there was no significant difference between SWR and ER.
Temperature and YYIRT Performance
Oral temperature increased significantly from the morning to the evening BR, and during SWR and ER (P < 0.001). This increase was greater BR than SWR (+0.71 ± 0.32°C vs. +0.4 ± 0.28°C; t = 2.3; P < 0.05) and ER (+0.71 ± 0.32°C vs. +0.41 ± 0.32°C; t = 2.1; P < 0.05) (Figure 1). However, there was no significant difference between SWR and ER (+0.4 ± 0.28°C vs. +0.41 ± 0.32°C; t = 0.07). During RF, in comparison with BR, oral temperature was not affected in the morning. However, there was a significant decrease in oral temperature at ER in the evening.
The results showed that performance during the YYIRT improved between the morning and the evening BR (P < 0.05). However, these diurnal variations were not found during SWR and ER ( 101.82 ± 415.06; t = 0.9). Moreover, performance during the YYIRT decreased significantly during SWR and ER (P < 0.01 and P < 0.001 respectively) only at 17:00 h. Furthermore, the results revealed that HRpeak decreased during SWR and ER in the evening (P < 0.05, Table 2).
Biochemical Measurements
Before RF, TC and TG levels increased significantly during the YYIRT and showed a significant diurnal variation with higher levels at 17:00 h compared to 07:00 h (P < 0.01). However, during the SWR, although the values were similar to BR at 07:00 h, they decreased in the evening to reach a value indicative of blunted diurnal variations. At the ER, morning and evening levels of TC and TG decreased significantly (P < 0.01) in comparison with BR (Table 3).
LDL-C, Apo-B, and Lp-a showed stable values throughout the daytime BR with a significant increase during exercise only for LDL-C and Apo-B (P < 0.001). During RF, these parameters displayed a decrease in 17:00 h values at rest and during exercise (P < 0.05). At the ER, while both morning and evening Apo-B and Lp-a levels were significantly reduced (P < 0.01), there was a significant diurnal variation in LDL-C with higher morning values (P < 0.01). HDL-C and Apo-AI values increased after the exercise and showed a significant time-ofday effect with higher evening levels BR (P < 0.001). The rise was greater at 17:00 than 07:00 h. Moreover, levels of these parameters tended to be higher during RF (P < 0.01) and the Hcy and hs-CRP levels increased significantly during exercise (P < 0.01) BR with evening higher values. During the SWR, the diurnal variation of these parameters was blunted by a reduction in the evening values with respect to BR (P <
Discussion
The aim of this study was to investigate the effect of RF on the diurnal fluctuations of lipoproteins, apolipoproteins, hs-CRP, and Hcy during the YYIRT in soccer players. The results demonstrated that HDL-C and Apo-AI tended to increase, while LDL-C, Apo-B, TC, TG, Hcy and hs-CRP decreased during RF. Moreover, the diurnal pattern of these biochemical measures was modified by the ER.
Body composition, dietary intakes, and exercise performance
The present study indicated that BM and FM decreased during ER. These findings are in agreement with previous researches [11,34] and could reflect an increased use of lipids during RF as evidenced by previous works in Tunisian subjects [4,16]. Moreover, the decrease in BM and FM in the present study's subjects could be related to changes in the daily energy intake since dietary records revealed a decrease in the subjects' nutritional outcome associated with a decrease in fat and cholesterol intakes.
Concerning the YYIRT, performance improved between 07:00 and 17:00 h BR in accordance with our previous findings [27]. Moreover, the present study's results are consistent with our group's previous ones in which we observed that RF modifies the diurnal pattern in aerobic performance by decreasing performance in the afternoon but not in the morning hours [27].
Biochemical Measurements
The lipid profile is an important indicator of cardiovascular health. Studies on the RF effect on blood lipids, lipoproteins, and apolipoproteins were scarce and present inconclusive results. Our results indicated that TC and TG levels increased significantly during exercise. Similar results have been shown in trained young men during submaximal exercise [17]. BR, the results indicated a diurnal fluctuation in TG and TC with higher levels observed at 17:00 h. In this context, it has been demonstrated that postprandial lipid, lipoprotein, and apolipoprotein concentrations were affected by circadian factors [21].
Moreover, during the SWR and ER, values of TC and TG were similar to BR in the morning, but decreased in the evening suppressing the diurnal variation observed BR. Accordingly, previous research indicated that TG and TC levels decreased or didn't change during RF [19]. The decrease in TG and TC shown in the present study at 17:00 h during RF could reflect the beneficial effects of fasting associated to exercise on the lipid metabolism.
According to previous research [35], the present results indicated that HDL-C and Apo-AI values increased after the exercise and showed a significant time-of-day effect during BR; the rise being greater at 17:00 h. This diurnal pattern of various biochemical markers was, recently, evidenced by our group at rest and during exercise in soccer players [19,20]. Concerning the exercise's effect on Apo-AI, it has been shown that Apo-AI may [36] or may not [37] increase after a physical exercise.
Moreover, HDL-C and Apo-AI levels tended to be higher during RF. In this context, Adlouni et al [3] suggested that the feeding behavior that occurs during RF beneficially affects serum apolipoprotein levels. In their study, Apo-AI increased by the day 29 of RF compared with the BR levels. Similarly, recent findings showed that Apo-AI levels were higher in the latter stages of RF and post-RF in trained athletes [11,12,38].
Concerning the RF effect on biochemical responses to exercise, HDL-C and Apo-AI levels increased after the exercise consistently with the findings of Bouhlel et al [17] who reported increased plasma HDL-C during a submaximal exercise during RF. Moreover, in the present work, the time-of-day effect on HDL-C and Apo-AI was suppressed by the ER with a significant decrease in the evening values at rest and during exercise. The delayed bedtime and shortened sleep during RF could reduce the amplitude of the diurnal fluctuation of performance and biochemical responses [39]. Moreover, the modified diurnal pattern of the biological markers could be explained by the reduction in evening core temperature and the modified dietary habits [39].
Alternatively, the present study's results indicated that mean LDL-C, Apo-B, and Lp-a levels didn't show any diurnal fluctuation at rest and during the exercise. However, other investigators identified a significant circadian variation in these parameters [22]. In this context, although Bremner et al [22] found that these markers peaked in the morning, other investigators indicated that acrophases were typically observed in the afternoon [35].
In the present work, significant increase during exercise was found only for LDL-C and Apo-B but not for Lp-a levels. To the best of the authors' knowledge, it seems that there has been no study investigating the acute effects of exercise on these biomarkers.
During RF, there was a decrease in LDL-C, Apo-B as well as Lp-a values in the evening at rest and during exercise. By the ER, there was a significant diurnal variation in these parameters with higher morning values. It has been established that a given nutrient ingested at an unusual time can induce different metabolic effects [24]. Moreover, we showed that both morning and evening levels of these parameters were significantly reduced. Similarly, Adlouni et al [3] showed that Apo-B levels fluctuated during RF but were lower during ER in healthy subjects. In addition, Chennaoui et al [12] found that Apo-B concentrations tended to decrease following RF in middle distance runners. However, concerning LDL-C levels, the present study's results did not agree with those of previous research which indicated an increase [11] or no change [13] in trained subjects. The decrease in fat and saturated fat intake observed in the present study during RF could explain these findings. The interaction between fasting and the effect of timeof-day on physical exercise during RF leads to a decrease in LDL-C, Apo-B, and Lp-a, and may beneficially affect the normal pre-RF diurnal pattern of these parameters. In fact, these biochemical markers were discriminative for cardiovascular risk. As concluded in previous research [40], it seems that beneficial changes in lipid markers are influenced not only by the timing of blood sampling and any changes in diet (calorie restriction), but also by the decrease in initial fat and body mass, and possible changes in physical activity during RF.
The present study's findings indicated that both Hcy and hs-CRP levels showed a significant diurnal variation BR with higher evening values. The diurnal increase in post-exercise Hcy levels could be explained by the daily variation in core temperature and resting Hcy values as previously shown by Hammouda et al [19]. Concerning hs-CRP, however, studies in healthy individuals have not demonstrated any significant circadian variation [41]. The divergence between the literature results and those of the present study on hs-CRP could be explained by differences between the investigated populations concerning the training status and the mean age [19]. Moreover, the increase in Hcy and hs-CRP during the exercise indicated increased acute-phase responses and activated inflammatory process [42]. Moreover, resting and post-exercise levels of Hcy and hs-CRP decreased significantly during RF. Similarly, previous research indicated that RF positively influenced the inflammatory status of the body explained by a decrease in the IL-6 concentrations associated with decreases in both hs-CRP and Hcy [10]. However, in judo athletes, recent findings observed that although hs-CRP increased during RF, Hcy remained stable [38]. The difference between these studies may be explained by the influence of the intense judo training in the study of Chaouachi et al [38]. The decrease in inflammatory status in the present study could be explained by the reduced daily energy and fat intakes during RF. Indeed, intermittent fasting is considered as an alternative to caloric restriction regimen, which has been shown to reduce oxidative damage to lipids, protein, and DNA which implies a protective effect against oxidative stress [43]. Furthermore, the morningto-evening difference in resting and post exercise Hcy and hs-CRP was suppressed during the SWR in comparison with BR. It seems that the beneficial effects of caloric restriction on the inflammatory status are more pronounced in the evening after an average of 13-h of daytime fasting. The decrease in evening Hcy values is maintained during ER which led to inversed diurnal pattern. However, since Vitamin A, B12, C, and E and Folate intakes showed normal values during RF, it seems that the influence of RF on daily activities and biochemical responses is more a matter of chronobiology than calorie restriction. Moreover, the modified circadian pattern of cortisol, melatonin, and core temperature, previously observed during RF [39,43] could explain the morning-to-evening difference in hs-CRP and Hcy. In this context, Bogdan et al [44] observed a shift in the onset of cortisol secretion during RF and a rise in serum cortisol levels in the afternoon, while the morning rise was apparently delayed. In addition, the circadian distribution of hepatic and myocellular enzymes (ie, gammaglutamyltransferase, alkaline phosphatase, creatine kinase, and glutamic transaminase), indicative of muscle injury, was also altered by RF [45].
A limitation of the present study is that we didn't use a control group of untrained subjects. Moreover, oral temperature was not measured after exercise to estimate its changes with exercise at the different times-of-day. Furthermore, biochemical parameters were recorded only before and after the YYIRT. Therefore, further studies should investigate a delayed blood sample to check peak values.
In summary, the present study showed that RF and exercise can be combined favorably to reduce body mass and body fat, as well as improving lipid profiles and inflammatory status in soccer players. Indeed, prolonged intermittent fasting may have positive effects on the cardiovascular risk factors (ie, Hcy, hs-CRP, Apo-AI, Apo-B, and Lp-a). Moreover, our results suggested that calorie restriction induced by RF could modify the diurnal pattern of resting and post-exercise lipoproteins, apolipoprotein AI and B, as well as hs-CRP and Hcy levels. Since many factors (ie, diet, physical activity, time-of-day) could contribute to the modified diurnal pattern of lipid profile and inflammatory markers, it would be difficult to identify each factor's contribution. In future research, it would be of interest to standardize diet and time-of-day of biochemical measures to detect the main concomitant effects of RF and physical activity on lipid profile and cardiovascular risk markers. | v3-fos-license |
2018-06-16T13:08:48.578Z | 2018-06-15T00:00:00.000 | 49215932 | {
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} | pes2o/s2orc | Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.
This study examined the effects and mechanisms of strontium ranelate (SrRn)-a drug used to treat osteoporosis-on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papillae cells; MDPs). It also determined whether topical application of SrRn to exposed dental pulp tissue promotes the formation of mineralized tissue in vivo. The MDPs were cultured with or without SrRn, and cell proliferation, odonto-/osteoblastic gene expression, mineralized nodule formation, and Akt phosphorylation were evaluated. The formation of mineralized tissue in SrRn-treated pulp tissue in rat upper first molars was evaluated histologically. The SrRn up-regulated cell proliferation and expression of Alp (alkaline phosphatase), Bsp (bone sialoprotein), Dmp (dentin matrix acidic phosphoprotein)-1, Dspp (dentin sialophosphoprotein), and Oc (osteocalcin) in a dose-dependent manner. Mineralized nodule formation was also enhanced by SrRn. NPS-2143, a calcium-sensing receptor (CaSR) antagonist, and siRNA against the CaSR gene blocked SrRn-induced proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation. SrRn induced Akt phosphorylation, and this was blocked by NPS-2143. Topical application of SrRn to exposed rat molar pulps induced the formation of osteodentin-like mineralized tissue. Our study revealed for the first time that SrRn promotes proliferation and odonto-/osteogenic differentiation/mineralization of MDPs via PI3K/Akt signaling activated by CaSR in vitro; mineralized tissue forms from the dental pulp in vivo.
hard tissue-inducing mechanisms are similar to those of CH 17 . Currently, CH, MTA, and their derivatives are generally used for vital pulp therapy, but more effective pulp capping agents/materials with active dentin-like tissue-inducing capacity might increase the success rate of vital pulp therapy.
Strontium ranelate (SrRn) contains two strontium atoms coupled by ranelic acid. It is used to treat osteoporosis 18 . Previous clinical trials have shown that SrRn significantly improves bone mass/quality and increases bone strength via changes in the bone matrix properties and bone mineral density in osteoporotic patients 18 . SrRn is promising in the treatment of symptomatic osteoarthritis 19 . SrRn has gained attention in osteoporosis therapy because it offers two mechanisms of action, i.e., induction of osteoblastogenesis and suppression of osteoclastogenesis 20,21 . Furthermore, local application of SrRn with a collagen sponge to artificial bone defects formed in rat calvaria promotes the regeneration of bone tissue 22 . These findings indicate the potent mineralized tissue-forming capacity of SrRn and suggest that SrRn can be used on dentin (indirect pulp capping) and dental pulp tissue (direct pulp capping) as a pulp-capping agent with a potent capacity to form mineralized tissue. Thus, the aims of this study were (1) to examine the effects and mechanisms of SrRn on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papilla cells; MDPs); and (2) to determine whether topical application of SrRn to exposed dental pulp tissue promotes mineralized tissue formation in vivo.
Materials and Methods
Cell culture and chemicals. The MDPs were derived from the incisor apical buds of ICR mice and were immortalized by transfection of human papillomavirus type 16 E6 gene missing the PDZ domain binding motif 23,24 . They were cultured in alpha-modified minimum essential medium (α-MEM, Wako Pure Chemical Industries, Osaka, Japan) with 10% fetal bovine serum (FBS, HyClone/GE Healthcare, UT, USA) and an antibiotic and anti-fungal solution (penicillin-streptomycin-amphotericin B suspension; Wako Pure Chemical Industries) at 37 °C/5% CO 2 /100% humidity. SrRn (LKT Laboratories, St Paul, MN, USA) and calcium chloride (CaCl 2 ; Wako Pure Chemical Industries) were dissolved in α-MEM directly and sterilized with a syringe filter (Millex-HA filter, Merck Millipore, Darmstadt, Germany). NPS-2143 hydrochloride (dissolved in dimethylsulfoxide, 1.0 µM; Cayman Chemicals, Ann Arbor, MI, USA) and LY294002 (dissolved in dimethylsulfoxide, 1.0 µM; Cayman Chemicals) were used as selective and potent calcium-sensing receptor (CaSR) antagonists 25 along with a potent pan-PI3K/Akt inhibitor 26 Odonto-/osteoblastic gene expression. MDPs (5 × 10 4 cells/well) were seeded in 12-well plates. After 24 h of culture, the media was changed to include the test agents, and cells were cultured for 72 h. The total RNA was extracted by QuickGene RNA cultured cell kit S (Wako Pure Chemical Industries). The cDNA was converted from extracted RNA using Primescript (Takara, Shiga, Japan). The qPCR assays were performed with specific primers (Table 1) and GoTaq qPCR Master Mix (Promega, Madison, WI, USA) using a CFX96 Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). Gyceraldehyde-3-phosphate dehydrogenase (Gapdh) was the internal control.
Mineralized nodule formation. MDPs (1 × 10 4 cells/well) were seeded in 48-well plates and cultured in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS. After 24 h of culturing, the media was changed to an osteogenic medium containing L-ascorbic acid (0.2 mM; Wako Pure Chemical Industries) and β-glycerophosphate (5.0 mM; Sigma Aldrich, St. Louis, MO, USA) with or without test agents. Mineralized nodules were stained with alizarin red S (Wako Pure Chemical Industries) after 7 d of culture. The density of the mineralized nodules was measured by ImageJ software v2 (https://imagej.net/ImageJ2).
Western blotting. MDPs (2 × 10 5 cells/well) were seeded in 24-well plates. Test agents were added to the media after 24 h of culture. Cells were lysed with a RIPA buffer (20 mM Tris/HCl pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1% NP-40, 5% glycerol) containing cOmplete Ultra (Merck Millipore) and PhosSTOP EASY (Merck Millipore). Cell lysates were applied to SDS-PAGE (10%), and the gel was transferred to a PVDF membrane (Immobilon-P, Merck Millipore) using a semi-dry transfer system (0.15 mA, 1 h; WSE-4040, ATTO, Tokyo, Japan). Transferred membranes were incubated with anti-Akt (1:1000; polyclonal, GTX121936, GeneTex, Los Angeles, CA, USA) and anti-p-Akt (1:500; polyclonal, GTX121936, GeneTex) antibodies overnight at 4 °C followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000; Jackson Immuno Research Labs, In vivo study. All animal experiments were approved by the Animal Care and Use Committee of TMDU, all surgical methods were performed in accordance with relevant ethical guidelines and regulations (#A2017-155A). Wistar rats (n = 12, male, 5-wk-old; Clea Japan, Tokyo, Japan) were given ad libitum access to food and water prior to the experiment. The rats were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The cavity preparation and pulp exposure were performed in the upper first molars of both sides with #1/2 round bars using a dental handpiece motor under a stereoscopic microscope (Dental Microscope Z; Mani, Tochigi, Japan). Bleeding in the cavities following the pulp exposure was removed with sterile cotton pellets. The SrRn (mixed with sterile water at 2 mg/µl), mineral trioxide aggregate (ProRoot MTA, Dentsply Sirona, Ballaigues, Switzerland; mixed according to the manufacturer's instructions), or CaCl 2 (mixed with sterile water at 2 mg/µl) was dressed over the exposed pulp (n = 4 in each group). No application of SrRn, MTA, and CaCl 2 was used as a control. The samples applied to each cavity were randomly chosen from SrRn, MTA, CaCl 2 , and no application; there was no rat in which the same samples were applied contra-laterally. The cavities were sealed with glass ionomer cement (Ionosit-Baseliner, DMG, Hamburg, Germany). The rats were sacrificed by CO 2 euthanasia after 3 weeks. The upper jaws were dissected from the maxilla and fixed with 4% paraformaldehyde/ PBS for 24 hours at 4 °C. Samples were then demineralized using 17% EDTA (Dojindo Molecular Technologies) for 3 weeks. After demineralization, the glass ionomer cement was removed, and the samples were embedded in paraffin. Hematoxylin and eosin staining was performed on 5 µm-thick sections, and the stained sections were observed under a light microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany).
Statistical Analysis. All in vitro experiments were carried out in triplicates. The data were submitted to one-way ANOVA followed by Tukey's test. The level of significance was established at * P < 0.05 or ** p < 0.001 using Prism software v7 (GraphPad, San Diego, CA, USA).
CaSR is involved in the up-regulation of cell proliferation and differentiation/mineralization of MDPs induced by SrRn.
Next, we investigated the possibility that CaSR acts as a targets of SrRn in MDPs, because Sr 2+ is known to activate CaSR 27,28 , which is involved in the control of many important cellular functions such as proliferation and differentiation 29 . The promoted cell proliferation and expression of Alp, Bsp, Dmp-1, Dspp, and Oc induced by SrRn on MDPs were disrupted by NPS-2143-a selective and potent CaSR antagonist ( Fig. 2A,B). The CaSR siRNA down-regulated the mRNA expression of CaSR in MDPs and also suppressed the expression of Alp, Bsp, Dmp-1, Dspp, and Oc induced by SrRn in MDPs (Fig. 2C). Mineralized nodule formation promoted by SrRn in MDPs was blocked by NPS-2143 (Fig. 2D).
SrRn promoted mineralization of the exposed rat molar pulp in vivo. Finally, the impact of topical application of SrRn on exposed dental pulp tissue including the formation of mineralized tissue was examined in vivo. The application of SrRn to the exposed pulp tissue of rat upper first molars induced mineralized tissue formation in the pulp tissue of all specimens (Fig. 5A,B). The newly formed mineralized tissue exhibited an atubular, osteodentin-like structure with sparse or no cellular inclusions in the inner (pulp side) portion. This new tissue completely separated the pulp tissue from the pulp chamber exposed to the oral cavity. The pulp tissue appeared mostly normal with a few inflammatory cells. MTA also induced mineralized tissue formation in the exposed pulp tissue in all specimens; the mineralized tissue had irregularly aligned tubules and fewer cellular inclusions compared to the SrRn-applied specimens. The underlying pulp tissue appeared essentially normal (Fig. 5C,D). In contrast, application of CaCl 2 failed to induce continuous mineralized tissue formation; some islets of mineralized tissues were formed in the dental pulp tissue. Abscess formation was detected in the exposed area of the pulp tissue (Fig. 5E,F). Some of the CaCl 2 -applied samples showed deposition of mineralized tissues on the surface of the root canal wall dentin. Controls without treatment (Fig. 5G,H) failed to form mineralized tissue in the exposed pulp tissue. Four specimens were examined, and the results were reproducible.
Discussion
In this study, SrRn up-regulated the proliferation of MDPs (Fig. 1A), which possess dental pulp cell properties 23,24 . SrRn also increased the production of human dental pulp cells (see Supplemental Fig. 2). This is consistent with prior findings showing that SrRn up-regulates the proliferation of MC3T3-E1 cells (a mouse calvaria derived osteoblastic cell line 28,31 ) and a primary osteoblast cell line 32 . However, the opposite results have also been reported for MC3T3-E1 cells 33 . The reason(s) for such discrepancy remains unclear, but there is a large variation among the sub-clones of MC3T3-E1 34 , and differences in the properties of different MC3T3-E1 sub-clones may result in different results during cell proliferation. The SrRn also promoted the odonto-/osteoblastic gene expression in a dose-dependent manner (Fig. 1B) and induced mineralization in MDPs (Fig. 1C). Osteoblastic differentiation by SrRn is reported in primary murine bone cells 21 , murine marrow stromal cells 35 , and MC3T3-E1 cells 36 . SrRn also induces commitment of osteoblastic differentiation from mesenchymal stem cells 37 .
One of the targets of Sr 2+ is CaSR-a Class C G-protein coupled receptor 29 expressed on various cells including osteoblasts and their precursors 38 . Osteoblasts from the conditional knockouts of CaSR exhibit delayed differentiation, reduced mineralization capacity, and altered expression of regulators of mineralization 39 . This suggests that CaSR signaling in osteoblasts is essential for their differentiation and mineralization. The enhanced cell proliferation, odonto-/osteogenic differentiation and mineralization induced by SrRn in MDPs were blocked by NPS-2143 (a potent CaSR antagonist) and CaSR siRNA ( Fig. 2A-D). The NPS-2143 also inhibited the SrRn-induced proliferation of human dental pulp cells (see Supplemental Fig. 2). Furthermore, SrRn promoted CaSR expression in MDPs (see Supplemental Fig. 3). These results indicate that SrRn may promote the proliferation and differentiation/mineralization of MDPs via CaSR signaling.
Involvement of the PI3K/Akt pathway is also reported in SrRn-induced osteogenic differentiation of human osteoblasts 30 . The PI3K/Akt signaling stimulates canonical Wnt signaling 37 , and this may be involved in promoted proliferation, differentiation, and mineralization of dental pulp cells due to SrRn. We previously reported that Wnt signaling upregulates odontoblast marker expression in MDPs 45 , and Wnt signaling activated by SrRn via CaSR/PI3K/Akt signaling may induce odonto-/osteoblastic gene expressions of MDPs. The PI3K/Akt signaling activates the production of BMP2 46 , which may also be involved in the differentiation and mineralization of MDPs. Ca 2+ is an agonist of the CaSR with a higher affinity than Sr 2+41 ; CaCl 2 (2 mM) promotes the proliferation of MC3T3-E1 cells 28 . However, CaCl 2 (1 mM) did not enhance the proliferation and differentiation/mineralization of MDPs in our experiment (see Supplemental Fig. 1) suggesting that 1 mM of Ca 2+ concentration may be too low to induce proliferation and differentiation/mineralization of MDPs.
In vivo application of SrRn to rat upper first molars induced tissue mineralization along the exposed pulp tissue (Fig. 5A,B). Mineralized tissues formed in CH-treated pulp tissue have been reported to be irregular with tubular openings or canalicular lumina containing vessels and cells 11 . The mineralized tissue induced by SrRn in this study had an atubular, osteodentin-like structure with sparse or no cellular inclusions in its mineralized structure indicating that it might have sufficient capacity to protect the underlying pulp tissue from the oral environment. The MTA induced dentin-like mineralized tissue (Fig. 5C,D) as previously reported 14,15 , and it offers slow-release of Ca 2+47 . In contrast, CaCl 2 failed to induce homogeneous mineralized tissue did form islets of mineralized bodies in the exposed pulp tissue (Fig. 5E,F). Ca 2+ is reported to have mineralized tissue formation capacity 28,47 , which agrees with these findings. However, CaCl 2 did not induce a continuous mineralized barrier to separate the intact pulp tissue from the oral cavity suggesting that an application method resulting in continuous and slow release of Ca 2+ might be essential to the formation of a mineralized dentin-like barrier.
Systemic side effects should always be considered in developing new treatment agents. Lithium chloride (LiCl 2 )-a GSK3b inhibitor and potent activator of Wnt canonical signaling-has recently been reported to induce mineralized tissue formation following direct application to the rat pulp tissue 48 ; however, a high intake of LiCl 2 can cause a risk of confusion and speech impairment including a risk of death at 20 mg/L of LiCl 2 49 .
The systemic side effects of SrRn are also of note and include cerebrovascular disease, peripheral vascular disease, and prior myocardial infarction. These were reported in patients given a 2,000 mg daily systemic dose treatment of SrRn for a year 50 . However, it is reasonable to suppose that topical application during vital pulp therapy may pose a much lower risk of side effects compared to systemic application 32,37,51,52 ; in vivo studies using rat models reported that systemic use of SrRn (625 g/kg) is well tolerated and safe with no adverse side effects 53,54 . However, it is necessary to evaluate the systemic side effects induced by the local application of SrRn before its clinical application.
In conclusion, we revealed for the first time that SrRn increases the proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation of dental pulp-like cells. This may be partially mediated by CaSR/ PI3K/Akt signaling (Fig. 6). Our in vivo study revealed that topical application of SrRn induced a continuous barrier of osteodentin-like mineralized tissue on the rat exposed pulp tissue highlighting the potential utility of SrRn as a new pulp-capping material. | v3-fos-license |
2020-08-20T10:12:41.714Z | 2020-08-19T00:00:00.000 | 221704624 | {
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} | pes2o/s2orc | Dataset on Insilico approaches for 3,4-dihydropyrimidin-2(1H)-one urea derivatives as efficient Staphylococcus aureus inhibitor
Series of anti- Staphylococcus aureus were studied via quantum chemical method and several molecular descriptors were obtained which were further used to develop QSAR model using back propagation neural network method using MATLAB. More so, the molecular interaction observed between 3,4-dihydropyrimidin-2(1H)-one Urea Derivatives and Staphylococcus aureus Sortase (PDB ID Code: 2kid) via docking was used as a screening tool for the studied compounds. The observed molecular compounds used in this work was also correlated to Lipinski rule of five and the developed QSAR model using selected descriptors from the optimized compounds was also examined for its predictability. Also, the observed molecular docking revealed the interaction between the studied complex.
Specification
Computational Chemistry Specific subject area
Type of data
Developed QSAR Model Equation Figure Table How data
Value of the data
• Datasets obtained in this research will help the scientists to know the molecular descriptors which describe the anti-Staphylococcus aureus properties of 3,4-dihydropyrimidin-2(1H)-one Urea Derivatives. • Data in this research will reveal the contribution of each calculated descriptor in the developed QSAR model. • It also helps in predicting library of efficient drug-like compounds via the developed QSAR model. • The ability of each observed compounds to inhibit Staphylococcus aureus via docking can also be understood.
Data description
The molecular compounds used in this work were displayed in Table 1 . In this work, sixteen molecular compounds were subjected to density functional theory via B3LYP with the standard 6-31G * * basis set for optimisation and the obtained molecular descriptors were reported for further investigation. 3,4-dihydropyrimidin-2(1H)-one Urea derivatives was extracted from the work done by Mukesh, 2015 [1] . Table 1 The Schematic diagram of 3,4-dihydropyrimidin-2(1H)-one urea derivatives [1] . Table 2 reveal the calculated molecular descriptors via density functional theory [2] . Series of calculated molecular parameter obtained were highest occupied molecular orbital (E HOMO ), lowest unoccupied molecular orbital energy (E LUMO ), band gap, molecular weight, Log P, Area, Ovality, polar surface area, polarisability, hydrogen bond donor (HBD), hydrogen bond acceptor (HBA) and number of rotatable bonds. Further investigation was conducted using Lipinski rule of five so as to determine the drug-likeness of the studied drug-like compounds [3] . Table 3 showed the developed QSAR model using the calculated molecular descriptors using back propagation neural network (BPNN) via MATLAB software [ 4 , 5 ]. The developed QSAR model involved molecular weight, volume, polarisability, E HOMO and Log P. This set of descriptors were chosen because they best described anti-Staphylococcus aureus activities of compounds used in this work than other calculated descriptors. The calculated correlation coefficient (R 2 ) for the developed QSAR model was 0.930. The developed QSAR model was validated by considering several parameters such as Adjusted R 2 , Cross validation (C.VR 2 ), P-Value, F-Value. Also, the molecular compounds used were divided in to two (Test set and Training set). The compounds used as training set were compound A2, A3, A5, A6, A7, A8, A9, A10, A11, A14, A15 and the compounds used as test set were A1, A4, A12, A13 and A16. Therefore, table 4 reveal the effectiveness of the developed model shown in Table 3 . Also, correlation between the observed and the predicted inhibition concentration was displayed in Fig. 1 . More so, five (5) molecular compounds were proposed and the IC 50 were predicted using the developed QSAR model ( Table 5 ). Table 6 Interactions between 3,4-dihydropyrimidin-2(1H)-one Urea Derivatives and Staphylococcus aureus sortase (PDB ID Code: 2kid ). Series of 3,4-dihydropyrimidin-2(1H)-one Urea Derivatives were docked against Staphylococcus aureus sortase and the binding affinity, inhibition constant as well as amino residues observed in the interaction between 3,4-dihydropyrimidin-2(1H)-one Urea Derivatives and Staphylococcus aureus sortase (PDB ID Code: 2kid ) [6] were displayed in Table 6 . The residues involved in the interaction were displayed in SI.
More so, the interaction between the proposed compounds and Staphylococcus aureus sortase (PDB ID Code: 2kid ) were displayed in Table 6 . The molecular interaction between the molecular compounds used and the receptor were displayed in SII.
Experimental design, materials, and methods
In this work, series of vital materials (Software) were used to accomplish this research [7] . Spartan'14 was used to optimised 3,4-dihydropyrimidin-2(1H)-one Urea derivatives studied in this work. The density functional theory used for the optimisation was achieved using three-parameter B3LYP that comprises Becke's gradient exchange correction [ 8 , 9 ], Lee, Yang, as well as Parr correlation functional [10] . It was through this that several molecular descriptors were obtained to develop QSAR model using BPNN via MATLAB software. Also, docking was accomplished using pymol 1.7.4.4 software. It was used for treating (removal of foreign compounds) downloaded Staphylococcus aureus Sortase (PDB ID Code: 2kid ) from protein data bank ( www.rcsb.org ). Also, the treated Staphylococcus aureus Sortase (PDB ID Code: 2kid ) was subjected to autodock tool 1.5.6 so as to locate the binding sites in the receptor and convert the receptor as well as the ligand to the format which will acceptable by autodock vina 1.1.2 that will do the docking calculation. The use of autodock tool 1.5.6 require the use of commands in order to accomplish the docking calculation; to execute the calculation, vina -config conf.txt -log.txt was used. Also, vinasplit -input out.pdbqt was used to split the calculated binding affinity according to the energy of each conformation. The observed grid box was as follows: centre ( X = 0.677, Y = 0.25, Z = −1.245) and size ( X = 64, Y = 52, Z = 56).
Funding
This research received no external funding .
Declaration of Competing Interest
The authors declare that they have no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | Methodology to compare costs of sanitation options for low-income peri-urban areas in Lusaka, Zambia
Urban slums and low-income peri-urban areas in developing countries are characterised by a lack of infrastructure. The absence of sustainable sanitation systems is one of the causes that can lead to a high level of water-borne diseases in these areas, especially during the rainy season. This paper presents a methodology for cost comparisons of sanitation system options with a focus on excreta management (a sanitation system consists of the household toilets, collection and transport of excreta, treatment and storage, and transport of sanitised excreta to reuse sites). Greywater collection and treatment are excluded from the analysis for simplicity reasons. We used three low-income peri-urban areas in Lusaka, Zambia, to demonstrate our proposed methodology. The population density in the three peri-urban areas ranges from 104 to 244 people/ha. Unlined pit latrines are the most common form of excreta management, even though drilled boreholes and shallow wells are used as sources for drinking water in the same areas. Based on four selection criteria (no use of water for transporting the waste, low costs, waste sanitisation, and no contribution to groundwater pollution from stored excreta), we have short-listed two options which meet most or all of the criteria: A conventional low-cost option (Option 5: VIP latrines with downstream processing) and an ecological sanitation option (Option 6: urine-diversion dehydrating (UDD) toilets with downstream processing). The concept designs for both options are based on the entire peri-urban population in Lusaka of approximately 1.23 m. people, and on the assumption that 12 residents who live on the same plot (or ‘compound’) would share one toilet. The paper details the assumptions used to create a set of default model input parameters which are used in the cost equations to calculate capital costs, annual operating costs and net present values (NVP). Based on this basic financial analysis, we calculated the following indicative costs: capital costs of 31 €/cap and 39 €/cap for Option 5 and Option 6, respectively. Annual operating costs per capita were estimated to be 2.3 €/a·cap and 2.1 €/a·cap) for Option 5 and Option 6, respectively. The NPV for Option 6 is about 14% higher than for Option 5 but the difference is not significant, given the accuracy of the cost estimate (about ± 25%). Overall, this paper shows that the two options are difficult to differentiate based on cost alone. The financial model allows examination of the relative contributions of the different components to the overall cost of the sanitation system. For example, the costs of urine storage and transport are significant contributors to the capital and operating costs of the Ecosan option, and ways to reduce these costs should be investigated.
Introduction Background
As a consequence of the rapid urbanisation process in many developing countries, communities of very poor people are now living in the inner city or periphery of those rapidly growing cities. These urban slums and unplanned low-income peri-urban areas are characterised by a lack of infrastructure.
Provision of safe water and sustainable sanitation for the urban poor is required as one of the factors to ensure public health, but is challenging for reasons such as insecure tenure, lack of political will, financing, cost recovery and choice of technical options. If municipalities and commercial utilities want to provide low-cost sanitation to peri-urban areas, which of the following technologies should they select for excreta management?
We propose a methodology for comparing costs of sanitation options, consisting of the following steps: • Analyse existing sanitation situation (we used three periurban areas in Lusaka as an example) • Define possible sanitation options and selection criteria • Short-list a small number of options (two in our case) based on the selection criteria • Prepare concept designs for the short-listed options • Prepare cost estimates based on the concept designs, using basic cost equations proposed in this paper • Compare results based on overall cost (net present value) and other sustainability factors (other sustainability factors are only touched upon in this paper).
In this paper, we focused on excreta management options and did not include greywater management -for reasons of simplicity and because in the peri-urban areas of Lusaka, excreta management is thought to have more urgency compared to greywater management with respect to public health protection.
Description of three peri-urban areas in Lusaka
Lusaka is the capital of Zambia in Southern Africa with a population of approximately 2 m. people in 2005 (annual growth rate of 3.5 %/a (CSO, 2003)); of which approximately 1.23 m. people live in low-income unplanned peri-urban areas, a majority of which are slum-like in character (GKW, 2005). The water supply service in the peri-urban areas of Lusaka is rudimentary, and sanitation service provision by Lusaka City Council to those areas is almost non-existent. We selected three typical peri-urban areas (Bauleni, Chawama and John Laing) to collect baseline information for the development of subsequent options. The fieldwork was carried out in Lusaka from November to December 2005 and is described in detail in Mayumbelo (2006). Field observations and informal discussions were used to investigate the current water and sanitation practices, state of the infrastructure and residents' health with respect to water-borne diseases. Table 1 summarises important characteristics of the three study areas, and Table 2 summarises the main research findings with regards to sanitation and water aspects for the three areas.
Sanitation provision in the peri-urban areas is generally left to the initiative of the residents who mostly use unlined pit latrines that they dig within their plot boundaries. The pits are covered with soil once they are full. The liquid fraction of the excreta percolates into the ground and ultimately reaches the groundwater. The groundwater table ranges from deep (approx. 30 m) to shallow (approx. 1 m). Karst features of the geological formations underlying Lusaka make it complicated to predict in which direction and at what velocity groundwater will flow, and makes it difficult to dig new pits (see Fig. 1).
Many residents in Chawama and John Laing use hand-dug shallow wells as a source of drinking water (see Fig. 2). This practice can be detrimental to their health since the groundwater quality in Lusaka fluctuates seasonally: it tends to deteriorate during the rainy season when pollution and recharge occurs due to pit latrines and the presence of preferential fast-flow mechanisms in the karst rock formations (Nkuwa, 2002). This problem is one of the causes of recurrent outbreaks of waterborne diseases in Lusaka's peri-urban areas (Mayumbelo, 2006). It is in fact a general problem for many areas in sub-Saharan
595
Africa with shallow urban groundwater, where the increase in diarrhoea incidents in the rainy season can be attributed to pit latrines in peri-urban areas (Lerner, 2004). Diarrhoeal diseases can be caused by many different factors and disease transmission routes; inadequate excreta management is an important factor and is the focus of this paper.
Sanitation system options for peri-urban areas in Lusaka
Options short-listing procedure We advocate applying a 'systems approach' to sanitation and as such, 5 major parts of the sanitation system ought to be distinguished ( Fig. 3): • Part A: Household toilets • Part B: Collection and transport of excreta from households to treatment site • Part C: Treatment and storage of excreta at (semi-) centralised location • Part D: Transport of sanitised excreta from treatment site to agricultural fields • Part E: Reuse of excreta in agriculture (sale of fertiliser).
For rural areas, the distances between Part A and Part E are very short, and they may therefore have negligible impact on the overall cost. But for urban situations, Part B to D may have significant cost implications. Another consideration is that whilst Part A can be designed for individual households, Parts B to E should be designed to cover many households to achieve economies of scale.
We considered six sanitation options, which are schematically presented in Fig. 4. Each of the six options is meant to cover the entire system (Part A to E). Options 1 to 5 are wellknown conventional sanitation options, whereas Option 6 is the still relatively unknown ecological sanitation (Ecosan) option (Drangert, 1998), which is described in more detail below.
Ecosan is a new paradigm in sanitation which aims to enable safe reuse of sanitised excreta and greywater (Winblad and Simpson-Hébert, 2004) and be sustainable in all aspects. The nitrogen, phosphorus and organic matter in sanitised urine and faeces can be used in agriculture as a fertiliser and soil conditioner, respectively. This aspect is particularly important for poor people living in areas of nutrient-depleted soils in sub-Saharan Africa who cannot afford to purchase artificial inorganic fertiliser. In general, Ecosan options do not rely on the soil for storage of excreta and infiltration of urine, and therefore significantly reduce the danger of leaching of nitrate and pathogens into groundwater (as may occur from the pits of pit latrines). A further advantage is that Ecosan technologies can typically be implemented at much lower costs than conventional waterborne sewers (UNEP, 2004).
Ecosan interventions also have the potential to contribute to a whole range of Millennium Development Goals (Millennium-Project (2005), Rosemarin (2003), Von Münch et al. (2006)), e.g. those related to basic sanitation provision, improvement of lives of slum dwellers, reduction of hunger, extreme poverty and child mortality. Higher agricultural yields of fields fertilised with Ecosan products can lead to a lower incidence of malnutrition and hence lower levels of morbidity.
The urine-diversion dehydrating (UDD) toilet is one of the numerous possible toilet types that can be used within an Ecosan approach. It separates the urine and faeces in the toilet, and the two substances are stored and treated separately from each other (GTZ, 2007). The faeces are air-dried in a ventilated single vault or double vault configuration (the second vault is used once the first vault is full), with the aim to achieve pathogen kill and volume reduction. The single vault system is used for the cost comparison in this paper because of its lower investment costs. Its main disadvantage is that some of the faeces are still fresh when the vault is being emptied; the associated health risk can be managed for example by ongoing hygiene education programmes for the workers who empty the toilet vault after one year (Moilwa and Wilkinson, 2006).
UDD toilets do not use water for flushing, which is important for areas with unreliable water supply, such as the peri-urban areas of Lusaka. UDD toilets are also quite simple to operate (compared to some composting toilet types), resilient to floods, and the toilets can be located on any level inside the house. The dried faecal matter from a UDD toilet is less offensive and odorous than faecal sludge from pit latrines because faeces are not combined with urine or water. For these reasons, the UDD toilet is used to represent Part A of the Ecosan option (Option 6) in this cost comparison.
In order to narrow down the available options for the purposes of a cost comparison, we can consider sustainability criteria. Based on the approach presented by Kvarnström and Af Petersens (2004), we applied the following 4 selection criteria for the short-listing: • Should not require water for transporting waste (poor water supply levels in peri-urban areas) • Have low capital, operation and maintenance costs to be financially sustainable • Should sanitise the waste to destroy pathogens to protect public health • Should not contribute to groundwater pollution via leaching of nitrate and pathogens from stored excreta, since shallow wells are being used as a drinking water source (it is unlikely that this practice will be abandoned in the foreseeable future in Lusaka and replaced by piped (treated) water from other sources, mainly due to lack of capital).
597
Options 1 to 4 (shown in Fig. 4) are discarded because they do not meet the first two selection criteria. Especially Option 1 (conventional sewer system) is comparatively expensive and requires a high level of institutional capacity and skilled work-force.
Of the six options considered, the only option that satisfies all the selection criteria is Option 6 (UDD toilets with downstream processing). Option 5 (VIP latrines with downstream processing) does not meet the last selection criterion: with the difficult ground conditions in Lusaka, pit latrines can contribute to groundwater pollution via leaching of nitrate and pathogens (pits are designed to allow infiltration of liquid; a 'lined pit latrine' only has lining at the top part of the hole to stabilise it).
In general, pit latrines are not appropriate when the groundwater table is shallow, the population density is high and groundwater is used for drinking water, the ground is underlain by pervious rock (e.g. karst geology) or rock that is difficult to excavate, the area has a potential for flooding, or the population has no means to either dig new pits or to safely empty full pits and treat the faecal sludge.
Even though Option 5 does not meet the fourth selection criterion (related to potential impact on groundwater), it is nevertheless included in the cost analysis in order to test the common perception that an Ecosan option (Option 6) is more expensive than a conventional low-cost on-site sanitation option (Option 5). It should be pointed out that groundwater contamination can also be caused by other factors, e.g. greywater infiltration (Carden et al., 2007), agricultural runoff, industrial pollution, or when residents use contaminated buckets to draw water from shallow wells. Hence, even with Option 6 implemented, groundwater pollution could still continue to be a problem in Lusaka if the other causes for groundwater pollution are not addressed as well.
Concept designs of two short-listed options
The concept designs of the two short-listed options are summarised in Table 3. They are based on the entire peri-urban population in Lusaka of 1.23 m. people, because certain components (e.g. treatment plant, vacuum tankers or trucks) are more economical on a larger scale. One toilet would be built per plot, and each toilet would be shared by three four-member households.
If the project was implemented, one would first begin with smaller pilot schemes to test the design. For the purposes of the cost estimates presented here, a full-scale implementation is assumed to demonstrate the approach for cost estimating (sanitation pilot projects typically have a higher per capita cost than full-scale projects).
As shown in Table 3, our concept design for Option 6 includes a centralised storage facility for the dried faecal matter and urine. Other treatment options (on plot or centralised) for the faecal matter could include: • Co-composting with organic waste (as assumed for Option 5) • Burial of the faecal matter in the ground provided the groundwater table is very deep and precipitation is not that heavy and frequent (Guness et al., 2006) Assume 5 workers managing the incoming and outgoing flows of material.
Part D: Transport of san. excreta Open trucks could be used but they are not included in cost estimate because we assume that the farmers who buy the fertiliser will organise the transport. Farmers will also need further urine storage of some form because nitrogen fertilisation is not carried out all year round; storage in the ground (soil) may be an option in some cases. Table 7). c For faecal sludge pumping to work, water has to be jetted into the pit to liquefy the faecal sludge sufficiently. The fertiliser produced from either option could be used in agriculture or in public gardens, parks, potted flowers or potted plants. It is likely that the solid fertiliser produced in Option 6 is of higher quality than the compost produced in Option 5 because the former is less contaminated with other substances and has a higher nitrogen content (no leaching of nitrogen into the ground). We used a conservative estimate for the sales price of compost or solid fertiliser in our calculations (2 €/t). Others have reported approximately 28 €/t for compost made from organic solid waste (Rothenberger et al., 2006) and 22 €/t for compost made from faecal sludge and organic solid waste (Vodounhessi and Von Münch, 2006). The land required to absorb all nitrogen in the excreta is approximately 39 000 ha for Option 6 (based on 5.7 kg N excreted/person·a and an application rate of 180 kg N/ha·a for maize (Jönsson et al., 2004)). Excreta collected from Option 5 could only fertilise a smaller area because nitrogen of the urine would seep into the ground at the location of the VIP latrine. The total area of Lusaka Province is 2 200 000 ha (CSO, 2003) and Lusaka City itself is approximately 36 000 ha. Hence, the area of 39 000 ha which would have to be set aside for (urban) agriculture represents about 2% of the total Lusaka Province area.
Financial model for short-listed sanitation options
Various authors have published cost estimates for sanitation systems (e.g. Hutton and Haller (2004), Rockström et al. (2005)) but the reported costs are often difficult to compare, e.g. because they only include Part A of the sanitation system, or only the first year of operation. A useful tool for a basic financial comparison of sanitation options is the Net Present Value (NPV) of the capital and annual operating costs of the entire sanitation system (Part A to E in Fig. 3). The option with the lowest absolute value for NPV is regarded as the most attractive option from a purely financial point of view.
We used a discount rate of 12% (equivalent to government borrowing rate) and a time period of 10 years in our NPV analysis; during this time-frame the options considered would not require replacements or major repairs. Where possible, we have formulated general cost equations for the financial model. The input parameters for the financial model (see Table 6) can be varied by the user of the model and should be verified for a specific application of the model.
The monetary benefits of Option 5 and 6 with respect to public health improvements are likely to be quite similar, and a cost-benefit analysis was therefore not carried out (the expected impacts on groundwater quality improvements for the two options are very difficult to assess in financial terms).
Capital costs
Capital costs for both options are summarised in Table 5, and the costs for Part A are explained in more detail below. The cost of a toilet increases with increasing volume of its 'substructure', i.e. the pit or vault. The minimum volume of the substructure is calculated by Eq. (1) below (parameter descriptions and values are provided in Table 6).
The substructure volume of the toilets in Option 5 is much larger than the substructure volume of toilets in Option 6 because of the longer time period between desludging events assumed for Option 5, due to access restrictions for the vacuum tankers (see parameter T d in Table 6). The capital cost for one toilet of Part A of Option 5 (see Table 4) consists of pit excavation, pit lining (to 1.2 m depth), cover slab, superstructure and a vent pipe. For Option 6, the cost items for one UDD toilet are a floor slab, faeces vault, cover slab, superstructure, a vent pipe, 2 x 200 ℓ plastic barrels for urine storage, a urine-diversion squatting pan and a bucket for sand or ash. The cost of a simple superstructure is identical for the two options. Details of the cost estimates (bill of quantities) are provided in Mayumbelo (2006).
Operating costs
The equations used in the financial model to predict the operating costs of Part B, C and E (C Part i, op ) are shown below (symbol names, parameter values and units are provided in Table 6). The operating costs of both options are summarised in Table 8. C Part B, op = F d ·N Lsk /N P/t · C ve + (F w,1 ·p f + p urine )·N Lsk ·CB t,1 /V tv (2) C Part C, op = F w,1 · p f · N Lsk · C tr,s + N w · C w,a C Part E, op = -ρ comp · F w,2 · F w,1 · p f · N Lsk · C comp -p urine · N Lsk · C urine (4) A); includes material and labour cost for superstructure and substructure (pit for Option 5, vault for Option 6); assuming cross-sectional area of 1.5 m 2 for both options, and unused pit depth (freeboard) of 0.6 m for Option 5. Zero freeboard assumed for Option 6. (in € unless otherwise indicated) -based on concept design in Table 3 Part Option 5 (VIP toilets + processing) Option 6 (UDD toilets + processing)
Comments
Part A Toilets with sub-and superstructure 35 650 000 38 010 000 Based on The default model parameter values shown in Table 6 are the result of a simplistic analysis, which does not take into account the fact that the residents will spend part of their day outside the peri-urban areas, the lower excreta production rates of children, nor the weight of the material added after defecation to cover the material and absorb moisture. The quantities of excreta to be moved in the two transport steps (Part B and Part D) are shown in Table 7. A summary of the financial model output values is provided in Table 9.
Discussion of cost estimates
The following observations can be made regarding the capital costs: • The accuracy of the cost estimate is expected to be ± 25% for a concept design of this nature. Hence, whilst the capital cost and NPV are higher for Option 6 than for Option 5, this difference is not significant compared to the accuracy of the estimate. • Part A (toilets) constitutes by far the largest contribution to the overall capital costs for both options. Hence the biggest potential for capital cost savings lies in Part A. • The second biggest contributor to the costs of Option 6 is the urine storage facility. Urine storage has two purposes: hygienisation (e.g. to reduce those pathogens that stem from cross-contamination with faeces); and storage (i.e. storing urine while it is not needed by the farmers). Longer urine storage times reduce health risks but also increase capital costs. We used two weeks as a minimum time needed to buffer farmers' demand but clearly, the practicalities of this assumption need further consideration.
The following observations can be made regarding the operating costs: • The operating costs are slightly lower for Option 6 than for Option 5, but the difference is not significant within the accuracy of the estimate • The largest contribution to the operating costs originates from Part B (excreta collection and transportation to treatment plant) for both options. The high transport costs of the urine barrels are a potential barrier to the adoption of Option 6. Transport in small-bore pipes, together with greywater, could be an alternative option in some cases but may be more capital cost intensive. • The sale of urine has potential to generate a significant income due to its nitrogen and phosphorus content whilst being virtually pathogen-free. The achievable sales price for urine requires further investigations (a conservative sales price was used here).
The NPV values of both options are close to each other (the NPV of Option 6 is only 14% higher than the NPV for Option 5). In summary, we can conclude that in the case of Lusaka, the Ecosan option (Option 6) cannot be ruled out based on cost, compared to the conventional VIP latrine-based option (Option 5).
Conclusions
Because Ecosan is still a relatively new and little-known approach to sanitation, many municipalities do not realise that it could be a viable and cost-effective alternative to sewer-based sanitation systems, septic tanks or pit latrines. Decision makers need adequate information regarding the costs of the entire sanitation system. Many previous publications that dealt with costs of sanitation only provided the capital costs of the toilet without the accompanying downstream processing infrastructure and annual operating costs. We have developed basic equations to calculate: • The minimum volume of the toilet's substructure; this has an impact on the capital cost of Part A (toilets); • Operating costs of Part B (transport), Part C (treatment/storage) and Part E (sale of fertiliser) The equations use a set of input parameters for which default values suitable for conditions similar to Lusaka are provided (we used 1.23 m. people living in peri-urban areas as a design basis). The capital cost and NPV for Option 6 (UDD toilets with processing) were found to be higher than for Option 5 (VIP toilets with processing), whilst the annual operating cost of Option 6 was slightly lower (but the difference was less than the expected accuracy of ± 25% for a concept design of this nature). The costs presented in this paper are of an indicative nature and serve to illustrate our cost comparison methodology. Further work is needed to refine the detailed design of the options as well as the values of the model input parameters, which will also vary from country to country. In order to contribute to public health improvements in periurban areas, it is necessary to have a sanitation system that is sustainable in all aspects, i.e. socially, technically, environmentally, institutionally and financially. This paper focuses on the financial aspects of sanitation systems. Financial sustainability is only one aspect in the decision making process amongst many others (e.g. user acceptance, cultural factors, institutional capacity to name but a few). One conclusion that can be drawn from our analysis of the entire sanitation system, is that the proposed Ecosan option is cost competitive compared to the commonly used option of VIP latrines with downstream processing -with the Ecosan option having the added benefit of a lower potential for groundwater pollution via leaching of nitrate and pathogens from stored excreta. | v3-fos-license |
2018-04-03T04:13:06.120Z | 2016-12-20T00:00:00.000 | 7206344 | {
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} | pes2o/s2orc | Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance
Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.
Introduction
The aldehyde dehydogenase (ALDH) superfamily consists of 19 isozymes, which catalyze the oxidation of endogenous and exogenous toxic aldehydes (short, long aliphatic and aromatic) into non-toxic corresponding acids [1,2]. Modest consumption of ethanol is believed to be beneficial for health, whereas large consumption can cause several complications [3,4]. Ethanol is first oxidized by alcohol dehydrogenase into the primary metabolite acetaldehyde, which is mutagenic and placed in group 1 carcinogens [5]. ALDH than detoxifies acetaldehyde by oxidizing it to acetic acid. More than 40% East Asian population (~560 million or~8% of the world population) cannot oxidize the toxic acetaldehyde into the non-toxic acid because of having a point mutation in the ALDH2 gene which results in almost no enzyme activity, leading to the accumulation of the toxic acetaldehyde [6][7][8]. ALDH2 deficient people are very sensitive to alcohol, a small dose of it can cause severe consequences like nausea, facial flushing, tachycardia and long-lasting headache [9]. There are many reports indicating the chances of occurrence of malignancy and other serious health problems due to ALDH polymorphism [10]. Particularly, the occurrence of squamous cell carcinoma in the upper aerodigestive track (UADT) among the middle aged East Asian populations is associated with the ALDH2 polymorphism [6,11]. There is more than 80 fold increased risk for squamous cell carcinomas in the UADT of heavy drinker heterozygotic individual (ALDH Ã 1/ Ã 2) compared with only about 4 fold increase in the wild-type (ALDH Ã 1/ Ã 1) heavy drinkers [6, [12][13][14].
Human salivary aldehyde dehydrogenase (hsALDH) found in the saliva is basically ALDH3 isoform of the ALDH enzyme. ALDH3 is highly expressed in the epithelial lining of the UADT, kidney, mammary gland, liver and stomach [15,16]. HsALDH catalyzes the oxidation of the aromatic aldehydes having bulky side chains, medium/long chain aliphatic aldehydes and α,β-hydroxyalkenal aldehydes, but not acetaldehyde under basal condition [17]. Moderate consumption of ethanol by ALDH2 Ã 1/ Ã 2 heterozygote individuals leads to a significant increase in the acetaldehyde concentration in the saliva which forms an adduct interacting with DNA, resulting in severe health issues [18]. There are reports on strategies to increase the rate of acetaldehyde elimination especially in ALDH2 Ã 1/ Ã 2 heterozygotes through the modulation of the activity of ALDH2 by chemical chaperones, and by inducing and recruiting other ALDH isoforms [4, 19,20].
The cruciferous vegetables which include broccoli, cauliflower, radish, kale, cabbage and brussels sprouts are a rich source of health beneficial secondary metabolites [21]. Much of the health benefits have been attributed to the physiological effects of the isothiocyanates, especially sulforaphane (SF) which has been shown to protect against various types of cancers, diabetes, atherosclerosis, respiratory diseases, cardiovascular diseases, neurodegenerative disorders and ocular disorders [22,23]. A number of mechanisms by which SF protects cells have been reported which includes induction of apoptosis, cell cycle arrest, anti-inflammatory effects, inhibition of phase 1 (cytochrome P450) enzymes and induction of phase 2 detoxification enzymes [23][24][25]. Therefore, modulation of important enzymes like quinone reductase, glutathione S-transferase, ALDHs and ribonucleoside diphosphate reductase by natural bioactive compounds like SF or synthetic chemical compounds may be a significant component of their anticarcinogenic action [26,27]. Recently it has been shown that SF accelerates acetaldehyde metabolism by inducing ALDHs and hence may protect individuals who are alcohol intolerant against acetaldehyde toxicity [20].
The present study was undertaken after getting very encouraging results from one of our previous studies where a chemical activator or chaperone (Alda 1) was designed which activated ALDH2 and restored near wild type activity of ALDH2 Ã 2 [4]. In this study, we aimed to investigate the effect of different types of cruciferous vegetables extracts and their bio-active compound SF on the activity of hsALDH. Since they activated this very useful detoxifying enzyme, we than evaluated the mechanism of activation of the enzyme by SF through kinetic measurements and different biophysical techniques. Molecular docking analysis was done to determine the binding site and the amino acid residues of the enzyme involved in the interaction with SF.
Materials and Methods Materials
SF, 6-methoxy-2-naphthaldehyde, NAD + , NADH, DTT and acetaldehyde were purchased from Sigma. Acetone, ethanol, acetonitrile, n-hexane and chloroform were obtained from SRL, India. Fresh cruciferous vegetables like cabbage, cauliflower, radish, turnip and broccoli were purchased from the local market of Medical Road, Aligarh, India in the month of November, 2015. These vegetables are grown in the agricultural field of the locality and made available to us through local vendors. EDTA was the product of Himedia chemicals, India. All other chemicals and reagents used were of analytical grade.
Preparation of crude extract of cruciferous vegetables
Fresh broccoli, cabbage, cauliflower, turnip and radish were obtained. The edible parts were collected in each case, washed and air dried in portions. The dried portions were combined and homogenized in 50 ml methylene chloride for 5 min. The crushed homogenate was left to autolyze at room temperature for 5 min. After autolysis, the homogenate was filtered and again extracted with 50 ml methylene chloride. The extracted fractions were combined and salted with anhydrous 5 mM sodium sulphate. The methylene chloride fractions were dried at 30˚C under vacuum on a rotatory evaporator. The residue was dissolved in acetonitrile and then filtered through a 0.22 μm filter and stored for further use.
HsALDH activity measurements
Preparation of crude saliva and purification of hsALDH by DEAE-Cellulose column was carried out according to our published procedure [28]. The collection of human saliva samples was approved by the institutional ethical committee of Interdisciplinary Biotechnology Unit (IBU), Aligarh Muslim University, Aligarh, India. The participants were the research students of IBU (MFA, AAL, LM) who gave a verbal consent to their research supervisor (HY) and the members of the ethical committee, which was approved. The activity assay of crude and pure hsALDH (ALDH3A1) was done using fluorogenic naphthaldehyde substrate. In particular, 6-methoxy-2-naphthaldehyde is used to measure selectively the activity of the ALDH3A1 isoform [29]. All the activity assays were performed in 50 mM sodium phosphate buffer, pH 7.5 at 25˚C, in the presence of 0.5 mM DTT and 0.5 mM EDTA. Substrate, 6-methoxy-2-naphthaldehyde (5 μM) and coenzyme NAD + (100 μM) were used to measure the activity of hsALDH [30,31]. The reaction was started by the addition of the enzyme at 25˚C and monitoring continuously for 5 min. Fluorescence background noise, if any, was measured prior to enzyme addition and subtracted from the final slope. Fluorimetric assays were run on a Shimadzu RF-5301PC instrument with excitation and emission wavelengths as 315 and 360 nm, respectively. The activity of hsALDH was expressed in terms of nanomolar (nM) concentration of flourogenic product or (NADH) formation per min per μg of the enzyme under the standard assay conditions using a standard plot of the respective product.
Effect of SF on the activity of hsALDH
The effect of the different vegetable extracts and SF on the activity of purified hsALDH was studied by incubating the enzyme for 1 min in presence of different concentration of the vegetable extracts (0-2.5 μg/ml) or SF (0-500 nM). Activity was then determined under standard assay condition as described above. Data were fitted in the case of SF into non-linear regression analysis curve of log (SF) vs activity to determine the EC 50 value under "Dose response Stimulation" using "Graphpad Prism 6.0".
Substrate dependent activity assay in absence/presence of SF
The activity of hsALDH was determined by varying the concentration of the substrate (0-20 μM) in absence and presence of 50 nM SF at standard reaction conditions. All the apparent enzyme kinetic parameters (V max and K m ) were determined by fitting the data in non-linear regression analysis curve of Michaelis-Menten plot and Line-weaver Burk plot under 'Enzyme kinetic' function in "Graphpad Prism 6.0".
UV absorption analysis
Absorption measurements were performed using a Perkin-Elmer (Lambda 25) double beam UV-VIS Spectrophotometer. Fixed concentration of hsALDH (0.2 mg/ml) and varying concentration of SF (0-300 nM) were taken and spectra were recorded from 250-350 nm at 25˚C.
Fluorescence quenching measurements
Fluorescence quenching measurements were done on a Shimadzu 5301 PC fluorescence spectrophotometer equipped with a constant temperature holder and the temperatures were maintained by a constant temperature water circulator (Julabo Eyela). The excitation and emission slit widths were set at 3 nm. Titration of SF (0-60 nM) with hsALDH solutions (0.2 mg/ml) was carried out in a dual-path length fluorescence cuvette (10 x 3.5 mm). Intrinsic fluorescence was measured by exciting the sample at 280 nm. The emission spectra were recorded in the range of 300-450 nm and the data were plotted by taking the emission intensity at 339 nm. The decrease in fluorescence intensity at 339 nm was analyzed using the following Stern-Volmer Eq (1): where, F 0 and F are fluorescence intensities in absence and presence of SF, K sv is the Stern-Volmer quenching constant, K q is the bimolecular rate constant of the quenching reaction and τ 0 is the average integral fluorescence life time of tryptophan, which is~10 −9 s. For the correction of inner filter effect of protein and ligand we used following Eq (2) [32,33]: Where F cor and F obs are corrected and observed fluorescence intensity, Aex and Aem are the absorption of the system at excitation (280 nm) and emission (339 nm) wavelength, respectively. Binding constant and number of binding sites were obtained from the following modified Stern-Volmer Eq (3): Where, K b is binding constant and n is number of binding sites.
Far UV-Circular Dichroism (CD) measurements
The CD studies of hsALDH in the absence and presence of SF were carried out with a JAS-CO-J815 Spectropolarimeter equipped with a Peltier-type temperature controller. The instrument was calibrated with D-10-camphorsulfonic acid. All the CD measurements were performed at 25˚C. Spectra were collected with a 50 nm min -1 scan speed, 0.1 nm data pitch and a response time of 2 s. Each spectrum was the average of 2 scans. The far UV-CD spectra were recorded in the wavelength range of 190-250 nm. All the spectra were smoothed by the Savitzky-Golay method with 25 convolution width. 0.2 mg/ml hsALDH concentration was used for the experiment.
Effect of SF on the activity of hsALDH towards acetaldehyde The activity of hsALDH towards acetaldehyde in the absence and presence of SF was determined using the above standard activity assay method using acetaldehyde (50 μM) as the substrate. NADH fluorescence was measured to determine the activity in terms of NADH formation. The instrumental settings for NADH were: Excitation at 340 nm and emission at 460 nm, with spectral bandwidths of 10 nm for both excitation and emission beams. The amount of NADH formed was determined from the standard curve of NADH at 460 nm.
Molecular docking studies
To determine the binding site and the amino acid residues of ALDH3A1 interacting with SF, in silico docking studies were performed by AutoDock Vina (http://vina.scripps.edu/) and further confirmed by commercially available docking software GOLD. The crystal structure of apoform of ALDH3A1 was obtained from Protein Data Bank (PDB ID: 3SZA), and sdf file of SF (CID: 5350) was obtained from PubChem database. Docking analysis was carried out with the grid size set as 60, 60 and 60 along the X, Y and Z axes with 0.375 Angstrom grid spacing. The 10 best solutions based on docking score were retained for further analysis. Discovery studio 3.5 was used for visualization and for the identification of residues involved in binding.
Results and Discussion
SF derived from its glucosinolate precursor contained in cruciferous vegetables has been reported to increase the phase II antioxidant enzymes in the human upper airway and has recently been shown to induce ALDHs [20,34]. The level of hsALDH was found to be high in the saliva of subjects who continuously ingest large quantities of broccoli [16]. Therefore, in the present study, the direct effect of different cruciferous vegetable extracts and SF on the activity of pure hsALDH has been determined.
Effect of different cruciferous vegetable extracts on the activity of hsALDH
The effect of five common cruciferous vegetables on the activity of hsALDH has been studied. The relative activity of the enzyme in the presence of varying concentration of each vegetable extract is shown in Fig 1. Each of the vegetable extract in the concentration used (till 2.5 μg/ ml) activated the enzyme. Maximum activation was observed at 1 μg/ml of cabbage, broccoli, cauliflower and turnip extract with 2.6, 2.0, 1.7 and 1.6 fold increases in activity, respectively. For these vegetable extracts, when the concentration of the extract was increased above 1 μg/ ml, the activating effect decreased gradually. However, the enzyme activity still remained higher than that in the absence of the vegetable extract even at 2.5 μg/ml of the extract. For the radish extract in the concentration range used, the relative activity kept increasing with increasing concentration of the extract. There was 1.3 fold increase in the activity at 1 μg/ml of the extract. Therefore, it is evident from the data that out of all the five cruciferous vegetables used in this study, cabbage exhibited the maximum activating effect on the activity of hsALDH. The level of glucoraphanin from which SF is derived is highly variable in different cruciferous vegetables and broccoli is a rich source of glucoraphanin [35]. However, still higher amounts are present in some cultivars of cabbage [36]. Therefore, the higher level of SF in cabbage extract may be the reason why this extract exhibited the maximum activating effect on hsALDH activity.
Effect of SF on the activity of hsALDH
The effect of SF on the activity of hsALDH was investigated. It was found that the activity of hsALDH remained the same till 20 nM concentration of SF (Fig 2). However, with further increase in the concentration of SF, the activity of the enzyme increased gradually upto 100 nM concentration of SF, after which the activity remained constant till 500 nM concentration of SF (Fig 2). With further increase in the concentration of SF (above 500 nM), there was a slight decrease in the enzyme activity which may be due to molecular crowding or non-specific binding of SF to the enzyme (data not shown). However, even at these high concentrations of SF examined, the activity was still higher than that in the absence of SF. Therefore, SF exerted an overall activating effect on the enzyme. The activity of hsALDH was increased by about 1.9 fold in presence of 100 nM SF. The concentration of SF at half maximum response (EC 50 value) was determined to be 52 ± 2 nM. Therefore, SF increases the activity of hsALDH to a good extent. However, crude cruciferous vegetables exhibited a greater stimulating effect on the activity of the enzyme than SF alone. This might be because of the presence of other beneficial compounds other than SF that shows cumulative effect to activate the enzyme. Ushida and Talalay (2013) similarly observed that out of the 20 compounds they examined, 15 of them including isothiocyanates such as SF, flavonoids, terpenoids, etc. increased the total ALDH specific activity in Hepa1c1c7 cells [20]. Some studies have shown that the ingestion of cruciferous vegetables lead to an increase in the activity of ALDH in human saliva and these vegetables were found to be inducers of the enzyme [16,20]. This in vitro study reveals that the binding of SF to hsALDH is strong because the binding constant (K b ) is high i.e 1.23 x 10 7 M -1 (see fluorescence quenching studies). Therefore, it is very likely that SF will bind with the enzyme in vivo and lead to its activation.
Substrate dependent activity assay in the absence/presence of SF
The activity of hsALDH with varying concentration of substrate (0-20 μM) was determined in the absence and presence of 50 nM SF. The apparent V max and K m were calculated by using the Michaelis-Menten plot ( Fig 3A) and the Lineweaver-Burk plot (Fig 3B). It was found that the K m of hsALDH towards the substrate (MONAL-62) decreased and the V max increased in the presence of SF (Table 1). Therefore, SF increases the affinity of hsALDH for the substrate. SF interacts with the enzyme and favours the binding with the substrate. It is proposed that SF due to its antioxidant property, protects the catalytic Cys 243 amino acid residue from oxidation, which leads to the increase in the catalytic efficiency of the enzyme.
UV-visible absorption studies
UV-visible absorption spectroscopy is an important tool for steady-state studies of proteinligand and DNA-ligand interaction as well as other biomolecules [37][38][39][40][41]. Changes in the far and near UV regions correspond to the secondary and tertiary structural changes, respectively. In proteins, we can discriminate the various internal chromophoric groups that give rise to electronic absorption bands. The aromatic amino acids contribute to bands in the range of 255-330 nm. Fig 4 shows that the absorption peak of hsALDH centers at 280 nm mainly due to the tryptophan residues. The formation of hsALDH-SF complexes is evident from the spectral data since absorbance decreases with the increase in SF concentration. The shift at 280 nm is not prominent. From these observations we can conclude that SF forms a complex with hsALDH and increases the compactness of the enzyme.
Fluorescence quenching studies
Fluorescence measurements give information about the molecular environment in the vicinity of the fluorophore molecules [42][43][44][45]. The fluorophores in protein are tryptophan, tyrosine and phenylalanine, however, tryptophan contributes maximally to the fluorescence [46]. Therefore, conformational changes of hsALDH were evaluated by the intrinsic fluorescence intensity before and after the addition of SF. The fluorescence intensity of hsALDH decreased gradually with increasing concentration of SF (Fig 5), which indicated that SF interacts with hsALDH. The decrease in fluorescence intensity upon addition of SF was analyzed according to the Stern-Volmer equation (Fig 6A). There is a linear dependence between F 0 /F and the molar concentration of SF in the Stern-Volmer plot. There are two types of quenching mechanisms, static and dynamic. In the dynamic mode of quenching, there is collision between ligand and protein which generally increases with increasing temperature, while in the case of static quenching, there is complex formation between ligand and protein which decreases with increasing temperature. When the value of K q is greater than the maximum scatter collision quenching constant (2.0 x 10 10 mol -1 sec -1 ), it shows that quenching is not initiated by dynamic diffusion but occurs by formation of a strong complex between ligand and protein [33,47]. Here, it was observed that the value of K q is greater than that of maximum scatter collision quenching constant (Table 2). Therefore, quenching is not initiated by collision but by complex formation between hsALDH and SF.
To determine the binding constant and the number of binding sites, log (Fo/F-1) vs. log [SF] was plotted (Fig 6B). From the slope and intercept of modified Stern-Volmer plots (Eq 3), the number of binding sites and the value of binding constant were calculated. The observed values of K sv , K q , K b and n are listed in Table 2. Therefore, there is one binding site on hsALDH for SF, and the binding between the two is quite strong.
Far UV-CD studies
CD is an important technique by which the secondary and tertiary structure of biomolecules is studied [48,49]. It also helps in the elucidation of intermediate states like molten globule (MG) during conformational alteration of the proteins [50]. In order to know the conformational changes in hsALDH after interaction with SF, far UV-CD spectra were recorded in absence and presence of SF (Fig 7). The CD spectrum of hsALDH exhibited two negative minima in the UV region at 208 and 222 nm, which is characteristic of the α-helix structure of the protein [51,52]. The binding of SF to hsALDH leads to a small increase in both the negative minima peaks, clearly indicating that the α-helical structure in the enzyme increases to a small extent upon interaction with SF. Further, the CD spectra of hsALDH in the absence and in presence of SF were found to be similar in shape, revealing that the structure of hsALDH is predominantly α-helical even after the addition of SF. Therefore, it appears that the binding of SF with hsALDH stabilizes the native structure of the enzyme without any significant conformational changes. The enzyme appears to become slightly more compact. Effect of SF on the activity of hsALDH towards acetaldehyde ALDH2 deficient alcohol consumers are exposed to high concentrations of salivary acetaldehyde and have been found to have an increased risk of upper digestive tract cancers [53]. Also, rinsing with ethanol-containing mouthwashes causes an increase in the acetaldehyde level in the saliva [54]. However, hsALDH is reported to be virtually inactive towards acetaldehyde [29]. Therefore, we examined whether SF can also activate hsALDH to oxidize acetaldehyde. The activity of hsALDH towards acetaldehyde as the substrate in the absence and presence of SF was studied (Fig 8). In the absence of SF, hsALDH showed virtually no activity towards acetaldehyde. However, in the presence of 50 nM SF, the enzyme exhibited significant activity towards acetaldehyde. Therefore, it is expected that SF should protect individuals from salivary acetaldehyde induced toxicity by activating hsALDH to oxidize acetaldehyde.
Molecular docking analysis
The docking results clearly showed that SF binds to the active site cavity of the ALDH3A1 and predominantly interacts with Ile 391, Thr 242, Tyr 115, Asn 114, Cys 243 and Tyr 65 (Fig 9A and 9B). The amino acid residues of the enzyme interact with SF mainly through hydrophobic interactions and hydrogen bonding. Tyr 65 is hydrogen bonded with SF while Ile 391, Thr 242 and Tyr 115 interact with SF through hydrophobic interactions. SF binds near the Cys 243 residue which acts as a nucleophile and plays a major role in the reaction catalyzed by the enzyme. It is very likely that SF due to its antioxidant property, protects the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Also, SF interacts with Asn 114 which is a highly conserved residue in the catalytic domain of ALDHs and is having a role in the stabilization of oxyanion form of the thiohemiacetal during catalysis [55]. Therefore, SF fits inside the catalytic center and occupies some portion of the active site and thus decreases the overall size of active site cavity. We speculate that the reduced cavity now becomes capable of fitting the small acetaldehyde molecule, and therefore hsALDH starts catalyzing it.
Conclusions
The present study clearly shows that the cruciferous vegetables and their key bio-active compound SF activate hsALDH which is a very important detoxifying enzyme in the mouth. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. The thermodynamic parameters indicate the formation of a spontaneous strong complex between hsALDH and SF. SF interacts with the enzyme and increases its affinity for the substrate. Molecular docking analysis revealed that SF occupies some portion of the active site of the enzyme and interacts with the important amino acid residues present in it, including the catalytic Cys 243 which acts as a nucleophile during catalysis. We propose that SF due to its antioxidant property, protects the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. In addition, the enzyme showed significant activity towards acetaldehyde in the presence of SF and hence can protect individuals who are alcohol intolerant against acetaldehyde toxicity. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements can lower the risk of acetaldehyde mediated toxicity and oral cancer development. | v3-fos-license |
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} | pes2o/s2orc | An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells
Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.
INTRODUCTION
Reliable isolation of high quality and high quantity RNA from peripheral blood mononuclear cells (PBMCs) and other cells is critical for a broad range of basic, preclinical, and clinical applications (1,2). RNA-based assays enable analysis of basal expression profiles and responses to antigen or mitogen stimulation (3,4). Human PBMCs are a common source of RNA as collection of blood is less invasive and allows in-depth monitoring of many aspects of immunobiology (1,5) including identification, classification and prognosis of cancers (6)(7)(8)(9)(10)(11) monitoring inflammation (12,13), and evaluating therapeutic efficacy (14)(15)(16)(17).
A range of RNA-based platforms are now available, all dependent upon high quality and high quantity RNA (1). However, an important requirement for many applications is both excellent analytical sensitivity (i.e., smallest number of cells detectable) and diagnostic sensitivity (i.e., smallest detectable response to stimulation) (18). Protein-level immunoassays (e.g., flow cytometry, cytokine bead-based arrays, ELIspot) (19)(20)(21)(22) are routinely used to detect PBMCs response to stimulation (23)(24)(25). Indeed, ELIspot has been used extensively as the "gold standard" immune assay given its sensitivity and has been optimized and validated as part of the global HIV/AIDS Comprehensive T Cell Vaccine Immune Monitoring Consortium (26)(27)(28). However, these protocols are limited by the relatively high number of cells required, especially when considering targets with low frequencies (24), when collection of large blood volumes is challenging (29,30), or when there are many experimental variables [e.g., vaccine/peptide (14,17,31,32) or epitope testing (33)(34)(35)]. Therefore, there is an unmet need for a robust RNA extraction and transcriptomic analysis protocol from limited input cell numbers (e.g., PBMCs) with high analytical and diagnostic sensitivity that meets or exceeds that of proteinlevel immuno-assays.
Reverse transcription quantitative PCR (RT-qPCR) remains the "Gold Standard" for assay of gene expression as an alternate readout to protein expression (36,37). RT-qPCR is more sensitive than traditional RNA quantification technologies (i.e., Northern blotting, nuclease protection assays, in-situ hybridization, RNA microarrays etc.) (38)(39)(40). More recent technologies such as Sanger and next-generation sequencing (i.e., RNA-Seq, single cell RNA-seq, NanoString) and advanced PCR methods (i.e., digital PCR) are similarly sensitive (41,42) but are relatively expensive or further require complex bioinformatical analysis (43,44). In contrast, our optimized RT-qPCR assay is designed specifically for cheap, robust, reproducible and sensitive analysis of gene expression, is available to almost any laboratory, and serves as a sensitive and specific alternative to protein expression. Additionally, by focusing on a limited number of genes, RT-qPCR is ideal for validation of genes of interest identified from more untargeted methods such as RNAseq.
However, there is an unmet need for a robust RNA extraction and RT-qPCR protocol with excellent analytical and diagnostic sensitivity, ideally to the single cell level. An important consideration for such a protocol is that RT-qPCR normalization can be achieved by either absolute quantification of copies per reaction using a standard curve, or by semi-quantitative foldchange of relative expression normalized to reference genes (39,45). However, in vitro stimulation has been shown to modulate the expression of many commonly used reference genes (46,47), and key assumptions underlying semi-quantitative analysis require consistent reference gene expression across experimental conditions within and amongst cell populations. An alternative is absolute quantification normalized to cell number, which minimizes this potential analytical bias (48)(49)(50).
To address this need, we developed a highly sensitive RNA extraction and RT-qPCR quantification strategy for analysis of gene expression from human PBMCs. We compared the efficiency of the latest generation of SYBR mastermixes and RNA extraction and reverse transcription kits, taking into consideration both total RNA yield and RNA concentration. We determined that ssoAdvanced TM Universal SYBR R Green Master-Mix provided optimal reaction efficiency, whilst SuperScript TM IV Reverse Transcriptase had the highest cDNA yields. We demonstrated significantly increased PBMC RNA recovery following extraction with the magnetic bead-based MagMAX TM mirVana TM kit, with no further enhancement of analytical sensitivity by including an additional step of RNA concentration. When testing the analytical sensitivity of our optimized protocol, we could detect RNA to the single cell level of highly expressed genes. Furthermore, by evaluating a hierarchy of CD8 + T cell epitope responses, we demonstrated diagnostic sensitivity with as few as 1 × 10 4 PBMCs. This optimized RNA extraction and RT-qPCR protocol, with high analytical and diagnostic sensitivity, provides a robust alternative to proteinbased immune assays. Table 1).
PBMCs
Blood was collected from healthy donors or buffy coats (n = 12) provided by the Australian Red Cross Blood Service, under a protocol approved by the James Cook University Human Research Ethics Committee (#H6702). PBMCs were isolated by density gradient centrifugation and cryopreserved in FBS 10% DMSO. Prior to use, samples were thawed rapidly at 37 • C, treated with DNAase I (1 µg/mL; StemCell), and rested for 18 h at 2 × 10 6 cells/mL in media (RPMI-1640, 10% FBS, 100 U/mL penicillin/streptomycin) at 37 • C and 5% CO 2 . Viable PBMCs were counted prior to downstream analysis.
HLA Typing
Genomic DNA was isolated from PBMCs using the QIAamp DNA Mini Kit (QIAGEN) according to manufacturer's instructions. High-resolution class I and class II HLA typing was performed by the Australian Red Cross Transplant and Immunological Services (Melbourne, Australia) using the MIA FORA NGS FLEX HLA typing kit (Immunocor) and Illumina MiSeq and MiniSeq platforms.
Quantitative PCR
Assay Setup qPCR was conducted using the QuantStudio 3 Real-Time PCR system running QuantStudio Design and Analysis Software (45).
SYBR Master-Mix Testing: cDNA Standard
The four SYBR master-mix kits were further evaluated with efficiency titrations of cDNA standards. Briefly, RNA was extracted from 1 × 10 6 unstimulated PBMCs using the RNeasy R Mini Kit (QIAGEN). Seven microliters of extracted RNA was converted to cDNA using the SuperScript TM III First-Strand Synthesis System kit (Invitrogen). Master-mix reaction efficiency was calculated from log 10 diluted cDNA (10 4 -10 1 cells/reaction) with RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.
Reference Gene Stability Testing 1 × 10 6 PBMCs were stimulated for 6, 12, 16, 24, or 48 h with or without PMA/Iono as described above. RNA was extracted with RNeasy R Mini Kit (QIAGEN). Seven microliters of extracted RNA was reverse transcribed with SuperScript TM III First-Strand Synthesis System kit (Invitrogen). qPCR was run with ssoAdvanced TM master-mix, RPL13a, SDHA, TBP and IFN-γ primers at 500 nM and samples at 10 2 cells/reaction.
Evaluation of RNA Extraction Kits
To evaluate RNA yield and quality, three RNA extraction kits: . qPCR was run with ssoAdvanced TM mastermix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM and sample diluted to 10 2 cells/reaction; except when considering concentration, when the samples were run undiluted.
Analytical and Diagnostic Sensitivity
For determination of analytical sensitivity, RNA was extracted from a log 10 serial dilution of unstimulated PBMCs (10 6 -10 0 cells/extraction), using the MagMAX kit with or without the RNeasy R MiniElute Cleanup Kit. A media-only extraction control was processed in parallel. For determination of diagnostic sensitivity, RNA was extracted using the MagMAX kit from titrated PBMCs (4 × 10 5 , 1 × 10 5 , 2.5 × 10 4 , and 1 × 10 4 ) incubated for 6 h with or without PMA/Iono or HLAmatched peptide. For sensitivity evaluations, 10 µL of RNA was converted to cDNA using the SuperScript TM IV First-Strand Synthesis System (Invitrogen). qPCR used undiluted sample with ssoAdvanced TM master-mix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.
Data Analysis
RT-qPCR, Bioanalyzer and NanoDrop data were analyzed using a repeated-measures two-way ANOVA with Bonferroni-corrected multiple comparisons test comparing test to control mean. Correlation between RT-qPCR and protein quantification was test with linear regression analysis. Analysis was conducted using GraphPad Prism version 7.0 (GraphPad). In all cases, P < 0.05 were considered significant.
ssoAdvanced TM Universal SYBR ® Green Master-Mix Provided the Highest Reaction Efficiency
Four master-mixes-ssoAdvanced TM Universal SYBR R Green Master-Mix (Bio-Rad), QuantiNova SYBR R Green PCR Kit (QIAGEN), PowerUp SYBR R Green Master-Mix (Applied Biosystems) and RT² SYBR R Green qPCR Master-Mix (QIAGEN)-were evaluated using two methods of preparing reference standards: (i) standards derived from log 10 diluted amplicon; and (ii) standards generated from log 10 diluted cDNA ( Figure 1A). Reaction efficiency was quantified using four primer sets: three sets targeted reference genes known to have high (60S ribosomal protein L13a; RPL13a), moderate (Succinate dehydrogenase complex, subunit A; SDHA) and low (TATAbinding protein; TBP) expression; and one set targeted a cytokine gene (interferon gamma; IFN-γ ) (47). When considering an acceptable reaction efficiency range (90-110%), 35.4% of the amplicon-derived standards ( Mitogen Stimulation Induced Changes in RPL13a, SDHA, and TBP Gene Expression The expression stability of three commonly used reference genes (47,53,54), RPL13a, SDHA, and TBP, previously reported as stable in PBMCs following stimulation (47), were evaluated by RT-qPCR within PBMCs stimulated with PMA/Iono for 6, 12, 18, 24, or 48 h. Expression of all three genes changed over time with cell culture, and significantly increased at 48 h post-stimulation as compared to baseline (P < 0.001, P < 0.01, and P < 0.01, respectively; Figure 1B). These data establish that the expression of common reference genes is significantly affected by stimulation, emphasizing the importance of absolute quantification normalized to cell numbers, rather than relative quantification.
Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration
Next, RNeasy R Mini and Micro silica columns (both QIAGEN) and MagMAX TM mirVana TM (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy R MiniElute Cleanup Kit (QIAGEN). In each case, PBMCs were incubated with or without PMA/Iono for 6 h. RIN assessment demonstrated that RNA integrity was high (>7) and consistent across all kits (Figure 2A
Single Cell Analytical Sensitivity Was Observed Following Magnetic Bead-Based RNA Extraction
We next evaluated the analytical sensitivity of our optimized protocol using MagMAX extraction kit ( Figure 4A) and MagMAX-RNeasy R MiniElute extraction-concentration kit ( Figure 4B). RNA was extracted from a log 10 serial dilution of unstimulated PBMCs and expression of IFN-γ , RPL13a, SDHA, and TBP determined by absolute quantification. The highly expressed RPL13a gene was detected at single-cell level from both MagMAX (0.88 log 10 copies/reaction; Figure 4A) and MagMAX-RNeasy R MiniElute combination (0.90 log 10 copies/reaction; Figure 4B) extractions. Extraction technique did not influence IFN-γ , RPL13a, or TBP expression; whilst variability in SDHA expression (P Ext < 0.05) was driven by increased RT-qPCR signal at 10 5 and 10 4 PBMCs per extraction. These data establish that our optimized protocol is capable of extracting and quantifying RNA of a highly expressed gene from a single cell. Importantly, the additional step of RNeasy R MiniElute Cleanup did not further enhance analytical sensitivity.
RT-qPCR Protocol Diagnostic Sensitivity Correlates Significantly With Protein Level
Quantification of Epitope-Specific Stimulation From as Few as 1 × 10 4 PBMCs Next, we determined the diagnostic sensitivity of our optimized RNA extraction and RT-qPCR protocol to confirm that it accurately reflected data generated using protein-level assays. We evaluated the epitope-specific stimulatory response for four CD8 + T cell epitopes restricted by different MHC molecules, quantifying IFN-γ production by flow cytometry, cytokine bead array and ELIspot; and IFN-γ mRNA by our optimized protocol. A limited epitope-specific IFN-γ response was demonstrated by flow cytometry (Figure 5A) and bead arrays ( Figure 5B) whereas all samples were observed to respond to stimulation by ELIspot ( Figure 5C). Importantly, our RT-qPCR protocol (Figure 6), was able to replicate the ELIspot results but with significantly reduced cell numbers (HLA-A1 2.5 × 10 4 , -A2 1 × 10 5 , -B7 1 × 10 4 , -B8 1 × 10 5 ; 48-fold, 12-fold, and 48-fold and 12-fold, respectively; Figure 5D).
Optimized RT-qPCR Assay Correlated With Best-Practice Protein-Based Immunoassays
Next, we assessed correlation between results obtained using our optimized RT-qPCR protocol and current best-practice immunoassays. The correlation between our RT-qPCR protocol and ELIspot was significant at all cell numbers tested [P = 0.0006, R 2 = 0.7063 (4 × (Figure 5E, left panel). Thus, data generated using our optimized RT-qPCR assay are consistent with best practice protein-based immunoassays. Furthermore, our assay is capable of defining an epitope-specific response hierarchy from as few as 1 × 10 4 cells, representing a clinically and diagnostically meaningful reduction in cell number. Taken together, we report here a highly sensitive RNA extraction and RT-qPCR quantification strategy using the MagMAX RNA extraction kit, Superscript TM IV reversetranscription kit and ssoAdvanced TM SYBR master-mix (Supplementary Table 3). This assay is sensitive to the single cell level, can define an epitope hierarchy of response from as few as 1 × 10 4 cells, and represents a sensitive and robust alternative to protein quantification for research, diagnostic and clinical applications.
DISCUSSION
Herein, we describe an optimized RNA extraction and RT-qPCR protocol requiring low PBMC numbers, with high analytical and diagnostic sensitivity, whilst maintaining high correlation to protein-level quantification that is typically reliant on much larger cell numbers for detection.
Precise RT-qPCR results are typically dependent on reactions maintaining efficiency close to 100% (45). Both assay design (e.g., primer concentration, master-mix) and sample (e.g., , n = 12 total) were stimulated with synthetic HLA-matched peptides representing CMV, Influenza or EBV CD8 + T cell epitopes "VTE," "GIL," "RPH," or "FLR" (black), respectively. All samples were cultured with media negative control or PMA/Iono positive control. IFN-γ mRNA expression was determined by absolute quantification RT-qPCR of titrated PBMCs (4 × 10 5 , 1 × 10 5 , 2.5 × 10 4 , and 1 × 10 4 ); gene copy number per reaction was quantified by standard curve and log 10 transformed. Single RNA extractions, with single reverse transcription reactions per n, were performed. qPCR performed in technical triplicate replicates. Flow cytometry and MagPIX performed in single wells. ELIspot performed in technical triplicate replicates. Sample mean calculated from the mean of the technical single or triplicates. Biological mean ± technical SEM above background shown. co-extracted inhibitors) may influence PCR efficiency. We made use of the open-access database PrimerBank TM since those primers have been designed for use under consistent conditions (i.e., optimal Tm 60 • C) and cover most known human and mouse genes (55). We found primer concentration titrations did not impact reaction efficiency, whereas the SYBR master-mix had a significant impact. PCR inhibitors, including hemoglobin, lactoferrin, anticoagulants, IgG, polysaccharides, and proteases, can be co-extracted in PBMC preparations (56,57). It is known that some DNA-polymerase variants and PCR buffer "enhancers" have improved reaction efficiency in the presence of such inhibitors (56,58). The ssoAdvanced TM master-mix, identified herein as optimal of those tested, appears to be one such master-mix facilitating PCR efficiency in the presence of co-extracted inhibitors. Optimization of mastermix reagents will likely continue to be important in improving blood-based PCR analysis and diagnostics (59,60); especially for accurate amplification of relatively low abundant targets, comparisons between populations with high variability, or amplification from inhibitor-enriched mediums (i.e., whole blood extractions) (10,59). We tested three RNA extraction kits by evaluating extraction quality and efficiency: RNeasy R Mini Kit, RNeasy R Micro Kit, and MagMAX TM mirVana TM Kit; in combination with the RNA purification and concentration kit: RNeasy R MiniElute Cleanup Kit. When extracting identically controlled samples, all kits yielded RNA with equivalent RIN scores and low technical variability between replicates. Importantly, RNA yield from PBMCs was significantly increased using MagMAX as compared to silica-column technologies. Additionally, when compared to silica-column extractions, we found MagMAX was more cost and time efficient when running larger number of samples (e.g., 96 samples in ∼2 h). We therefore expect magnetic bead-based extractions will become increasingly common within bloodbased nucleic acid isolations (59,61,62). In addition to extraction techniques (e.g., silica column, phase separation), other factors that could impact RNA quality, yield and concentration include sample collection, storage, and transportation.
Four reverse transcriptase (RT) kits were also evaluated: SuperScript TM III First-Strand Synthesis System, SuperScript TM IV First-Strand Synthesis System, iScript TM Advanced cDNA Synthesis Kit and High-Capacity RNA-cDNA Kit TM . Of those, Superscript TM IV was associated with the highest qPCR signal. A previous study evaluating RNA extracted from PBMCs using earlier-generation RT kits reported >128-fold increased qPCR signal between kits (1). We speculate that the reduced variability that we observed between RT kits tested in our study reflects consistent kit quality, purity of the RNA extracted by MagMAX, or a combination thereof.
Both analytical sensitivity and diagnostic sensitivity are key criteria for any RT-qPCR protocol. We show that the analytical sensitivity of our assay is to the level of single cell RNA detection for relatively highly expressed RPL13a. Sample concentration and clean-up has been suggested to remove inhibitors and increase sensitivity (63,64). Unexpectedly, we found this step did not improve our analytical sensitivity, and was timeconsuming, expensive and reduced sample volume. Nevertheless, if concentration is warranted under specific experimental situations, our data suggest that it is technically feasible while retaining high analytical sensitivity. Diagnostic sensitivity determined using MagMAX showed that an epitope response hierarchy could be detected with as few as 1 × 10 4 PBMCs. It is well-known that there is no absolute correlation between RNA expression and protein translation. Indeed, correlations between transcript and protein expression would be markedly reduced under situations of epigenetic, post-transcriptional or post-translational modification of the gene of interest (65). Nevertheless, we correlated our optimized RT-qPCR assay with commonly used protein-level immunoassays (flow cytometry, cytokine bead arrays, and ELIspot) and showed a very high correlation with the gold-standard protein-level assay, ELIspot, as well as the commonly used MagPIX bead-based cytokine assay, at all tested PBMC concentrations. This highlights that our protocol represents a robust alternative to protein-based assays (e.g., when measuring changes in cytokine mRNA expression in PBMCs in response to specific in vitro stimulation). This work will significantly improve analytical capacity of studies relying on irreplaceable, relatively small or costly human samples (e.g., neonatal PBMCs) (66,67).
Another important outcome of our work is the finding that absolute quantification of transcripts and subsequent normalization to cell numbers is the most appropriate analysis strategy for RNA/RT-qPCR quantification from PBMCs (45,48). We observed significant alterations in gene expression of commonly used reference genes RPL13a, SDHA and TBP following stimulation. This is not unexpected as reference genes have been described as variable across cell types, tissues, and experimental and stimulatory conditions (25,47,68,69).
In summary, we report herein the development of an optimized PBMC RNA extraction and RT-qPCR protocol. We employed a qPCR strategy of absolute quantification utilizing PrimerBank TM primers and ssoAdvanced TM Universal SYBR R Green Master-Mix. PBMC RNA was isolated with MagMAX TM mirVana TM Total RNA Isolation Kit and reverse transcribed with SuperScript TM IV First-Strand Synthesis System. Our assay provided single cell analytical sensitivity and a diagnostic sensitivity that could define response hierarchy from 1 × 10 4 cells. This assay offers an alternative to current best practice protein-based immunoassays, especially for limited PBMC numbers. This work has broad applicability for both clinical and primary research practice.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by James Cook University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.
ACKNOWLEDGMENTS
We are grateful to all of the donors who kindly provided samples in support of this study. Figure 6 was created using BioRender.com. | v3-fos-license |
2020-06-02T21:38:00.970Z | 2020-01-01T00:00:00.000 | 219168165 | {
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} | pes2o/s2orc | Preparation of skeletally diverse quinazoline-2,4(1H,3H)-diones using Na2SiO3/SnFe2O4 catalytic system through a four-component reaction
Sodium silicate with the formula of Na2SiO3 was used for the four-component reaction of ketones, aldehydes and cyanoacetamide. The effect of SnFe2O4 nano-particles on the catalytic potential of Na2SiO3 was investigated. The desired products synthesized by this method are 5-amino-7-aryl-2,4-dioxo-2,3,4,4a,7,7a,8,9,10,11-decahydro1H-benzo[i]quinazoline-6-carbonitriles, 5-amino-2,4-dioxo7-aryl-1,2,3,4,4a,7,7a,8,10,11-decahydropyrano[3,4-i] quinazoline-6-carbonitrile, 5-amino-9-methyl-2,4-dioxo7-aryl-2,3,4,4a,7,7a,8,9,10,11-decahydro-1H-pyrido[3,4-i] quinazoline-6-carbonitrile and 5-amino-2,4-dioxo-7-aryl1,2,3,4,4a,7,7a,8,10,11-decahydrothiopyrano[3,4-i]quinazoline-6-carbonitrile derivatives. This method has advantages of high yields, simple procedure and easy work-up.
Quinazoline-2,4(1H,3H)-dione moiety could be constructed by cyclization of 2-aminobenzonitrile with DMF (Rasal and Yadav, 2016), the reaction of CO 2 and 2-aminobenzonitriles (Ma et al., 2013), carbonylation and then cyclization of anthranilamide (Li, 2009), and treatment of substituted anthranilic acid with potassium cyanate (El-Brollosy, 2007). However, quinazoline-2,4(1H,3H)-diones can be synthesized via many methods but most of them suffer from structure diversity and scope expanding. A new method for the preparation of important scaffolds with a core of quinazoline-2,4(1H,3H)-dione has been developed by Jiang et al. through a multicomponent reaction of aldehydes, cycloketones, and cyanoacetamide under MW irradiation conditions using K 2 CO 3 and Cs 2 CO 3 as basic catalysts (Jiang et al., 2009(Jiang et al., , 2016. Sodium silicate with a general formula of SiO 2 (Na 2 O) n is a white powder that dissolves easily in water and provides an alkaline solution which is stable in neutral and alkaline media. Depending on their prepared ways and purity, sodium silicate has a unique chemistry and has own industrial and consumer applications. One of the important uses of sodium silicate is its use in the manufacture of detergents and ceramic and tile industries. The participation of sodium silicate in the composition of detergents will control the corrosion and alkaline attack. It also acts as a melting aid and prevents cracking of biscuits (Ebnesajjad, 2011;Lagaly et al., 2005). The use of sodium silicate increased stiffness and reduced permeability in predominantly granular soils. Impregnation of concrete with sodium silicate reduces porosity and increases its longevity. Sodium silicates are known to reduce lead, copper and other heavy metals in Scheme 1: Four-component reaction of aldehydes, cycloketones, and cyanoacetamide. drinking water and reduce the calcium and magnesium hardness (Ebnesajjad, 2011;Lagaly et al., 2005). Their function is used as a corrosion inhibitor to form a microscopic film on the inside of the water pipe and to prevent the formation of bells. Silicates are used to remove oil, wax, and soot from cotton in the weaving industry. In agriculture, soluble silicates are a vital nutrient for plant health and, in small quantities, increase plant resistance to disease and increase yields. The use of sodium silicate reduces the friction resistance to 5%, which results in less fuel consumption for trucks and trucks (Ebnesajjad, 2011;Lagaly et al., 2005).
In this, we aim to investigate the effect of SnFe 2 O 4 nano-particles on the catalytic activity of Na 2 SiO 3 and to prepare a series of quinazoline-2,4(1H,3H)-dione through a multi-component reaction of aldehydes, cycloketones, and cyanoacetamide under solvent-free conditions using a mixture of SnFe 2 O 4 /Na 2 SiO 3 (Scheme 1).
Results and discussion
X-ray diffraction patterns (XRD) of pure SnFe 2 O 4 nanoparticles and sodium silicate combined with different ratios of SnFe 2 O 4 nanoparticles are shown in Figure 1. Accordingly, SnFe 2 O 4 nanoparticles have amorphous particles.
The morphology of the synthesized nanoparticles was studied through the FESEM test ( Figure 2). Observations show that homogeneous spherical nano-particles of SnFe 2 O 4 with dimensions of less than 50 nm were formed. The images taken from mixed samples indicate the formation of almost homogeneous samples in which SnFe 2 O 4 nanoparticles are distributed homogeneously in the sodium silicate structure.
First the preparation of 5-amino-2,4-dioxo-7-phenyl-2, 3,4,4a,7,7a,8,9,10,11-decahydro-1H-benzo[i]quinazoline-6-carbonitrile (1d) through the four-component reaction of cyanoacetamide, cyclohexanone, and benzaldehyde using NaOH and Na 2 SiO 3 as basic catalysts under solventfree conditions (100°C) was investigated (Table 1). Na 2 SiO 3 shows a higher yield (Table 1, entry 2). By increasing the temperature to 130°C, the product yield was increased up to 75% (Table 1, entry 3). Subsequently, SnFe 2 O 4 nano-particles were added to sodium silicate with three different ratios as 0.5, 1, and 2% w/w. The catalytic activity of three samples was examined under solvent and solventfree conditions and the results are summarized in Table 1 (entries 4-11). The results show that solvent suppressed the reaction process and all solvent media are inferior and no product yield was formed. Notably, the addition of SnFe 2 O 4 nano-particles increased the productivity of Na 2 SiO 3 in solvent-free conditions. The higher yield was achieved when sampling with 2% w/w of SnFe 2 O 4 nanoparticles was used. Finally, Na 2 SiO 3 containing 2% w/w of SnFe 2 O 4 was selected as the most effective catalyst for the preparation of 1d.
The effect of catalyst dosage on the product yield is shown in Table 1. Accordingly, the reaction of cyanoacetamide, cyclohexanone and benzaldehyde was investigated at 130°C in the presence of different amounts of catalyst (Na 2 SiO 3 (with 2% of SnFe 2 O 4 )). The results show that 7.1% mol of the catalyst has the highest efficiency to produce a high yield of product 1d. No notable increase was done on the product yield with increasing the catalyst dosages to higher than 7.1% mol. Temperature is one of the effective parameters on the productivity of multi-component reactions. Thus the reaction of cyanoacetamide, cyclohexanone, and benzaldehyde in the was investigated at different temperatures (Table 1). Giving access to 1d as the expected product is not effective at below 100°C. The reaction has a reasonable high yield at 130°C and no notable change in reaction yield was observed with further increasing of the reaction temperature. Finally, the optimum condition for the preparation of 1d was chosen as Na 2 SiO 3 with 2% w/w of SnFe 2 O 4 as catalyst (7.1% mol), temperature (130°C) and solvent-free condition.
Next, the reaction conditions were further modified and the reactions were performed under ultrasonic radiation conditions and high speed ball milling (HSBM) technique. In all reaction conditions, high yield products were obtained. Generally, aromatic aldehydes substituted at para and meta positions with halogen atoms show more reactivity compared to those of electron-donating groups (e.g. CH 3 , OCH 3 ). ortho-Substituted aldehydes showed longer reaction times than their para-counterparts.
The proposed mechanism responds that the initiation of the reaction will include the Knoevenagel condensation reaction of cyanoacetamide with benzaldehyde and cyclohexanone to form 2-cyano-3-phenylacrylamide (1) and 2-cyano-2-cyclohexylideneacetamide (2) intermediates which can be catalyzed by Na 2 SiO 3 . Compound 2 reacts with a base to form an anion intermediate which subsequently undergoes a nucleophilic 1,4-addition to form 3. The next steps consisted of cyclization, intramolecular reactions, and finally 1,3-H shift to form the desired products (Scheme 2).
Conclusion
A mixture of Na 2 SiO 3 with three different proportions of SnFe 2 O 4 nano-particles was prepared, characterized by XRD, FE-SEM techniques and subsequently, was used for the preparation of 1d-5d, 1e-7e, 1f-7f, and 1g-3g in high yields. The high basic power of sodium silicate is decreased by adding SnFe 2 O 4 nanoparticles which reduce the adverse reactions and disorientation, and increased the selectivity of the catalyst.
Experimental
The solvents, reactants and chemicals used in this study are from the Merck and Sigma-Aldrich Chemical Companies. The XRD patterns were taken in a PHILIPS PW3040 device. NMR spectra were recorded in a Bruker Avance DPX 400 MHz instrument in DMSO-d 6 as solvent relative to TMS (0.00 ppm). A Heraeus CHN-O-Rapid analyzer was used for elemental analysis. FE-SEM photographs are taken using the FEI NOVA NANOSEM 450 photographic apparatus.
Preparation of SnFe 2 O 4 nanoparticles
Two different solutions were prepared. The first solution was made up of SnCl 2 (1 mmol) and FeCl 3 ‧6H 2 O (2 mmol) in 100 mL of ethanol. The second solution contains 20 mL ammonia in 40 mL of distilled water. The second solution is added slowly to the first solution under stirring. When the sedimentation process was completed, the solution is released for 30 min. The desired precipitate is then separated by a filter paper and washed with distilled water. Finally, the mixture was calcined at 500°C.
Preparation of SnFe2O4/sodium silicate powder
SnFe 2 O 4 nanoparticles are added to a solution of sodium silicate (1g Na 2 SiO 3 in 10 mL H 2 O) with a ratio of 0.5% w/w SnFe 2 O 4 /sodium silicate and heated up to 100°C for 60 min, until a solid homogeneous mixture is obtained. Two additional samples was prepared with 1% and 2% w/w of SnFe 2 O 4 by the same procedure.
Synthesis of 1H-benzo [i] quinazoline-6carbonitrile derivatives
Method A: A mixture of cyanoacetamide (3 mmol), cyclohexanone (1.2 mmol), benzaldehyde (1 mmol) and nanocatalysts (7.1% mol) are heated at 130°C until the reaction is complete (TLC monitoring). After completion of the reaction, the mixture is cooled to ambient temperature and mass was dissolved in hot ethanol and the insoluble catalyst is separated off by the filter paper. The resulting solution is concentrated until the evaporated solvent and sediments are recrystallized in cold watery ethanol and subsequently washed with diethyl ether to yield the pure product. The reaction was also carried out with benzaldehyde derivatives.
Next, the reaction was carried out with all of the conditions described above with tetrahydro-4H-thiopyran-4-one (1.2 mmol) and cyclopentanone (1.2 mmol) instead of cyclohexanone.
Method B:
The mixture was ultra-sonicated at 120°C. The procedure is similar with method A.
Method C:
The mixture was milled at 120°C. The procedure is similar with method A.
The mixture was milled in a stainless steel grinding vial (10 mL volume) equipped with a stainless steel ball (diameter: 6 mm; mass: 1.04 g) for appropriate times at 25 Hz and 120°C. | v3-fos-license |
2021-05-17T00:02:57.508Z | 2020-11-05T00:00:00.000 | 234681396 | {
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} | pes2o/s2orc | PHB Produced by Bacteria Present in the Argan Field Soil: A New Perspective for the Synthesis of the Bio-Based Polymer
: Bio-based plastics, i.e., non-synthetic polymers produced from renewable resources are gaining special attention as a feasible solution to the environmental issues caused by concerns re-garding the impact of waste plastics. Furthermore, such materials can also represent an alternative to petroleum-derived polymers, due to the scarcity of this raw material in the near future. In the polyhydroxyalkanoates (PHA) family, polyhydroxybutyrate (PHB) was the first to be synthesized and characterized. PHB soon gained great attention from industrial and academic researchers since it can be synthesized from a wide variety of available carbon sources, such as agro-industrial and domestic wastes. The aim of this original research has been the identification of the presence of PHB synthetizing bacteria in some soils in a Moroccan region and the production of the bio-based PHB. In particular, the soils of the argan fields in Taroudant were considered. Taroudant is a southwestern region of Morocco where the argan oil tree Argania spinosa is an endemic and preserved species. Starting from rhizospheric soil samples of an argan crop area, we isolated heat-resistant bacteria and obtained pure cultures from it. These bacteria present intracellular endospores stained by the Schaeffer-Fulton method. The presence of intracellular endospores is a very important starting point to verify the effective production of PHB as a compartmentalized material. Further analyses are currently ongoing to try to extract and characterize PHB granules.
Introduction
The increasing worldwide consumption of coal, oil, and natural gases is inducing a rise in these fossil prices, and an increase in carbon dioxide emissions that cause the world's climate change. In addition, in the following decades, because of the continued need for fossil fuels, petroleum resources will be depleted in a short time. It is necessary, therefore, to reduce and replace these non-renewable resources with alternative "green" resources for the sustainable development of the next generations. As an example, biomass represents an energetic resource to solve economic, political, and environmental issues [1].
Biomass means non-fossilized and biodegradable organic material originating from plants, animals, and microorganisms. Biomass is considered a renewable resource as long as its exploitation rate does not exceed its replenishment by natural processes; it can, therefore, be conveniently used to synthesize bio-based polymers [1]. Among bio-based polymers, polyhydroxyalkanoates (PHA) are a group of polyesters synthesized by prokaryotic Gram-positive and Gram-negative enterobacteria [2,3], cyanobacteria [4,5], and archaea organisms living in extreme environmental conditions [6,7]. PHAs are thermoplastic, biodegradable, biocompatible, and non-toxic bio-based polymers. They have a high degree of polymerization, are highly crystalline and isotactic, possess optical properties, and are insoluble in water.
Microbial-derived polymers belonging to the PHA family are entirely synthesized by bacteria; at the same time, they can be completely bio-degraded by living organisms, which means that they can undergo a decomposition process that leads to small compounds, such as methane, carbon dioxide, and water, due to the microorganism's activity in particular composting conditions. The microbial species that are able to synthesize PHA acts in the presence of a renewable feedstock of pentoses, hexoses, starch, cellulose, sucrose, lactose, CO2, and CH4 under unfavorable growth conditions due to unbalanced nutrient availability. Generally, these conditions include a sufficient carbon source and a reduced number of other substrates such as nitrogen, phosphate, and oxygen. During starvation periods, in the absence of external sugars, the PHA producers can use the carbon source stored in the granules to provide the cell with the necessary amount of energy to survive in this uncomfortable condition, particularly for osmoregulation, motility, and metabolic pathways [8].
The Poly-3-hydroxybutyrate (PHB) is the most studied and characterized member of the PHA family. It is a thermoplastic homo-polyester consisting of hydroxybutyrate monomers which give the polymer 80% crystallinity. PHB has an extremely regular structure: it is almost completely isotactic (like synthetic polypropylene), with the chains forming helical structures. The chains are able to closely pack to form crystals, obtaining a stiff polymer [9].
The processing of PHB is usually carried out by extrusion in melt state for the production of polymeric films or other items [10]. PHB displays a melting temperature of 175-180 °C and thermal degradation temperature around 240 °C, which means a narrow temperature window for the processability of this material, therefore, making it difficult to process [11]. Furthermore, unfavorable conditions during processing, e.g., too high levels of humidity/temperature and/or too long permanence in the machine, can cause polymer degradation, corresponding to lower performance of the final products.
To overcome problems during processing, the bacterial production process can be modified to produce co-polymerized PHB. The crystallization of PHB can be interrupted by adding different building blocks, such as 4-hydroxybutyrate (4-HB), 3-hydroxyvalerate (3-HV) [10], or 3-hydroxyhexanoate (3-HH) [11]. Co-polymerization lowers the melting temperature down as far as 75 °C, thus facilitating the processing and production of PHB; as a consequence, the degree of the crystalline phase is reduced, depending on the percentage of PHB in the co-polymer.
The tensile strength of PHB is close to that measured on isotactic polypropylene (iPP); on the other hand, its elongation at break is significantly lower (6%) compared to that of iPP, being about 400%. This means that PHB is much more brittle compared to iPP. To overcome this weakness, PHB can be also blended with different synthetic and bio-based (such as starch, lignin-derivate) polymers with a similar melting temperature range or modified upon the addition of fillers or additives: in this way it is possible to improve the characteristics of PHB with the aim of widening its field of applications, possibly reducing also the costs of the final products [9].
The high costs of carbon substrates can limit the production of PHB on a large scale. To overcome this problem, alternative substrates rich in carbohydrates are proposed, like biomass from green spaces, wastes, secondary products of industrial processes, such as glycerol, sugarcane bagasse, and lignocellulose from agricultural and forestry residues [12].
In the last years, it is becoming interesting to study the possibility of blending PHB with industrial biowastes for two reasons: first, because the disposal of industrial bioproducts is an environmental issue, and second because it has been reported that this blending process can improve the mechanical properties of the natural polymer by creating a matrix with multiple applications [13].
The high costs of carbon substrates represent also an ethical problem for PHB production, particularly in those regions where the nutritional situation is a difficult issue to be solved. With the biotechnology techniques, it is possible to synthesize PHB from cheap renewable resources such as agricultural and industrial wastes [8,14,15], representing promising alternatives to produce PHB at competitive costs, without causing ethical conflicts [16].
The aim of this work was to isolate, for the first time, PHB synthesizing bacteria from the argan field soil and to identify the presence of the bio-based polymer granules by adopting the Schaeffer-Fulton endospore staining methods. The intracellular granules accumulation was first examined by adopting Malachite Green. Afterward, a second identification test was implemented by adopting the variation of the Schaeffer-Fulton staining, where the Malachite Green dye was replaced by a Methylene Blue solution. The presence of endospores was observed by the compound light microscope using oil immersion. This experimental work represents the first step of the synthesis of PHB bio-based polymer starting from argan wastes, resulting from the fruits and the pressing process for the argan oil extraction.
Samples Collection
The soil samples for the isolation of the PHB producing bacteria were collected from rhizospheric soil in the agricultural crops of argan oil (Argania spinosa) in Taroudant, a southwestern region of Morocco where the argan tree is endemic. All samples were collected from six different locations of the same area of the argan crop to screen the physical characteristics that best suit the PHB producing bacteria growth. The physical characteristics of the soil were the presence compared to the absence of water, the presence of domestic pollutants in the urban location compared to the absence of pollution in the rural area, and the proximity to either dead trees or healthy trees. Soil samples were collected in sterile conditions by using ethanol 70%, preserved into sterile vials, and stored at 22 °C temperature for 48 h. Afterward, all samples were stored at 4 °C for 3 weeks for the bacterial isolation.
Pretreatment: Isolation of Microorganisms
One gram of each soil sample was dispersed in 10mL of sterile water. These samples were homogenized at 200 rpm for 3 h to ensure homogeneity of bacterial organisms in a culture, and after heated at 80 °C for 10 min to isolate endospore-forming bacteria. All samples were serially diluted to 10-8 using sterile water and plated by spreading 100 µL of the dilutions −5, −6, −7, −8 on sterile nutrient agar plates, composed as follows: Peptone 5 g/L, Yeast extract 3 g/L, Sodium chloride 5 g/L, Glucose 1 g/L, Agar 18 g/L, in 1L of distilled water, at pH 7.0. Thereafter the plates were incubated at 30 °C for 48 h. All grown bacteria were isolated on modified agar plates consisting of: Beef extract (0.3%), Peptone (0.5%), Sodium Chloride (0.8%), Glucose (1%), and Agar (1.5%) [17].
Schaeffer-Fulton Endospore Staining Using Malachite Green and Safranin
The detection for PHB-producing bacteria was performed by using the Schaeffer-Fulton method for staining endospores. The staining was prepared by dissolving 0.5 g of Malachite Green in 100 mL of distilled water. The Safranin counterstain was prepared by dissolving 2.5 g of Safranin powder in 100 mL of 95% ethanol. After fixing the microorganisms on the glass slide, the specimen was covered with a square of blotting paper and saturated with Malachite Green stain solution for 5 min by steaming over boiling water and adding more dye when it dried off. After washing the slide with distilled water, the specimens were counterstained with safranin for 30 s, and once again washed with distilled water. The slides were examined under oil immersion at 1000× for the presence of endospores. The vegetative cells appear red to pink, while the endospores are bright green [18].
Schaeffer-Fulton Endospore Staining Using Methylene Blue Solutions
The newly alternative Schaeffer-Fulton endospore staining using Methylene Blue solutions was implemented to confirm the presence of endospores already identified in the staining with Malachite Green. In this proposed study, the alternative staining method resulted in coloring endospores of the species Bacillus subtilis and Clostridium tetani. A solution of Methylene Blue stain 0.5% at pH 12 was prepared by diluting 0.1 g of the dye with 20 mL of a buffer solution at pH 12. A bacterial smear of the positive endospore producing bacteria, already identified using Malachite Green staining, was prepared and fixed over low heat. The Methylene Blue stain 0.5% at pH 12 was added to the specimens and heated over a steam bath for 5 min. Afterward, the slide was washed with distilled water and counterstained with Safranin for 30 s. Before proceeding with the microscopic observations, the slides were again washed with distilled water to remove the counterstaining dye. According to this work, the warming process allows the Methylene Blue solution to penetrate the endospores' wall, and the alkaline pH of the staining solution allows the dye to penetrate the alkaline bacterial cytosol [19].
Results and Discussion
In this work, we investigated the potential presence of microorganisms able to synthesize the biopolymer PHB from Argania spinosa crop soil in a unique environmental area of Morocco where this species is endemic and preserved.
Isolation and Screening of PHB Producing Bacteria
Six soil samples were collected from the agricultural crops of argan oil (Argania spinosa) in Taroudant, a southwestern region of Morocco. Four thermo-resistant bacterial species were isolated from all six soil samples. Only one different bacterial species, represented in Figure 1a,b, was isolated from a location in proximity of an urban area where wastewater and garbage contaminate the argan field. Recent works indicated the presence of six different bacterial strains of PHB producers isolated from sewage samples. Among all the isolated species, one Strain 11 produced 45.9% of PHB by using glucose as the sole carbon source [20]. From this perspective, this work aimed to isolate new bacterial strains able to synthesize the biopolymer PHB, to subsequently optimize the synthesis, modification, and biodegradation processes of the polymer by using the argan waste biomass. Morphological analysis of the pure isolated colonies of the PHB-producing bacteria described the whole colony appearance as irregular to flat, with undulate margins of a white, opaque color, and a rough surface. Similar morphological characteristics were discussed by Kaliwal and colleagues. Of all the six PHB-producing strains isolated from fodder field soil, the researchers identified a strain called BBKGBS6, classified as a member of the genus Bacillus, that showed similar morphological characteristics and was able to accumulate 60% of intracellular PHB [21].
Identification of PHB-Producing Bacteria by Schaeffer-Fulton Endospore Staining
For rapid detection of PHB-producing bacteria, the Schaeffer-Fulton staining method was used. The pure colonies isolated from the modified agar medium were stained with Malachite Green, a preliminary screening agent for lipophilic molecules. Under microscopic observations at 100X on oil immersion, the bacterial endospores showed a green coloration as reported in Figure 2a. This positive result is explained by the activity of the lipophilic endospore membranes that allow the Malachite Green to cross the membrane and to retain the green coloration [19] (Ruth, 2009).
The endospore was also stained using a variation of Methylene Blue, which was used as a coloring alternative to Malachite Green in staining the bacterial species Bacillus subtilis and Clostridium tetani by Okatari et al. In this recent work, the endospore staining properties of Methylene Blue were investigated using differently concentrated solutions of the dye at different pH levels. Their work demonstrated that the species Bacillus subtilis are well stained at an optimal stain concentration of 0.5% and pH 12, while in staining the species Clostridium tetani, the optimal concentration was 0.5% at pH 11 [19]. In following this alternative, this study adopted confirmatory staining that was performed by using the Schaeffer-Fulton staining method where the conventional Malachite Green dye was substituted by an alcoholic solution of Methylene Blue 0.5% at pH 12. The pH and concentration parameters selected for this work correspond to the optimal staining conditions of the bacterial species Bacillus subtilis since the morphological analysis previously conducted may suggest that the species isolated belongs to the genus Bacillus. After staining with Methylene Blue 0.5% at pH 12, the specimens were counter-stained with Safranin. The colored specimens were observed under the compound light microscope at 1000× in oil immersion. Figure 2b shows how the bacterial endospores retained the blue color of the Methylene dye, which passed through the endospore membranes and colored the internal granules.
Conclusions
Top and rhizospheric soil samples collected from agricultural crops, industrial contaminated fields, and uncultivated fields have been selected as favorable environmental areas for the PHB producing bacteria growth. Notably, olive oil, vegetable oil, and sunflower oil crops have been identified as good reservoirs for the isolation of PHB synthesizers as well as for the optimization of the polymer synthesis and modification by using the corresponding biomass waste material like oil wastewater and natural solid wastes.
In this work, the soil samples were collected from the argan field in the southwestern region of Morocco, where the Argania spinosa species is endemic and preserved. Among all the soil samples collected, five different bacterial thermo-resistant specimens were isolated and tested for the identification of intracellular endospores. Only one bacterial species was identified as positive. This selected species was isolated from an area of the argan field exposed to human contaminations, such as wastewater and solid wastes. The positivity of this work was confirmed by the Schaeffer-Fulton staining methods for the identification of endospore producing bacteria. The conventional method of using Malachite Green evidenced the presence of intracellular material that retained the green dye in contrast with the red color of the vegetative compartments that retained the Safranine. Furthermore, the variation method that used the Methylene Blue stain instead of Malachite Green confirmed the presence of intracellular compartments colored in blue because they retained the first colorant, while, also in this method, the rest of the bacterial cell appeared in red. After this first identification, further biochemical and biomolecular analysis is necessary to identify the bacterial species isolated. Moreover, further research is necessary to understand the role of the microorganism in the synthesis of the PHB and how the argan waste products can be valuable for the PHB synthesis, blending, and biodegradation.
Conflicts of Interest
The authors declare that they have no conflict of interest. | v3-fos-license |
2019-01-13T04:10:11.082Z | 2018-11-04T00:00:00.000 | 104450153 | {
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} | pes2o/s2orc | HPLC Method of Analysis for Determination and Standardization of Luteolin and Vanillic acid in Dry Extract of Paronychia argentea Lam
The main objective of this study was to establish a chromatographic method for analysis, determination and standardization of the two main components vanillic acid and luteolin as major components in Paronychia argentea Lam dry extract. This analytical method was designed to be a simple and fast with an appropriate separation of the two main components of the extract. High pressure liquid chromatography (HPLC) method of analysis was developed to quantitatively determine, identify and standardize the two main active constituents in the pharmaceutical dry extract against luteolin and vanillic acid as primary reference standards as it is the major active constituents of the dry extract of P. Argentea, where the linearity obtained was higher than R2 = 0.99981 and 0.99908 respectively. Although the method was proven to be suitable, further specific analysis validation was conducted to include the following: linearity, precision, range, limit of detection, limit of quantitation and filter compatibility. The luteolin and vanillic acid were completely separated from the other components in the herbal dry extract with an Rf value of 1.3 and 5.7 min. respectively. The concentration of Luteolin is 0.4% while vanillic acid content is 0.1% in the dry extract. Keyword: Paronychia argentea, HPLC, Analysis, Vanillic acid, Luteolin, Extract, Quantitation, Validation, Linearity, Precision and Repeatability. Abbreviations: HPLC, High Pressure Liquid Chromatography, AchE, Acetyl Cholinesterase, RS, Reference Standard, LOD, Limit of Detection, LOQ, Limit of Quantitation.
INTRODUCTION
Paronychia argentea Lam.(Locally known as Rijl El Hamameh) is a perennial herb, distributed widely throughout in Jordan 1 .Several studies showed that Paronychia argentea has hypoglycemic activity [2][3][4] , and it has been proved to be useful as gastric analgesic, bladder, prostate, abdominal ailments treatment, and stomach ulcers treatment 5 .It also showed significant alpha amylase 6 and acetyl cholinesterase (AChE) enzyme inhibitory activity 7 , the plant extract of Paronychia argentea showed antioxidant activity 8 .Other in vivo and in vitro studies on different extracts from Paronychia argentea revealed the immunomodulating activity of the plant 9 .
Accurately weighed a 20 mg of Vanillic acid RS and transferred to a 50-ml volumetric flask then 30 ml of mobile phase was added, and the solution was sonicated for 10 minutes and made up to volume with the mobile phase(Final Concentration 0.4 mg/ml).
Standard solutions were injected using equal volumes of 20ml of at a flow rate of 1.0ml\min.and a wavelength of 260 nm.
To calculate the percentage of both vanillic acid and luteolin in the sample taken of the dry extract, the following formula was used.Sample preparation for HPLC analysis 4.0 g of dry extract were transferred to a 25 ml volumetric flask, and 15 ml diluent (mobile phase) was added and sonicated for 20 minutes.The volume was completed with diluent (mobile phase) and filtered through 0.45 µm Nylon filter.
Method Validation
The analytical method used for the quantification of Luteolin and Vanillic acid in the
Materials
Plant material of P. argentea, aerial part, was collected on April 2016 from Ajloun area, Jordan.The dried plant material (600g) was grounded and soaked in ethanol (90%) for three weeks with frequent agitation, the alcohol solution was and then filtered and evaporated using Rota Vapor to obtain a solid residue (48 g).
Standard luteolin Primary Reference
Standard, batch number HWI01784 was purchased from HWI Pharma services GmbH-Germany.Standard vanillic acid Primary Reference Standard, lot number STBD6012Vwas purchased from Sigma Aldrich -Germany, Distilled water, Acetonitrile HPLC grade, Methanol HPLC grade, Acetic Acid 99% HPLC grade.
High Pressure Liquid Chromatographic analysis Preparation of Mobile phase
Distilled water, methanol and acetic acid were mixed at a ratio of (700:300:10) ml respectively then passed through a nylon membrane filter having a pore size of 0.45µm and sonicate to degas.
Preparation of Reference Standards
3.0 mg of Luteolin RS were accurately weighed and transferred to a 5 ml volumetric flask P. argentea extracts was validated for linearity, LOD and LOQ, precision, as previously described.
Optimization of Chromatographic Conditions
The HPLC conditions were optimized for the mobile phase composition, column temperature, wavelength, and flow rate (Table below).Detection wavelengths were set according to the ultraviolet (UV) absorption maxima of the compounds (260 nm).
Peak of Luteolin and Vanillic acid standard preparations appear at retention time of 1.3 min 5.7 min.respectively as shown in Fig. 5 and Fig. 6 respectively, and a sample preparation was injected into the chromatograph and two peaks appear at retention times of 1.6 min.5.7 min.respectively as shown in Figure 7.
Determination of major components in Paronychia argentea Lam dry extract
It was found that the Luteolin content is 0.4% while vanillic acid content is 0.1%
Method Validation Linearity
The linearity of the assay method of vanillic acid was determined in the range from 25, 40, 50,60,75, 85 and100%, proportional to the concentration relative to the prescribed standard concentration 0.4 mg/ml and the curve was linear over a this large number of concentration and exhibited a linear regression (R 2 = 0.99981) and slope of 201133.9864.
Precision System repeatability
In this test, system repeatability tests were examined and standard solutions containing Vanillic acid were Prepared, and injected 10 times into the HPLC system.Average of peak areas and % RSD values were calculated and shows to be within acceptable limits of 0.98%.The resulting areas and RSD values shown in Table 3.
Analysis Repeatability
For the determination of the repeatability of the extract, six samples obtained from multiple sampling of extract were analyzed, in a single laboratory on a single day.Assay percentage, and RSD values were calculated.The results obtained are listed in Table 4.
Range
Range is the concentration interval between the upper and lower concentration of analyte for which is shown that the method has suitable level of linearity.Range for determination is extended from 25% to 100% vanillic acid.
Limit of Detection
Limit of detection (LOD) is the lowest concentration of analyte in a sample, which can be detected, but not necessary quantified, under the stated experiment conditions.
For vanillic acid it is estimated by extrapolation of regression line through Y axis from standard curve at low concentration as in the following expression.
The LOD was found to be 0.40 (µg/ml) for vanillic acid which is equivalent to 0.2% and 1.2(µg/ ml) which is equivalent to 0.6% of nominal value of Luteolin respectively.
Limit of Quantitation
The limit of quantitation (LOQ) is the lowest concentration of analyte in a sample, which can be determined with an acceptable accuracy and precision detected.
For Vanillic acid is estimated by extrapolation of regression line through Y axis from standard curve at low concentration as in the following expression Figure 3 and 4. The LOQ was found to be 1.2 (µg/ml) for vanillic acid which is equivalent to 0.6% of nominal value and 9.7(µg/ml) which is equivalent to 4.8% of nominal value of Luteolin respectively (Tables 6, 7 and 8).
Filter compatibility
Variation of the filter type is described below and the results are reported in Table 9.
Several attempts were used to develop a reverse phase HPLC method of analysis for the separation of the two main components luteolin and vanillic acid available in dry extract of Paronychia.
A suitable mobile phase was established to separate the two major compounds of the extract in one run with a suitable and reasonable time, where both vanillic acid and luteolin were detected at a wavelength of 260 nm, and a sample preparation was injected into the chromatograph and two peaks appear at retention times of 1.3 min.5.7 min.respectively and a runtime of 15 min.and at a flow rate of 1.0 ml\min.using ODS-3 (150X4.6)mm, 5 µm.
A baseline resolution was obtained under the testing of the analysis conditions, as well as the chromatograms of the standards and samples preparation are shown in Fig. 5, 6 and 7. this method has been validated as per ICH guidelines for linearity, precision, limit of detection and limit of quantitation for both main active components, and a linear relationship was noticed for the two compounds and the correlation coefficient was R 2 = 0.9998.method developed and used is precise, repeatability LOD, LOQ and filter compatibility, it can be concluded that the method developed and used is precise, sensitive accurate and reproducible, and facilitate quantitative determination of this extract and will accomplish the objective of this study and consequently utilizing a local Jordanian plant since the active materials in the extract are variable.The concentration of luteolin and vanillic acid in the dry extract was 0.
CONCLUSION
The two major peaks of Luteolin and Vanillic acid were simultaneously separated and determined successfully from the other components in the Paronychia argentea Lam dry extract collected from the plant located in Jordan and was found that the Luteolin content is 0.4% while vanillic acid content is 0.1% using a validated chromatographic method of analysis.These two compounds are considered as a standardized reference of the plant material.
Statistical analysis are shown in Table1 and a plot of area under the curve versus concentration can be seen in Fig. 1.The standard curve was plotted and evaluated for linearity.The obtained equation for the standard curves was: Equation: Y = AX -B, Where B is the intercept with Y-axis and A is the slope
Fig. 3 .
Fig. 3. Regression for concentrations vs. area of vanillic acid standard for LOD and LOQ
Table 3 : System precision for vanillic acid standard
Area under the curve for 10 replicate injections
: Standard solution of luteolin prepared for calculation of LOD and LOQ
4 and 0.1% respectively. | v3-fos-license |
2018-11-06T00:00:48.539Z | 2018-11-03T00:00:00.000 | 53207408 | {
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} | pes2o/s2orc | Redox nanoparticles: synthesis, properties and perspectives of use for treatment of neurodegenerative diseases
Oxidative stress (OS) and nitrative stress (NS) accompany many diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Antioxidants have been proposed to counteract OS/NS in these diseases. Nevertheless, the effects of antioxidants are limited and new, more efficient antioxidants are searched for. Redox-active nanoparticles (RNPs), containing antioxidants create a new therapeutical perspective. This review examines the recent literature describing synthesis and potential applications of cerium oxide RNPs, boron cluster-containing and silica containing RNPs, Gd3N@C80 encapsulated RNPs, and concentrates on nitroxide-containing RNPs. Nitroxides are promising antioxidants, preventing inter alia glycation and nitration, but their application poses several problems. It can be expected that application of RNPs containing covalently bound nitroxides, showing low toxicity and able to penetrate the blood–brain barrier will be more efficient in the treatment of neurodegenerative disease, in particular AD and PD basing on their effects in cellular and animal models of neurodegenerative diseases.
Introduction
Age-related diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) constitute a considerable socioeconomic burden for contemporary societies. As human mean lifespan increases, growing incidence of these diseases has features of a pandemic. AD is one of the main causes of insufficiency and need of care by other persons, which significantly deteriorates the quality of life of both the affected persons and their caregivers. It should be emphasized that current AD and PD treatments alleviate only the symptoms of the disease without halting its progress. Oxidative stress (prooxidative disturbance in the equilibrium between prooxidants and antioxidants; OS) is observed in the course of many diseases and plays a role in the etiopathogenesis of some of them. Neurodegenerative diseases, including AD and PD are characterized by increased levels of protein nitration, which is mainly the result of the reactions of peroxynitrite (ONOO − ) primarily with residues of such amino acids as tyrosine, phenylalanine and histidine [1][2][3]. Peroxynitrite is formed in vivo under conditions of OS/NS, in the diffusion-controlled reaction between nitric oxide (NO • ) and superoxide radical anion (O 2 •− ). Production of NO • and O 2 •− intensifies as a result of immune response, inter alia, by macrophages. Peroxynitrite has multiple actions on various molecules, especially proteins [4], including one-and two-electron oxidations, nitration (attachment of NO 2 ) and nitrosylation (attachment of NO).
It is generally assumed that increased level of nitration, evidencing enhanced ONOO − production, is a universal marker of inflammation; it was also found in such pathologies as AD, PD, amyotrophic lateral sclerosis, diabetes, rheumatoid arthritis, lupus, atherosclerosis, hypertension, as well as liver and cardiovascular diseases [5].
Page 2 of 16 Sadowska-Bartosz and Bartosz J Nanobiotechnol (2018) 16:87 Antioxidants have been proposed to counteract the development of neurodegenerative diseases. However, the effects of antioxidant intervention, including increased consumption of dietary antioxidants, are generally modest in the best case [6,7]. These findings suggest that new antioxidant compounds, of increased efficacy, should be searched for. Synthetic antioxidants may be more efficient than naturally occurring ones. Apart from the antioxidant function, synthetic compounds have other beneficial actions; e.g. they can interfere with protein aggregate formation or inhibit undesired enzymatic activities. Free nitroxide radicals (nitroxides) are promising in this respect. They are many-faceted antioxidants, which have pseudo-superoxide dismutase (SOD) activity, inhibit Fenton chemistry by the ability to oxidize transition metal ions, terminate radical chain reactions by radical recombination and accept electrons from the mitochondrial electron transport chain. They react also with protein tyrosyl and tryptophanyl radicals [8].
Nitroxides have been demonstrated to inhibit oxidation and, especially, nitration by reacting with ONOO − and/or its decomposition products. Our comparative study of 11 nitroxides of various structure demonstrated structuredependent differences in their efficiency, suggesting the possibility of choice and design of optimal compounds. Nitroxides were more effective in preventing nitration than oxidation reactions. Concentrations corresponding to IC 50 for prevention of nitration of model compounds for most nitroxides were lower than 100 nM, which, in combination with the lack of changes of ESR signal of the nitroxides, speaks for catalytic decomposition of ONOO − or its decomposition products by these compounds. No prooxidant effect of nitroxides was seen in prevention of dihydrorhodamine 123 (DHR123) oxidation induced by 3-morpholino-sydnonimine (SIN-1, OONO − donor). Interestingly, nitroxides showed a concentration window for preventing DHR123 oxidation by ONOO − , exerting a prooxidant effect at both high and low concentrations. Nitroxides turned out to be much more effective in protecting human serum albumin (HSA) against nitration of tyrosine residues than against oxidation of thiol groups. Most nitroxides, except for the most hydrophobic ones (especially 4-nonylamido-TEMPO and 3-carbamoyldehydro-PROXYL) protected cells from the cytotoxic action of SIN-1 and SIN-1-induced protein nitration [9]. TEMPO, as well as an N-arylnitroxide and an N, N-diarylnitroxide, react with alkylperoxyl radicals, the propagating species in lipid peroxidation. They are significantly more reactive than Vitamin E, the most efficient natural antioxidant trapping peroxyl radicals. Nitroxides reduce peroxyl radicals by electron transfer with rate constants of 10 6 -10 7 M −1 s −1 . This reaction is catalytic; NADPH in the aqueous phase can be used as a hydride donor to promote nitroxide recycling. In cell culture, nitroxides are potent inhibitors of ferroptosis [10]. We found that nitroxides are glycation inhibitors: 2,2,6,6-tetramethylpiperidin-1-yl-oxyl (TEMPO), 4-carboxy-TEMPO and 4-hydroxy-TEMPO offered significant protection against glycoxidation induced by glucose and fructose, while 3-carbamoyl-PROXYL enhanced glycoxidation [11].
Nitroxides are not toxic to breast cancer MCF-7 cells (human breast adenocarcinoma cell line) at concentrations of up to 500 µM, with the exception of 4-cyano-TEMPO (significant reduction of survival at a concentration of 500 µM) and 4-nonylamido-TEMPO (significant reduction of survival in the concentration range of 250-500 µM) after 72-h incubation in the presence of these compounds. Preincubation of cells with nitroxides at a concentration of 100 µM enhanced survival of MCF-7 cells treated subsequently with 100 µM SIN-1. These compounds reduced also the level of intracellular protein nitration induced by SIN-1 [9]. Nitroxides have been shown to have antitumor activity and, consequently, they prolonged the lifespan of mice with an increased tendency for malignancy [12,13]. It is important that nitroxides can be administered in vivo chronically. In fact, long-term administration of 4-hydroxy-TEMPO increased latency to tumorigenesis and doubled the lifespan of Atm-deficient mice and p53-deficient mice [13]. Nevertheless, it must be noted that low-molecular-weight (LMW) nitroxides pose several problems such as nonspecific dispersion in normal tissues, and preferential renal clearance resulting in short life time in vivo.
Covalent attachment of nitroxides to nanoparticles (NPs) of optimal structure can be a solution of this problem, increase the efficacy of nitroxide action and allow for selective delivery. Nanoparticles are natural or synthetic particles whose size is within the range of 1-100 nm. Nanoparticles are widely used in nanomedicine, playing an important role as diagnostic tool and being also applied for treatment, monitoring and control of many diseases, including neurodegenerative disorders. Many studies are focused on development of new drug delivery systems, which are necessary to increase the therapeutic effectiveness of NPs [14,15]. Nowadays there are many approaches in drug delivery to the central nervous system (CNS). The most common strategies are based on polymeric NPs and lipid-based NPs; inorganic NPs (especially iron oxide and quantum dots) are also used. Neurotoxicity of NPs and their effect on neurons, cellular components and the blood-brain barrier (BBB) are equally important [16]. In order to prevent or at least limit adverse effects to normal organs, tissues and cells, disturbance of normal redox reaction in healthy tissues must be prevented. It can be achieved by optimal design and targeting of NPs.
In this review we concentrate on advantages of biocompatible and colloidally stable NPs, in which nitroxide radicals are covalently conjugated to a polymer structure (nitroxide-based redox nanoparticles, NRNPs). These NPs effectively scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) with a characteristically prolonged bioavailability and tissue-residence time much longer than that of conventional LMW antioxidants. The confinement of the nitroxide radicals in the polymer core prevents their rapid metabolism and excretion out of the blood circulation. The nano-sized formulation limits internalization of NRNPs in healthy cells, thereby preserving the normal redox status of these cells.
Structure of redox nanoparticles and synthesis methods
Nanoparticles are generally composed of three layers: (a) the surface layer, which may be functionalized with a variety of small molecules, surfactants, metal ions and polymers, (b) the shell layer, which is chemically different material from the core, and (c) the core, which is the central portion of the NP and usually is referred to as the NP itself [17,18]. There are several methods for producing NPs, including gas condensation, attrition, chemical precipitation, ion implantation, pyrolysis and hydrothermal synthesis.
Nowadays many types of NPs are used. This review focuses on redox nanoparticles (RNPs), in particular NRNPs. Redox nanoparticles are biocompatible NPs that can scavenge ROS with high efficiency [19]. Redox nanoparticles are not internalized in normal cells by the intact cell membrane, so almost no cellular dysfunction is observed in normal cells after their administration [20]. Moreover, some RNPs (NRNP N , see below) disintegrate under acidic conditions due to the protonation of amino groups, because they possess amine linkages in the core. Accordingly, NRNP N s disintegrate into individual polymers within the stomach and can be absorbed as dissociated polymer into the bloodstream across the intestinal epithelium [21]. Most nanomedicines are not used as orally administered drugs for systemic diseases, since NPs of the size of 10-100 nm are not absorbed via the gastrointestinal tract. Redox polymers are absorbed by the blood following oral administration of RNPs, thereby rendering them an ideal medication for chronic diseases. Nanoparticle formulation can be drunk easily due to its low viscosity. This advantage is practically very important, considering the long-term treatment of chronic diseases [22]. Owing to large surface area-to-volume ratio of nanomaterials, higher electron densities can be found on the outer surface, resulting in increased catalytic activity [23]. For instance, metal oxide nanomaterials can potentially be involved in effective ROS scavenging and/or deoxygenating reactions [22,24]. In recent years, an increasing number of studies have emphasized that nanomaterials can mimic the properties of antioxidant enzymes (nanomaterial-based artificial enzymes, socalled nanozymes), to inhibit apoptosis and improve the cell survival [25].
A few examples of RNPs are presented below.
Cerium oxide (CeO 2 ) nanoparticles (CNPs)
Cerium dioxide (ceria/CeO 2 ), popularly known as nanoceria, has been reported to be the most stable oxide of the cerium. Cerium is the most reactive element in the lanthanide series due to its electropositive nature. Cerium exists in two different oxidation states (Ce 3+ and Ce 4+ ) or in a mixed valence state. The Ce 4+ oxidation state is more stable than Ce 3+ . Cerium has two main types of oxides-cerium dioxide (CeO 2 ) and cerium sesquioxide (Ce 2 O 3 ), nevertheless the more stable CeO 2 is used rather than Ce 2 O 3 [24]. This interchangeability of Ce 3+ and Ce 4+ makes them regenerative ( Fig. 1) [23]. Cerium oxide nanoparticles (C-NPs) have been synthesized using many different methods such as thermal decomposition, sol-gel microemulsion methods, solvothermal oxidation, flame spray pyrolysis and microwave-assisted solvothermal process or mixing cerium sulphate and ammonia solution at room temperature [26]. All C-NPs contain the same core elements (Fig. 2a), but do not display similar biological effects. Prooxidant toxicity of C-NPs was demonstrated in some cases and antioxidant protective effects in others that could be attributed to different physicochemical parameters of the various C-NPs. The method of C-NP synthesis, type of the stabilizing agent used, and the Ce 3+ /Ce 4+ surface ratio have been reported 16:87 to play major roles in producing CeO 2 -NPs with different physicochemical properties [23,[26][27][28]. CeO 2 -NPs mimicking catalase and SOD activities or displaying peroxidase-like activities have been reported [29]. Oxidaselike activity of CNPs originated from surface Ce 3+ atoms as the catalytic centers. CeO 2 -NPs with lower Ce 3+ density on the surface show catalase or peroxidase mimetic activity. CeO 2 -NPs can also induce angiogenesis in vivo by modulating the intracellular oxygen environment and stabilizing hypoxia inducing factor 1α (HIF-1α) [30]. CeO 2 -NPs of high surface density of Ce 3+ inhibit the growth of both gram-negative and gram-positive bacteria [28]. It was reported also that C-NPs can trigger neuronal survival in AD model, beta-amyloid [Aß (25-35)-treated SH-SY5Y cells, twice-subcloned cell line derived from the SK-N-SH neuroblastoma cells], through modulation of the extracellular signal-regulated kinases 1/2 and 5 (ERK1/2, ERK5) and the brain-derived neurotrophic factor (BDNF) signaling pathways [31]. These factors are involved in the signal transduction pathways of neuronal survival and differentiation [32]. There are many reports on the neuroprotective effect of engineered CeO 2 -NPs. Arya et al. [33] reported that CeO 2 -NPs promoted neurogenesis and modulated hypoxia-induced memory impairment through the 5′-adenine monophosphateactivated protein kinase-protein kinase C-cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP) protein pathway. Using polyethylene glycol-coated 3 nm nanoceria (CeO 2 -NPs), it was shown that NPs efficiently penetrated the BBB, were localized in rodent brain in adult male Sprague-Dawley rats and decreased OS [32,34,35]. Using an in vivo model of mild lateral fluid percussion brain injury in the rat, Bailey et al. [35] demonstrated the beneficial antioxidant action on C-NPs on traumatic brain injury outcome. These authors found that C-NPs improved neural survival as well as cognitive function by decreasing macromolecular free radical damage and promoting preservation of endogenous antioxidant systems [35]. Nanoceria formulation enhances the proliferation and migration of fibroblasts, keratinocytes and vascular endothelial cells, which further accelerate the wound healing process. Nanoceria can also protect regenerating tissue by reducing OS in the wounded region [36]. Nanoceria acted as antioxidants against a free radical-mediated autoimmune degenerative disease in the brain [37]. Nevertheless, cerium cations erode from NPs, which causes strong toxicity to normal tissues and cells in vitro. The increased ROS in cultured human bronchial epithelium, normal (BEAS-2B) cells by C-NPs trigger activation of cytosolic caspase-3 and chromatin condensation, which means that C-NPs can exert cytotoxicity inducing the apoptotic process [38].
Silica-containing redox nanoparticles (Si-RNPs) are a special type of NPs, which can have medical applications as novel nano-sized adsorbents for peritoneal dialysis, and orally administrable drug carriers for the treatment of gastrointestinal inflammation. These RNPs consist of silica NPs and amphiphilic block copolymers with nitroxide radicals (Fig. 3a). Electrostatic interactions between the cationic segment of a polymer in the core and the entrapped silica nanoparticles form a crosslinked structure that provides siRNP stability in vivo, even under harsh conditions in the gastrointestinal tract. Si-RNPs can be applied not only as adsorbents of body wastes, but also as drug carriers with high loading capacity due to their excellent adsorption properties [40]. Gd 3 N @ C 80 encapsulated redox nanoparticles (Gd 3 NPs) is a novel contrast agent for magnetic resonance imaging (MRI). These RNPs contain gadolinium ions in the core. Because free Gd(III) ions are toxic, it is necessary to make Gd-chelate compounds or encapsulate the ions inside RNPs. Gd 3 N @ C 80 encapsulated RNP structure (Fig. 3b) is based on typical poly(ethylene glycol) shell with encapsulated fullerene. Inside the hollow sphere structured from 80 carbon atoms, three atoms of Gd are placed. This NP is obtained by reaction of PEG-b-PMNT with Gd 3 N @ C 80 . Gd 3 N @ C 80 particles are characterized by low toxicity, high relaxivity and solubility in water. Gd 3 NPs have the ability to scavenge ROS. In addition, Gd 3 NPs have also colloidal stability, long circulation time and can be used as nano-contrast agent in MRI technique for clinical diagnosis [41].
Nitroxide radicals-containing redox nanoparticles (NRNPs)
Nitroxide radicals-containing redox nanoparticles confine nitroxide radicals to their core. For this reason, their antioxidative nature is not fully expressed when they have the full, spherical form. Nitroxide radicalscontaining redox nanoparticles are thought to have the potential to be used as high-performance bio-nanoparticles in vivo [42]. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) groups as a side chain of the PMNT segment are known to be stable radicals not reacting with each other. As mentioned before, TEMPO molecules have a pseudosuperoxide dismutase activity; they directly react with both carbon-centered and peroxy radicals preventing the conversion of hydrogen peroxide to hydroxyl radical. Hence, NRNP N may attenuate the formation of hydroxyl radical. Because the PMNT segment possesses repeating amino groups, it protonates in response to pH decrease and becomes hydrophilic to result in disintegration under acidic conditions (pK a , 6.5) (Fig. 5). The sensitivity of NRNP N s micelles to pH changes is an important considering the low pH extracellular environment of tumour tissues, which could affect treatment efficacy. pH-insensitive redox nanoparticles (NRNP O s), which are not disintegrated in any area of the gastrointestinal tract, maintain a micellar form in the stomach and intestine. After oral administration, NRNP O s are accumulated on the surface of the intestinal epithelium, but not inside the villi, because diffusion of NRNP O s across the intestinal mucus layer to reach the epithelium is difficult, given its size of 40 nm. Contrary to NRNP O , the redox polymer after disintegration of NRNP N is internalized deeply in the villi across the intestinal epithelium [21]. A few examples of synthesis methods of selected NRNPs are described below.
Poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl) aminomethylstyrene] (MeO-PEG-b-PMNT)
The synthesis of NRNPs is a several-step process. At first, poly(ethylene glycol) monomethyl ether is used to prepare poly(ethylene glycol)-b-chain transfer agent (PEG-b-CTA). In the next step, block copolymer is synthesized by the method of the radical polymerization of chloromethylstyrene (CMS). The product of the reaction is poly(chloromethylstyrene) (PCMS). In result, methoxy-poly(ethylene glycol-b-poly(chloromethylstyrene) is obtained and its main segments are a hydrophilic PEG and a hydrophobic PCMS. To obtain PEG-b-PMNT, chloromethyl groups on the PCMS segment of the block copolymer are converted to nitroxide radicals via ami- (Fig. 4a). The last step relies on preparation of RNP N micelles. MeO-PEG-b-PMNT is used to create micelles using a dialysis method. At first, this polymer is dissolved in dimethylformamide and the solution is dialyzed against distilled water. As a result, NPs with a diameter of 40 nm are formed. The driving force for micelle formation are hydrophobic interactions of NH 2 -TEMPO, which is the active segment. MeO-PEG-b-PMNT, possessing the cationic PMNT segment, may be internalized in the brain via the endocytosis pathway. Interestingly, it can prevent efflux of chemotherapeutic drugs, e.g. doxorubicin, by inhibiting P-glycoprotein, the main protein conferring multidrug resistance [43].
Furthermore, the long-term access of redox polymers coupled to serum protein to the brain vessel wall by extended blood circulation time might increase internalization tendency in the brain. Since redox catalytic species are covalently conjugated to redox polymers as stated above, they are internalized together with the polymers. It was reported that 5-6% of the injected in mice dose of the redox polymers (NRNP O ) was absorbed into the bloodstream and circulated over 24 h while orally administered TEMPOL was eliminated within 1 h from the blood [20].
TEMPO-containing acetal-PEG-b-PCMS
The anionic ring-opening polymerization of ethylene oxide was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)-methanesulfonate (acetal-PEG-Ms) (Compound 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl as well as acetal terminal groups (acetal-PEG-SH) (Compound 2). Poly(ethylene glycol)block-poly(chloromethylstyrene) (acetal-PEG-b-PCMS) (Compound 3) was synthesized by the free-radical telomerization of CMS using Compound 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (Compound 3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) groups via the amination of Compound 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (Compound 4) (Fig. 4c). The obtained Compound 4 formed core-shell-type NP in aqueous media when subjected to dialysis: the semi-invariant average diameter of the NP was about 40 nm, and NP emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of NPs were resistant to reduction even by 3.5 mM ascorbic acid. This means that these NPs are high-performance NPs that can be used in vivo [45].
The NRNP showed almost no cytotoxicity up to the 8 mM level, which is in sharp contrast to LMW TEMPO [46]. The compartmentalization of the TEMPO radicals in the NRNP core improves their stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PC-TEMPO segment, the disintegration of the NRNP is caused by protonation of the amino groups in response to the acidic pH environment (pH < 6.0) [46]. The polymer structure is similar for the majority of NRNPs: it is an amphiphilic block polymer with nitroxide radicals inside the core [45,[47][48][49] (Fig. 4c).
Potential applications NRNPs are based on their several features: • The disintegration of NRNP N in response to low pH; NRNPs disintegrates after intravenous administration in response to low pH environments, such as ischemic, inflamed and tumor tissues, owing to protonation of amino groups of the hydrophobic segment, resulting in increased ROS scavenging activity, because of an exposure of the nitroxide radicals from the NRNP N core. Orally administered NRNP N s are disintegrated to redox polymers in the stomach; redox polymers are absorbed in the blood stream through the intestines and circulate for ca. 24 h, and ultimately reach the liver. Contrary to LMW antioxidants, NRNPs and disintegrated polymers cannot be internalized into normal cells [48]. Additionally, NRNPs suppress liver inflammation and reduced the infiltration of neutrophils, monocytes and macrophages [42]. • RNPs are characterized by long blood-circulation time; The EPR signals in the blood stream are hardly observed even after 2 min, when LMW TEMPOL is administered via tail-vein injection. The half-life of TEMPOL in blood has been reported to be approximately 15 s [50]. On the contrary, when NRNPs prepared from PEG-b-PMNT (NRNP N ) and PEG-b-PMOT (NRNP O ) are administered, the ESR signal in the blood stream was observed even 2 h after the tailvein injection of NRNPs. The half-life of the NRNP N s was 60-times longer (15 min) than that of LMW TEMPOL. RNP O s show much longer circulation. Actually, the half-life of the NRNP O s was 600 min, 2400-times longer than that of LMW TEMPOL [42]. • Low toxicity: The extremely low toxicity of the NRNP N is probably due to the fact that the outer PEG layer constitutes an excellent stealth shield around the amino TEMPO moieties in the NRNP core [42]. • NRNPs are excellent free radical scavengers. • NRNPs are also potential ESR imaging tool [46]. To visualize, monitor or record spatiotemporal distribution of molecular and cellular processes, imaging modalities such as optical imaging (luminescence and fluorescence), radionucleotide-based imaging [positron emission tomography (PET), single photon emission computed tomography (SPECT)], magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound need specific molecular probes to target molecules, cells or tissues and allow them to be imaged. A desired molecular imaging probe should provide high binding affinity and specificity to target, high sensitivity and high stability in vivo as well as less toxicity. More recently, chlorin e6 (Ce6) and alpha-tocopherol succinate (TOS) were conjugated to LMW heparin via cysteamine as the redoxsensitive linker, forming amphiphilic Ce6-LMWH-TOS (CHT) polymer, which could self-assemble into NPs in water and encapsulate paclitaxel (PTX) inside the inner core (PTX/CHT NPs). The enhanced near-infrared (NIR) fluorescence intensity and ROS generation of Ce6 were observed in a reductive environment, suggesting the cysteamine-switched "ON/ OFF" of Ce6. The redox-responsive PTX/CHT NPs successfully integrated the chemotherapeutic effect of PTX, photodynamic therapy and NIR imaging capacities of Ce6, displaying great potential for smart NIR imaging-guided tumor combinational therapy [51]. A summary of properties of various redox nanoparticles is given in Table 1.
Redox nanoparticles as a novel treatment approach for treatment of neurodegenerative diseases
AD is a primary degenerative disease of the CNS, characterized by progressive deficit of memory and other cognitive functions, such as the learning ability, thinking, problem-solving and language communication. In 2015, there were approximately 48 million people worldwide with AD and the abundance of this disease has a growing tendency due to the increase in the lifespan in the developed countries. Alzheimer's disease is one of the most financially costly diseases [56]. The most characteristic hallmarks for this disease during a neuropathological examination of the brain are β-amyloid (Aβ) plaques and hyperphosphorylated τ (tau) deposits and fibrillar degeneration [57]. β-amyloid acts as a prion-like protein, which means that Aβ seeds can be intracellular oligomers. More recently, Olsson et al. [58] established that intracellular inclusions of Aβ can be seeded and stably maintained in [21]; biocompatible, stable, colloidal; long-term circulation ability in the bloodstream; protection against radiation-induced weight loss; reduction of the ROS-associated skin aging and skin damage caused by UV exposure [52]; potential use for oral treatment of non-alcoholic fatty liver disease; absorption to blood after disintegration in stomach; reduction of inflammation and liver fibrosis [22]; disintegration in acidic environments through protonation of amino groups in the RNP N core after oral administration; inflammation reduction; inhibition the development of colitisassociated colon cancer [53] Methoxy-poly(ethylene glycol)-block-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)oxymethyl- [59]. Primary motor signs of PD include tremor of the hands, arms, legs, jaw and face, bradykinesia, rigidity or stiffness of the limbs and trunk, impaired balance and coordination. PD is characterized by progressive degeneration and decay of dopaminergic neurons of the nigrostriatal path and, to a lesser extent, of the mesocorticolimbic and hippocampal path and, with variable intensity, noradrenergic, serotoninergic as well as cholinergic neurons, due to the deposition of insoluble protein aggregates, such as α-synuclein (αSyn) and parkin, forming so-called Lewy's bodies in the cytoplasm [60]. Till now, there is no cure for AD and PD, with the currently recommended treatments causing only transient alleviation of symptoms of these diseases. The need for new medicines is enormous, taking into account the socioeconomic dimension of these neurodegenerative diseases. Evidence for the involvement of OS and NS in the progress of AD and PD is compelling. The brains of patients suffering AD present a significant extent of oxidative damage associated with the abnormal marked accumulation of Aβ and the deposition of neurofibrillary tangles. Metals such as iron copper and zinc seem to play an important role in the induction of OS [61]. There are high affinity binding sites for copper and zinc on the N-terminal, metal-binding domains of Aβ and its APP.
Copper is a potent mediator of the highly reactive hydroxyl radical (OH·) formation, and consequently contributes significantly to the generation of OS characteristic of AD brain. High concentrations of copper have repeatedly been found in amyloid plaques [62]. In addition, high concentrations of zinc were associated with memory and cognitive regions of the brain, including the neocortex, amygdala and hippocampus, which are mostly affected in AD pathology. The binding of zinc induces a highly ordered conformational state of Aβ (1-40), leading to the production of toxic fibrillary Aβ aggregates. Moreover, the immunological/inflammatory response to insoluble Aβ plaques involves the disruption of zinc homeostasis followed by uncontrolled cerebral zinc release, which is typical for OS. Thus, the uncontrolled accumulation of zinc and Aβ leads to zinc-induced and Aβ-mediated OS and cytotoxicity [63]. Brain cell membrane phospholipids are characterized by a high content of polyunsaturated fatty acids, which are particularly vulnerable to free radical attacks. Increased lipid peroxidation is one of the most prominent features in the AD brain [64]. Proteins, which are the most abundant organic compounds in the cells, are the main target of ROS and RNS. Protein oxidation is markedly increased in AD. The oxidation of brain proteins can affect enzymes critical to neuron and glial functions. This is the case for two enzymes especially sensitive to oxidative modification, glutamine synthetase and creatine kinase, which are markedly reduced in AD brains [65]. Inactivation of glutamine synthetase results in alterations of glutamate concentrations and enhancement of excitotoxicity, whereas oxidative impairment of creatine kinase may decrease energy metabolism [66]. The pathologic aggregation of protein leads to fibril formation and insolubility. Neurofibrillary tangles are characterized by the aggregation and hyperphosphorylation of the τ protein into paired helical filaments. Phosphorylation is linked to oxidation through the microtubule-associated protein kinase pathway and through activation of the transcription factor nuclear factor-κB, thus potentially linking oxidation to the hyperphosphorylation of the τ protein. Protein oxidation is also capable to facilitate advanced glycation end products as a posttranslational modification of proteins. Furthermore, OS in the brain affects DNA, producing oxidation of guanine and other DNA bases, strand breaks, sister chromatid exchange, and DNA-protein crosslinking. Hence, the overproduction of ROS may have a deleterious effect and can be an important mediator of damage to cell structures and consequently significantly contribute to the progress of AD [2]. In the brain of PD patients, elevated iron levels are also observed. The effects of the disease are most marked in the nigrostriatal dopaminergic system, which can be explained by formation of potent redox couple formed by iron and dopamine itself [67].
Copper dyshomeostasis has also been demonstrated in the PD brain [68]. Reduced activity of Complex I of the respiratory chain in the substantia nigra pars compacta neurons of patients with PD has been demonstrated; it may contribute to the generation of excessive ROS. Mitochondrial Complex I deficiency in the frontal cortex, fibroblasts, and blood platelets have also been reported in patients with PD. Furthermore, the relationship between mitochondrial dysfunction and PD is supported by the findings that the Complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP). 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine metabolite, 1-methyl-4-phenylpyridinium (MPP + ), may exert cytotoxic effects on the dopamine neurons, resulting in clinically parkinsonian phenotype, and induce nigral degeneration with cytoplasmic α-Syn [69]. It has been also reported that genetic mutations in proteins including α-Syn, parkin, phosphatase and tensin homolog-induced putative kinase (PINK) were linked to the familial forms of PD. Mutations of these genes have been known to affect mitochondrial function and increase OS [70]. Changes in the levels of antioxidant molecules have also been reported even in the early stage of PD. For example, the levels of glutathione, a major cellular antioxidant, have been reduced in the substantia nigra pars compacta in PD [71]. It has been suggested repeatedly that both AD and PD patients can benefit from antioxidant supplementation and, since transition metals are important factors in the generation of OS in these diseases, from treatment with metal chelators. However, metal chelation may deplete the body of metals essential for the vital functions of the organism. The problems with BBB penetration by many chelators can make them active mainly outside the brain. The use of nitroxides may be a solution to both these needs. Unlike most antioxidants, which reduce metal ions facilitating their participation in the Fenton reaction, nitroxides are efficient antioxidants, but they oxidize transition metal ions preventing them from being involved in the Fenton reaction [8]. In view of the existence of common features between AD and diabetes [72] the anti-glycating activity of nitroxides may be also of value [11]. Free nitroxides are short-lived in vivo and part of them may have difficulties in penetrating the BBB. Covalent linking of nitroxides to nanoparticles able to penetrate BBB can prolong their lifespan and enable them to reach the brain. Appropriate design may create nitroxides combining the antioxidant and metal-oxidizing properties with another therapeutically useful functionality, e.g. inhibition of protein aggregation.
Neuroprotective role of NRNPs against AD-in vitro studies
Overproduced ROS can be stopped thanks to ROSscavenging properties of RNPs [6]. Chonpathompikunlert et al. [73] [74]. Although most of the previous studies have reported that PI is highly toxic at high concentrations [75][76][77] no cytotoxicity was observed up to the PI concentration of 100 mM, when the PI was coupled with 1 mM NRNPs. The compartmentalization of piperine in the core of the NRNPs might play an important role in the reduction of the toxic effect of PI.
In vivo studies of neuroprotective effects of RNPs in neurodegenerative diseases
The employment of antioxidants in neurodegenerative diseases has been proposed to ameliorate OS/NS. Nevertheless, the effects of antioxidants, among them natural components of the diet are limited and new, more efficient antioxidants are searched for. Vitamin E, LMW antioxidant, was reported to show slight efficacy such as slowing of functional decline of AD in clinical trials although complete recovery was not observed [78]. More recently, drug delivery systems using nanomedicines have attracted much attention in various areas of therapy. Nevertheless, most nanomedicines are not used as orally administered drugs for chronic diseases, such as AD and PD, because nanoparticles between the sizes of 10 and 100 nm are not absorbed via the gastrointestinal tract, as discussed before [21,55]. An orally administered drug with nanomedicine-like characteristics absorbed in the blood can be an ideal oral medication for such systemic diseases. Nagasaki group (2011, 2012) have proposed "redox polymer nanotherapeutics" using amphiphilic block copolymer, poly(ethylene glycol)b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (MeO-PEG-b-PMNT) (10 kDa, referred to as a redox polymer) [42,79]. This redox polymer possesses nitroxide radicals (NRNP N ) in bound covalently the hydrophobic segments and forms a polymeric micelle under physiological conditions, which confines the nitroxide radicals in its core and is 40 nm in diameter. As discussed above, NRNP N disintegrates after intravenous administration in response to low pH environments, such as inflamed, ischemic as well as tumor tissues, resulting in increased ROS scavenging activity [48]. Until now, the therapeutic effect of intravenously administered NRNP N was confirmed for various OS/NS-induced injuries including intracerebral hemorrhage and ischemiareperfusion injuries of the kidney, heart and brain) [21,82]. The administration of TEMPO in NRNP exerted cardioprotective effects against ischemia and reperfusion injury in canine hearts without exerting unfavourable hemodynamic effects [80]. Moreover, NRNP N do not cause adverse effects, because of no internalization in healthy cells such as blood cells and colon mucosal cells [20,53]. Nagasaki group [53] reported that orally administered pH-sensitive NRNP N (prepared by the self-assembly of MeO-PEG-b-PMNT (MW [PEG] = 5500 Da; MW [PMNT] = 4500 Da) using the dialysis method revived the cognition in 17-week-old the senescence accelerated mouse (SAMP8) mice [21], which are a spontaneous animal model of overproduction of APP and oxidative damage [81]. Small, but evident, amounts of NRNP N were internalized in the brain of normal mice. After treatment with NRNP N , ROS levels were decreased significantly in the brain of SAMP8 mice, probably because of the long access of redox polymers to blood vessel in brain. The scavenging of ROS in the brain prevented OS and resulted in recovery of endogenous antioxidant enzyme activities, thus protecting neuronal cells effectively. Furthermore, orally administered NRNP N did not show any detectable toxicity to main organs [21]. More recently, Hosoo et al. [82] reported that an intra-arterial injection of a novel NRNP N , containing 4-amino-TEMPO in the core, after cerebral ischemia-reperfusion injury reduced BBB damage and infarction volume by improving multiple ROS scavenging capacities. Therefore, NRNPs can provide neurovascular unit protection [82]. Boonruamkaew et al. [83] used the polymer PEG-b-PMNT as a neuroprotector. The PEG segment is the water-soluble part, while PMNT is the water-insoluble part. The TEMPO groups catalytically react with ROS. These authors confirmed that oral administration of NRNP N prevents against Aβ accumulation in Tg2576 mice overexpressing a mutant form of APP. It was shown that NRNP N -treated mice had significantly attenuated cognitive deficits of both spatial and non-spatial memories. NRNP N treatment increased inhibition of superoxide dismutase and glutathione peroxidase activity, neuronal densities in the cortex and hippocampus, decreased Aβ(1-40), Aβ and gamma (γ)-secretase levels, and reduced Aβ plaque [83]. In summary, RNP N may be a promising candidate for AD therapy because of its antioxidant properties and reduction in Aβ aggregation, thereby suppressing its adverse side effects (Fig. 6).
Scope of future applications
Neurodegenerative diseases, like AD and PD, are a group of disorders that have an increasingly high prevalence along with the shortage of effective treatments. Based on results of current studies, RNPs may be useful in the therapy of various diseases. Because of their nano-size and surface hydrophobicity, they are capable of penetrating some cells (e.g. cancer cells) and crossing BBB. Their antioxidant properties can help to decrease cell damage caused by free radicals. These particles are biocompatible; moreover, NRNPs have long-term blood circulation and low toxicity. Their surface properties can be modified to obtain better targeting. All these unique properties make RNPs very promising for the treatment for neurodegenerative diseases and their application in animal models and clinical studies may be expected.
Conclusion
Neurodegenerative diseases, like AD and PD, are a group of disorders that have in common their increasingly high prevalence along with the shortage of effective treatments. The use of nanomedicine, nanoscale structures for drug delivery, exhibits a really high therapeutic potential in the field of neurodegenerative diseases therapy.
Based on results of current studies, redox nanoparticles have many potential applications in nanomedicine. They could be used especially as drug delivery systems. Because of their nano-size and surface hydrophobicity, they are capable of penetrating altered cells (e.g. cancer cells) and crossing BBB. Their antioxidant properties can help remove ROS and thus decrease cell damage caused by free radicals. These anti-inflammatory and potentially anti-ageing particles are biocompatible; moreover, RNPs have long-term blood circulation and low toxicity. They Page 14 of 16 Sadowska-Bartosz and Bartosz J Nanobiotechnol (2018) 16:87 also could protect against ionizing radiation. All these unique properties make RNPs the foundation of innovative methods in treatments for neurodegenerative diseases. | v3-fos-license |
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} | pes2o/s2orc | New acetate-intercalated CoNiFe, ZnNiFe and CoZnFe layered double hydroxides: Synthesis by forced hydrolysis in polyol medium and characterization
New layered double hydroxides (LDHs) CoNiFe, ZnNiFe and CoZnFe with M II /M III molar ratio of 3, with acetate ions in the interlayer region have been prepared by forced hydrolysis in polyol medium. The physico-chemical properties of all products were characterized by the methods of powder X-ray Diffraction, Infrared Spectroscopy, UV-Spectroscopy, Elemental analysis, Thermal analysis, Transmission Electron Microscopy and Scanning Electron Microscopy. It was found that all compounds present the typical features of hydrotalcite-like structure and exhibit a turbostratic character. The insertion of acetate anions into LDHs was confirmed and the interlayer spacing values were respectively 12.47, 13.64 and 14.69 Å for CoNiFe, ZnNiFe and CoZnFe. We notice a difference between the interlayer spacing for all synthesized phases with and without zinc. This can be explained by a structural modification on the level of the layers and/ or different arrangement from the inserted species (anions + water) in the interlayer space for zinc materials.
INTRODUCTION
Hydrotalcite-like compounds, also called anionic clays or layered double hydroxides (LDHs) are very attractive materials for a broad range of applications [1,2].They can be presented by the general formula [M II 1-x M III x (OH) 2] +x/n (A -n ) x/n.mH2O. The positive layer charge is created by substituting a divalent cation with a trivalent one in the octahedral hydroxide layer, with anions (A -n ) and water in the interlayer space [3].
LDHs are elaborated by several synthesis methods: coprecipitation at fixed pH and exchange reactions [4], reconstruction [5], mechanochemical process [6] and reverse microemulsion method [7]. However the coprecipitation is the most commonly used method with carbonate as the intercalated anion in the most cases. Despite its widespread use, this method presents two main disadvantages. First, it needs a precise control of several parameters mainly the pH. Second, the carbonate anion has a great affinity toward the inorganic layer charged positively. This limits exchange reactions and thus the obtention of LDHs with desired intercalated anions.
Recently an alternative synthesis method has been proposed. It takes advantages from the various properties of polyols. Indeed it has been shown that two main reactions can be easily conducted in such medium: namely reduction and forced hydrolysis along with inorganic polymerization [8] [9]. The competition between these two reactions can be easily controled by the hydrolysis ratio defined as h=nH2O/ nM. This led to the synthesis of various inorganic materials: metal, oxides, layered hydroxide salts (LHS) and more recently LDH compounds: LDH-Ni-Al [10] and LDH-Ni-Fe [11] with acetate as the intercalated anion.
In this paper, we want to put this novel route, efficiency for synthesis of a series of layered double hydroxides containing three transition metals CoNiFe, ZnCoFe, and ZnNiFe with acetate anions intercalated in the interlayer space. The obtained LDH are characterised by several complementary analyses (X--ray diffraction, TEM, DTA-TGA, IR, UV and Mossbauer spectroscopy). Such LDHs based on transition metals are of great interest as precursors for metal-spinel oxides composites with potential magnetic and catalytic properties [12].
Synthesis of LDHs Samples
The samples were synthesized with a molar ratio (M/Fe) of 3 by forced hydrolysis in polyol medium according to the method described by Poul et al. for elaboration of layered hydroxysalts [8]. It consists in the hydrolysis at 130°C of mixture of acetate salts dissolved in DEG with a total molar concentration of 0.1 mole/L.
The precipitation of the corresponding LDHs occurred when the hydrolysis and alkalinity ratios h and b were fixed at 100 and 2 respectively, where h=nH2O/n (M+Fe) and b= nNaOH/n (M+Fe).
The suspensions were kept under continuous stirring for 6 hours. After separation by centrifugation, the solids CoNiFe-Ac, ZnNiFe and ZnCoFe-Ac were dried under air at 60°C.
Characterization
Powder X-ray diffraction (PXRD) patterns were obtained at room temperature using a Inel diffractometer with Co-Kα1 radiation (λCoα1 = 1.7889 Å). The crystallite size was calculated using the Scherrer equation. Scanning electron microscopy study was performed using LEICA STEREOSCAN 440 instrument. Electron microscopy and diffraction studies were performed on a JEOL-100 CX II microscope. Infrared spectra were recorded by transmission on PERKIN ELMER 1750 spectrometer on pressed KBr pellets with 4cm -1 resolution between 400 and 4000cm -1 . Elemental analysis (metal, carbon and hydrogen) was performed by ICP method at the centre national de la recherche scientifique, service central d'analyse à SOLAIZE (France). Differential thermal and thermogravimetric analyses (DTA and TGA) were carried out on a Setaram TG 92-12 thermal analyzer with heating rate 1°C/mn under argon, in alumina crucible. UV-Visible spectra were recorded between 200 and 2500 nm, with Cary 5/Varian spectrometer. Polytetrafluoroethylene (PTFE) was used as a reference. The 57 Fe Mössbauer spectra were recorded in a transmission geometry using a 57 Co/ Rh -ray source. They were analyzed by a least-squares fitting method to lorentzian function. The isomer shifts () were referred to that of -Fe at 300 K. The sample (area: 3 cm 2 ) has been prepared by dispersion of the compound (40 mg) in a specific resin.
X-ray diffraction study
The X-ray diffraction patterns of all the synthesized compounds ( Fig. 1) have typical features of turbostratic lamellar compounds [13]. Indeed, three (00l) sharp and symmetric peaks at low diffraction angles, and two broad, less intense and asymmetric peaks can be clearly identified. The patterns were indexed in a hexagonal lattice with rhombohedral symmetry (R-3m), commonly used as a description of the layered double hydroxide structure [14]. The value of the crystallographic parameter a which is the average metal-metal distance in the brucite-like layers of the LDH compounds, has been N o v e m b e r 1 6 , 2 0 1 4 calculated from d110 (a=2d110), whilst, the value of parameter c which is related to the thickness of the brucite-like layers, the nature of the interlayer anion and water content, has been also determined.
The insertion of acetate anions into LDHs was confirmed and the interlayer spacing values were respectively 12.47, 13.64 and 14. 69 Å for CoNiFe, ZnNiFe and CoZnFe (Table 1) We notice a difference between the interlayer spacing for all synthesized phases. This can be explained by a structural modification on the level of the layers and/ or different arrangement for the inserted species (anions + water) in the interlayer space for zinc materials. The correlation length along c deduced from the XRD analysis (Table 1) reveals a better cristallinity for ZnCoFe-Ac in comparison with CoNiFe-Ac and ZnNiFe-Ac compounds. In this case, it corresponds to the stacking of approximately 33 layers by crystallite.
2. Morphology
SEM images of all synthesized materials show the low level of cristallinity and confirm the turbostratic character of these compounds. Fig. 2(a) and (b) depict the SEM images of CoNiFe-Ac and ZnNiFe-Ac systems respectively, it is clearly seen that these particles are precipitated as small, pseudo-spherical aggregates of platelets; they are also very similar to the morphology of other LDHs [15].
The particles of ZnCoFe-Ac (Fig. 2(c)) sample are formed by the aggregation of small platy crystallites without any definite shape. They appear to be denser compared to those of the CoNiFe-Ac and ZnNiFe-Ac. For TEM images, the particles of all compounds appear as aggregates of thin crumpled sheets with irregular size and shape (Fig. 3). This aggregation is similar to that described by Taibi et al. for NiFe-Ac LDH [11]. Electron diffraction pattern of these samples confirm the hexagonal symmetry of the brucite structure, to which all the hydrotalcite-like materials belong. The preferential orientation of the crystallites along the (00l) heaxogal plane is clearly established for CoNiFe sample (Fig. 4).
Infrared Spectroscopy
IR spectra for all samples are shown in Figure 5. The broad absorption bands at high frequency (3600-3400 cm -1 ) indicate the presence of OH groups and water molecules in the brucite-like layer. The absorption band around 1640 cm -1 for CoNiFe-Ac and ZnCoFe-Ac is assigned to δH2O vibration of the water molecules [16,17]. In the case of ZnNiFe-Ac, this band is obscured by the strong band due to the νas(-COO -) of the acetate anion [18]. for CoNiFe-Ac and ZnCoFe-Ac and 1579 cm -1 for ZnNiFe-Ac. νs (-COO -) = 1410 cm -1 for CoNiFe-Ac and ZnNiFe-Ac and 1386 cm -1 for ZnCoFe-Ac [19].
The absorption bands around 1344 cm -1 are attributed to CH3 vibrations [19]. The existence of a low-intensity bands between1300 and 1000 cm -1 indicate the presence of DEG molecules as adsorbed species [11]. Bands at lower wavenumber (ν < 800 cm The band close to 1360 cm -1 is absent in all spectra, confirming the intercalation of all compounds with acetate anions without any trace impurity of carbonate anions.
4. Thermal analysis
The TG/DTA analysis for all samples is shown in Figure 6. Three weight losses are observed for each sample. The first weight loss corresponds to the removal of adsorbed and interlayer water molecules, the second weight loss is due to dehydroxylation of the brucite-like layers, while the third one is due to the removal of interlayer anions [22].
For all compounds, three endothermic effects can be distinguished in the DTA curve. The first endothermic peak due to the loss of surface and interlayer water was observed at 116°C with loss of mass of 6.3% for CoNiFe-Ac, at 83°C (7.3%) for ZnNiFe-Ac and at 82 and 127°C (9.4%) for ZnCoFe-Ac.
5. Chemical analysis
First it should be noted that under synthesis conditions, Fe 2+ was oxidized to Fe 3+ as confirmed by Mössbauer analysis (as shown hereafter). This is due to the presence of a sufficient amount of water able to oxidize ferrous ion. In Table 2, are summarized the results of elemental chemical analysis of the compounds and their corresponding formulae. It can be observed that in CoNiFe-Ac compound, the calculated M II /Fe III ratio is in a good agreement with the experiment data, but in ZnNiFe-Ac and ZnCoFe-Ac this ratio is larger than in the starting solution.
For all samples, the results show that the observed carbon amount is higher than that expected in the acetate anions; this is due to the adsorption of polyol (DEG) on the surface of compounds as has been seen in the IR study.
Similar results have been observed for Ni-Co hydroxyacetates prepared in polyol medium [23], where the amount of adsorbed polyol has not been taken into account in the chemical formula. N o v e m b e r 1 6 , 2 0 1 4 Table 2 for exact formula).
6. UV-Visible Spectroscopy
For CoNiFe-Ac and ZnCoFe-Ac, the absorption bands at around 1200 and 500 nm ( Fig. 7(a) and (c)) are attributed to the d-d transitions of octahedral coordinated Co 2+ [24].They are assigned as ν1(Co [23,25,26]. The UV-Visible spectra of the CoNiFe-Ac and ZnNiFe-Ac (Fig. 7(a) and (b)) show that Ni 2+ ions are also located in octahedral sites with the presence of absorption bands at around 380, 665 and 1115 nm due to ν2 (Ni Table 3). Each spectrum shows only one doublet with isomer shift (δ) ranging from 0.33 to 0.34 mm/s and quadrupole splitting (Δ) 0.45 -0.56 mm/s indicating Fe 3+ nature of the Fe atom, and no evidence of Fe 2+ signal was found [32]. The small value of quadrupole splitting (Δ) corresponds to high spin Fe 3+ ions in octahedral site.
CONCLUSION
In the current study, a novel series of ternary layered double hydroxides with acetate anion intercalated in the interlayer region has been prepared by forced hydrolysis in polyol medium, in the ratio range 3.17 ≤ M II Fe III ≤ 4.24.
X-Ray diffraction investigations show that these materials derive from the brucite structure with variable basal spacing, the Zinc based compounds having the more higher interlamellar distances.
IR study shows that acetate anions are located in between the brucite sheets as free species. No trace of carbonate CO3 anion was detected.
Because of the turbostratic character of CoNiFe-Ac, ZnNiFe-Ac and ZnCoFe-Ac, it was hard to get more insight into their intralayer structural organization by X-ray diffraction analysis. Thus two complementary analysis techniques namely UV-Visible and Mössbauer Spectroscopy have been used.
For all samples, UV-Visible spectra present characteristic bands of Co 2+ and Ni 2+ in octahedral sites, and 57 Fe Mössbauer measurement reveals that Fe atoms are present in the LDHs lattice in Fe 3+ state and no such Fe 2+ ions are present.
Owing to their chemical compositions, these based 3 d-transition LDHs are belevied to be very versatile precursors for a wide range of metal-spinel nanocomposites. | v3-fos-license |
2021-05-08T00:03:00.529Z | 2021-02-24T00:00:00.000 | 233951759 | {
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} | pes2o/s2orc | Palladium Nanoparticles Functionalized with PVP-Quercetin Inhibits Cell Proliferation and Activates Apoptosis in Colorectal Cancer Cells
: Nanotechnology is focused on the development and application of novel nanomaterials with particular physicochemical properties. Palladium nanoparticles (PdNPs) have been used as antimicrobials, antifungals, and photochemicals and for catalytic activity in dye reduction. In the present investigation, we developed and characterized PdNPs as a carrier of quercetin and initiated a study of its effects in colorectal cancer cells. PdNPs were first functionalized with polyvinylpyrrolidone (PVP) and then coupled to quercetin (PdNPs-PVP-Q). Our results showed that quercetin was efficiently incorporated to PdNPs-PVP, as demonstrated using UV/Vis and FT-IR spectroscopy. Using transmission electron microscopy, we demonstrated a reduction in size from 3–14.47 nm of PdNPs alone to 1.8–7.4 nm of PdNPs-PVP and to 2.12–3.14 of PdNPs-PVP-Q, indicating an increase in superficial area in functionalized PdNPs-Q. Moreover, hydrodynamic size studies using dynamic light scattering showed a reduction in size from 2120.33 nm ± 112.53 with PdNPs alone to 129.96 nm ± 6.23 for PdNPs-PVP-Q, suggesting a major reactivity when quercetin is coupled to nanoparticles. X-ray diffraction assays show that the addition of PVP or quercetin to PdNPs does not influence the crystallinity state. Catalytic activity assays of PdNPs-PVP-Q evidenced the chemical reduction of 4-nitrophenol, methyl orange, and methyl blue, thus confirming an electron acceptor capacity of nanoparticles. Finally, biological activity studies using MTT assays showed a significant inhibition ( p < 0.05) of cell proliferation of HCT-15 colorectal cancer cells exposed to PdNPs-PVP-Q in comparison to untreated cells. Moreover, treatment with PdNPs-PVP-Q resulted in the apoptosis activation of HCT-15 cells. In conclusion, here we show for the first time the development of PdNPs-PVP-Q and evidence its biological activities through the inhibition of cell proliferation and apoptosis activation in colorectal cancer cells in vitro. very and a and triggers mechanisms as a control Our data showed that a small increase (8%) in apoptosis relative to control. This slight effect may be due to (i) the low effective concentration of quercetin delivered in the cellular cytoplasm, (ii) the effect of temperature in the release of quercetin from PdNPs in the cellular microenvironment, and (iii) the potential agglomeration of PdNPs-PVP-Q in the culture media. Thus, further measurements of PdNPs-PVP-Q and quercetin in cellular compartments are required. PdNPs-PVP-Q after 36 h. We also found that PdNPs-PVP-Q induced apoptosis of HCT-15 cells at 36 h relative to the control, suggesting a potential antitumoral effect in vitro. We are aware of the limitation of our study, as we observed that PdNPs-PVP-Q induced only a small increase (8%) in apoptosis of colorectal cancer cells relative to control, which may be due to the experimental conditions tested here which influenced the effective concentration of quercetin delivered in the cellular cytoplasm and the effect of temperature in the release of quercetin from PdNPs-PVP in the cellular microenvironment. Additionally, we did not discard the potential agglomeration of PdNPs-PVP-Q in the culture media and other unknown mechanisms involved in the cancer cell response to NDPs such as autophagy induction and cell cycle arrest. Therefore, further studies of PdNPs-PVP-Q and quercetin in cellular compartments are required to fully understand its utility in vitro and in vivo. In conclusion, the result of this study reveals that PdNPs-PVP may be used as a carrier of molecules with biological activity such as quercetin. Further studies are needed to increase the quercetin liberation from PdNPs-PVP and to use these palladium-based nanoparticles as carriers for different therapeutic molecules in cancer and other human diseases. Author Contributions: Conceptualization, H.A.P.-C. and E.O.-B.; methodology, H.A.P.-C., and R.M.-C.; formal writing—original draft H.A.P.-C.; writing—review R.R.-M., C.L.-C., L.A.M.-N., L.N.M.-C., H.A.P.-C., R.M.-C.; visualization, H.A.P.-C., R.M.-C.; supervision, E.O.-B.; project administration, H.A.P.-C., E.O.-B.; funding acquisition, R.R.-M., E.O.-B.
Introduction
Nanotechnology has become a very wide and diverse research area with rapid development and application. An objective of nanotechnology study is nanoparticles (NPs) with different compositions. Metallic nanoparticles have been of great interest due to their specific physicochemical characteristics. These characteristics include a small size (between 1-100 nm), a large superficial area, high reactivity, and the extraordinary quantum effect [1,2]. Due to their physical-chemical characteristics, NPs have incredible pharmacokinetic properties and can be used as carriers of drugs or molecules for a specific purpose (antibiotics, antibodies, or organic molecules) [3,4]. However, nanoparticle stability remains challenging, due to the electrostatic attraction in naked NPs, which tend to agglomerate. PdNPs superficial modification to avoid agglomeration has been reported [5,6]. Molecules as glucosamine [7], sodium dodecyl sulfate (SDS) [8,9], tween [10], and PEG [5,6,11] have been used to superficially modified the NPs. The mechanisms reported to avoid agglomeration were the addition of negative charges to NP surfaces, which increases the repulsion forces inhibiting the union of several NPs [12], and molecule additions to surface NPs generate a steric stabilization [13]. Palladium nanoparticles (PdNPs) have been added to carbon nanofibers using SDS as a surfactant. The ends of SDS bind to palladium ions and the fast ion reduction suppresses the agglomeration of PdNPs [14]. Palladium is a platinum group element (PEG). In this group, it is possible to find the platinum derivate cisplatin, a metal-based drug used in cancer therapy. Nevertheless, cancer therapy with platinum showed high secondary effects, and many cancer types have developed resistance to it. In this respect, the palladium complex derivate and PdNPs show promising antitumor activity. A few PdNP antitumor effects have been reported. PdNPs were used in a transferrin platform, loaded with paclitaxel, and used to study inhibitory effects in an MCF-7 cellular line. The results showed that a combination of PdNP and paclitaxel in polymeric nanoparticles increased cytotoxicity more than PdNPs or paclitaxel alone. The use of lasers increased cytotoxicity activity [15]. Phan et al. [16] synthesized PdNP coatings with chitosan, which showed a 20% inhibition in MDA-MB-231 cells when irradiated with infrared light. Moreover, PdNPs have been used for bacterial [17] or fungal inhibition [18], showing effective inhibition. Different PdNP synthesis methods have been validated. The use of such plant extracts as Ananas comosus [19], Spirulina platensis [20], and Sapium sebiferum [21], among other reducing agents, have shown that metabolites from natural sources can be used as reducing and stabilizing agents. The presence of proteins and phytochemicals such as steroids, flavonoids, alkaloids, and other molecules in these extracts favors metal reduction and nanoparticle stability [22,23]. The activity shown in these nanoparticles can be mediated by the functional groups adhering to the PdNP surface. Among the molecules adhering to the NP superficies are flavonoids. Flavonoids are molecules that show important antioxidant (activating enzymes involved in oxidative stress response), antitumor, and anti-inflammatory activity. Anti-inflammatory activity inhibits cytokine production, among other activities [24]. It has been reported that this flavonoid affects cell proliferation in different cancer cell line types [25,26]. One of the most studied flavonoids is quercetin due to its antioxidant, anti-inflammatory, and antiproliferative effects [27]. Quercetin is the most abundant flavonoid and is ubiquitously distributed in plants. Dietary sources of quercetin include apples, strawberries, onion, tea, and wine [28]. Moreover, the role of quercetin in iron regulation through inhibiting lipid oxidation and reducing iron valence is well described [29]. It has been stated that quercetin inhibits cell proliferation and triggers apoptosis by deregulating the genes Bax and Bcl-2, and this generates an arrested cell cycle at G2/M phases in glioblastoma U251 cells [30]. It was reported that in metastatic ovarian cancer cells, quercetin increases the level of proapoptotic molecules such as cytochrome c, Bid, Bad, Bax, caspase-3, and caspase-9, indicating the mitochondrialmediated apoptotic pathway [31]. Similar results have been described in prostate cancer; quercetin activates caspase-3 and caspase-9 triggering apoptotic mechanisms via the mitochondrial pathway [32].The NPs used as carriers for the delivery of quercetin into target cells have been evaluated in different diseases [33,34]. On the one hand, NPs alone offers a system that causes damage to specific cells by mechanisms such as oxidative stress, lipids and proteins oxidation, and high toxicity due to their physicochemical characteristic as the superficial charge [35]. In some cases, like cancer, central nervous degenerative, and cardiovascular diseases damage is generated by oxidative stress [36]. Two strategies to respond to oxidative stress have been described: (i) endogenous detoxifying systems including the enzymatic activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and (ii) nonenzymatic activity of exogenous molecules such as quercetin, vitamin C, and lycopene which also show potent antioxidant activities [34]. However, the exogenous antioxidant molecules are light, temperature and pH sensible, and enzymatic degradation susceptible [36]. An efficient form to inhibit this degradation is the encapsulation or transportation of these molecules by organic or metallic nanoparticles [36]. Gold NPs have been used as quercetin carriers, the NPs inhibit epithelial-mesenchymal transition, angiogenesis, and metastasis in breast cancer cell lines [37]. Quercetin has been encapsulated in a self-assembled chitosan system. The system proved to be efficient in the inhibition of breast cancer cell proliferation [38]. Metallics and organic nanoparticles coupled to quercetin have been also proved to be useful in the inhibition of oxidative stress in degenerative diseases [37,39]. Ultrasmall bimetallic Cu2-xSe-based bimetallic nanoparticles modified with quercetin promoted the microglia polarization and downregulate IL-6 and upregulate IL-10 playing anti-inflammatory activity [34]. However, as was describe before, PdNPs play an important role in nanomedicine and nanobiology due to their activity against a broad spectrum of organisms, and their chemical properties make the PdNPs a desirable candidate for the functionalization with quercetin.
Previously, we have developed PdNPs and tested their effects as a fungicide, showing that treatments induce cell wall damage and oxidative stress in Aspergillus niger and Candida albicans [18]. Here, we extend these initial findings in fungus; we functionalized PdNPs with PVP (PdNPs-PVP) and with quercetin (PdNPs-PVP-Q). The physicochemical characteristics were evaluated, and their biological activity was tested in colorectal adenocarcinoma HCT-15 cells, as quercetin is a well-known antitumor naturally occurring agent.
Although several studies have reported the anti-tumor effects of quercetin in HCT-15 cancer cells, no previous approaches using palladium nanoparticles coupled with quercetin to deliver cargo into colon cancer cells have been reported.
Nanoparticles Synthesis
The PdNPs, PdNPs-PVP, and PdNPs-PVP-Q were synthesized using the reduction method described by Creighton and Eadont [40] with some modifications. The modifications were made in a reaction of K 2 PdCl 4 (1 mM) with NaBH 4 (3 mM). PVP was added in a final proportion of 5% (p/v). The reaction was stirred for 24 h at room temperature. Afterward, PVP-PdNPs were centrifuged for 20 min at 37,567 RCF. They were washed with distilled water twice and left to dry at 60 • C. The PVP-PdNPs were used for quercetin functionalization. A known weight of PVP-PdNPs was suspended in water and mixed with a quercetin solution in a 2% final concentration (p/v). The suspension was then agitated 12 h in light absence. PdNPs-PVP-Q were recovered by centrifugation and characterized. The synthesis of all nanoparticles was monitored by a UV/visible spectrophotometer in a range from 200 to 1100 nm (Evolution 220 Thermo Scientific).
Nanoparticles Characterization
The size, morphology, and agglomeration of all nanoparticles were analyzed by electronic microscopy. All the samples were suspended in ethanol, and the suspensions were then sonicated for 30 min using a bath sonicator (Cole-Parmer). Afterward, the samples were placed in an aluminum support for Scanning Electron Microscopy (JEOL JSM model 7401F) and carbon-coated copper grid for Transmission Scanning Microscopy (JEOL model TEM-2100F). An average of 300 nanoparticles was counted to obtain the distribution size. The hydrodynamic size and zeta potential were determined in a Malvern Zetaziser Nano ZS instrument operating at a light source wavelength of 532 nm, and a fixed scattering angle of 173 • was used. A flow rate of 0.5 mL/min was used, and dynamic light scattering was analyzed every 3 s. Zeta potential was determined with laser Doppler electrophoresis using the same equipment. Changes in structure and confirmation of PVP and quercetin addition were measured via X-ray diffraction and FTIR-IR analysis. The crystallinity structure of all synthesized NPs was determined in an X-ray diffractometer model XDR, DP Phillips Xpert PRO, with Cu-Kα radiation (λ = 0.15406 nm) in a 2θ range from 20 • to 80 • . FTIR-IR analysis was done in a range from 399 to 3999 cm-1 in a FTIR-IR Afinnity-1S spectrophotometer (Shimadzu).
Dye Degradation
All nanoparticles were employed to evaluate their degradation dye evaluation. The dyes employed were methylene blue (MB), methyl orange (MO), and 4-nitrophenol (4N; all at 10 µM) with sodium borohydride (NaBH 4 ; 5 mM). All dyes were exposed to NPs at 10 ppm and were incubated in the dark for 60 min. After that, the absorbance in a UV-visible spectrum was measured from 300 to 800 nm (Varioskan Lux Thermo Scientific, Waltham, MA, USA).
Quercetin Liberation
The nanoparticles were suspended in a phosphate buffer, collocated in a dialysis membrane (repligen ® MWCO, 300 Kd), and stirred for 24 h at 4 • C at a pH of 6.5 and 7.0. Samples were taken at 12 and 24 h for quercetin determination. The quercetin concentration was determined in a UV-visible spectrum (Varioskan Lux Thermo Scientific, Waltham, MA, USA), using quercetin as the standard curve.
Cell Proliferation Assays
HCT-15 cells were plated at a density of 1 × 10 4 cells/well into 96-well dishes in a complete medium. The HC-T15 cells were treated with PdNPs, PdNPs-PVP, and PdNPs-PVP-Q at 1000 ppm for 0 and 36 h. For cell proliferation analysis, the MTT reagent ([3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide] was added to HCT-15 cells (1 × 10 4 ) and incubated for 3 h at 37 • C. Afterward, isopropanol was added to the cells, and the cells were incubated for 10 min. Absorbance was recorded at different time points using a spectrophotometer (model Evolution 220; Thermo Scientific, 570 nm). Assays were performed three times, and data were expressed as mean ± standard deviation (SD).
Apoptosis Assays
Death of the HCT-15 colorectal cancer cells was quantified by fluorescence-activated cell sorting (FACS) assays using the annexin V method. An annexin-V/propidium iodide (PI) double assay was performed using the apoptosis detection kit (Roche). For assays, HCT-15 cancer cells were treated with PdNPs, PdNPs-PVP, and PdNPs-PVP-Q at 1000 ppm and were then incubated at 37 • C in a 5% CO 2 atmosphere for 36 h. Cells were detached and resuspended in 100 µL of incubation buffer with 2 µL of Annexin and 2 µL of propidium iodide according to the manufacturer's recommendations. Stained cells were analyzed in a FACSCALIBUR flow cytometer (BD Biosciences). Quercetin and cisplatin controls were added at 40 and 50 µM concentrations, respectively. The assays were repeated three times, and data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using a Student's t-test, and a p-value <0.05 was considered significant.
Statistical Analysis
Experiments were performed three times in triplicate, and results were expressed as mean ± standard deviation (SD). A p < 0.05 value was considered statistically significant.
Nanoparticles Synthesis
Due to their physicochemical activity, NPs have been proposed as carriers of drugs, biological molecules, and antibodies. In our previous work, PdNPs demonstrated antifungal activity [18]. In this work, we added PVP to the PdNP synthesis as a surfactant to modify their surface, and this worked as a link between quercetin and PdNPs. PdNP modification by PVP and the further addition of quercetin were evaluated by UV/Vis spectrometry ( Figure 1). The reaction showed a color change from yellow-orange to brown dark, indicating Pd ion reduction. These color changes have been reported in other works [41,42]. UV/vis spectrometry analysis showed peaks of absorption in a range from 199 to 414 nm in the PdNPs-PVP-Q, suggesting incorporation of both components in the PdNPs system ( Figure 1). These results are consistent with the green synthesized PdNPs mediated by carboxymethylcellulose [41] and an extract from Euphorbia granulate [43].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of method synthesis in size and morphology has been shown in previous works [47,48].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of method synthesis in size and morphology has been shown in previous works [47,48].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of method synthesis in size and morphology has been shown in previous works [47,48].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of method synthesis in size and morphology has been shown in previous works [47,48].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of method synthesis in size and morphology has been shown in previous works [47,48]. The TEM analysis showed that the nanoparticles have a semispherical shape regardless of the PdNP type evaluated (Figure 2a-f). Similar results were found with the reduction of palladium chloride by tetraethylene glycol with KOH [45] and with reduction mediated by heat and the use of carboxymethylcellulose [41]. Nevertheless, other authors have reported PdNP synthesis as spherical, using methanol as a reducing agent [47], square-shaped, with citric acid as a reduction agent, heat, and PVP as a stabilizing agent [49], and flower-shaped and porous [16].
TEM analysis showed the differences in size with the PVP and quercetin addition (Figure 2g-i). The PdNPs alone showed a size between 3.7 and 14.47 nm, with almost 30% of PdNPs in a range from 6.42 to 7.42 nm (Figure 2g). The surfactant addition produces nanoparticles with a size range between 1 and 8.74 nm, with 35% of PdNPs in a size from 2.12 to 3.14 nm (Figure 2h). When the quercetin was added to PdNPs-PVP, the nanoparticles showed a tighter distribution from 2.3 to 8.29 nm (Figure 2i). Additionally, the PdNPs- The TEM analysis showed that the nanoparticles have a semispherical shape regardless of the PdNP type evaluated (Figure 2a-f). Similar results were found with the reduction of palladium chloride by tetraethylene glycol with KOH [45] and with reduction mediated by heat and the use of carboxymethylcellulose [41]. Nevertheless, other authors have reported PdNP synthesis as spherical, using methanol as a reducing agent [47], square-shaped, with citric acid as a reduction agent, heat, and PVP as a stabilizing agent [49], and flower-shaped and porous [16].
TEM analysis showed the differences in size with the PVP and quercetin addition (Figure 2g-i). The PdNPs alone showed a size between 3.7 and 14.47 nm, with almost 30% of PdNPs in a range from 6.42 to 7.42 nm (Figure 2g). The surfactant addition produces nanoparticles with a size range between 1 and 8.74 nm, with 35% of PdNPs in a size from 2.12 to 3.14 nm (Figure 2h). When the quercetin was added to PdNPs-PVP, the nanoparticles showed a tighter distribution from 2.3 to 8.29 nm (Figure 2i). Additionally, the PdNPs-PVP-Q showed 50% of PdNPs in a size range from 3.6 to 5.09 nm (Figure 2i). Quercetin helps nanoparticles to have a more uniform size, even more so the surfactant (PVP). Change in the distribution size could be attributed to the addition of quercetin on Appl. Sci. 2021, 11,1988 7 of 16 the PdNPs-PVP surface, which avoids nucleation and increases in size. It has been reported that NP size decreased when surfactant was added to the nanoparticle synthesis due to the NPs' stabilization [48,50] [49,51,52]. This stabilization depends on the molecule size employed, the charge, and the molecule's nature [9,12]. Regarding NP synthesis mediated by an extract from plants, it was suggested that the reduction of metallic salts could be carried out by flavonoid molecules such as apigenin and luteolin [51]. Additionally, these molecules and phenols provide stability in the nanoparticle formed [52,53].
Nanoparticles agglomerate in a solution depending on the solution's ionic strength and the nanoparticles' superficial charge [54]. Because of this, determining the size of the nanoparticles in suspension is important. To determine these nanoparticle characteristics, hydrodynamic size and zeta potential were analyzed (Figure 3). In each part of the NP synthesis, the addition of PVP or quercetin changes the hydrodynamic size and zeta potential (Figure 3a and 3b, respectively). Hydrodynamic size showed a diminution depending on the NP type (Figure 3a). This diminution showed an exponential correlation between the type and the hydrodynamic size form (R 2 = 0.999; Figure 3a). PdNPs alone showed a size 2120.33 ± 112.53 nm, three times larger than PdNPs-PVP and 16 times larger than PdNPs-PVP-Q (Figure 3a). The size diminution with PVP addition in silver nanoparticles [55], antimony [56], or bismuth [57] has been shown. Surfactant and quercetin addition generated a diminution in size and zeta potential. molecules and phenols provide stability in the nanoparticle formed [52,53].
Nanoparticles agglomerate in a solution depending on the solution's ionic strength and the nanoparticles' superficial charge [54]. Because of this, determining the size of the nanoparticles in suspension is important. To determine these nanoparticle characteristics, hydrodynamic size and zeta potential were analyzed (Figure 3). In each part of the NP synthesis, the addition of PVP or quercetin changes the hydrodynamic size and zeta potential (Figure 3a and 3b, respectively). Hydrodynamic size showed a diminution depending on the NP type (Figure 3a). This diminution showed an exponential correlation between the type and the hydrodynamic size form (R 2 = 0.999; Figure 3a). PdNPs alone showed a size 2120.33 ± 112.53 nm, three times larger than PdNPs-PVP and 16 times larger than PdNPs-PVP-Q (Figure 3a). The size diminution with PVP addition in silver nanoparticles [55], antimony [56], or bismuth [57] has been shown. Surfactant and quercetin addition generated a diminution in size and zeta potential.
The index of the interaction between colloidal nanoparticles is estimated by the zeta potential. Thus, this parameter measures the nanoparticles' capacity to agglomerate or its dispersion stability. Nanoparticles with a zeta potential larger than +30 mV or −30 mV normally are considered stable [55]. The addition of PVP or PVP-Q addition to PdNPs showed a change in the zeta potential (Figure 3b). PdNPs-PVP-Q showed the smallest value with a charge of −7.9 ± 0.73 mV. In both attributes (hydrodynamic size and zeta potential), the quercetin contributes to the size of nanoparticles favorably. These results are coincident with the result shown by Vergara-Castañeda et al. [58]. However, this result is not consistent with the zeta potential increase from Sb2S3 [56] or Bi2S3NPs [57], where the increase was related to the hydrodynamic size diminution. Two mechanisms have been reported for NP stabilization: (1) electrostatic stabilization, where the nanoparticles are repelled by the NPs' surface charge, and (2) steric stabilization, where NPs are coated by macromolecules, which generates repelling forces [13,50]. X-ray diffraction analysis was performed to describe the crystallinity of the nanoparticles ( Figure 4). PdNPs showed a centered cubic structure (fcc) with five pics at 40.1, 46.4, The index of the interaction between colloidal nanoparticles is estimated by the zeta potential. Thus, this parameter measures the nanoparticles' capacity to agglomerate or its dispersion stability. Nanoparticles with a zeta potential larger than +30 mV or −30 mV normally are considered stable [55]. The addition of PVP or PVP-Q addition to PdNPs showed a change in the zeta potential (Figure 3b). PdNPs-PVP-Q showed the smallest value with a charge of −7.9 ± 0.73 mV. In both attributes (hydrodynamic size and zeta potential), the quercetin contributes to the size of nanoparticles favorably. These results are coincident with the result shown by Vergara-Castañeda et al. [58]. However, this result is not consistent with the zeta potential increase from Sb 2 S 3 [56] or Bi 2 S 3 NPs [57], where the increase was related to the hydrodynamic size diminution. Two mechanisms have been reported for NP stabilization: (1) electrostatic stabilization, where the nanoparticles are repelled by the NPs' surface charge, and (2) steric stabilization, where NPs are coated by macromolecules, which generates repelling forces [13,50].
X-ray diffraction analysis was performed to describe the crystallinity of the nanoparticles (Figure 4). PdNPs showed a centered cubic structure (fcc) with five pics at 40.1, 46.4, 67.9, 82.7, and 86.7 2-tetha, indicating the presence of (111), (200), (220), (311), and (222) planes. The PVP and quercetin addition in the nanoparticles caused a diminution in intensity compared to PdNPs alone (Figure 4b,c). However, such an addition does not change the NPs' crystallinity. Previous studies have reported that the addition of PVP to PdNPs showed no change in the NPs' crystallinity, only in the intensity [59]. 67.9, 82.7, and 86.7 2-tetha, indicating the presence of (111), (200), (220), (311), and (222) planes. The PVP and quercetin addition in the nanoparticles caused a diminution in intensity compared to PdNPs alone (Figure 4b,c). However, such an addition does not change the NPs' crystallinity. Previous studies have reported that the addition of PVP to PdNPs showed no change in the NPs' crystallinity, only in the intensity [59]. To further confirm the incorporation of quercetin with PdNPs, we used FT-IR spectroscopy ( Figure 5). Our results indicated that quercetin showed characteristic transmittance bands. The OH group stretching was detectable at 3406 and 3283 cm-1 , while the OH bounded to the phenol group was detected at 1379 cm-1 . The aryl group was detectable at 1666 cm-1 , and the C=C from the aromatic ring was found at 1610, 1560, and 1510 cm-1. The C-H in aromatic hydrocarbon in the plane was detectable at 1317 cm-1, whereas the C-H out of the plane was observed at 933, 820, 679, and 600 cm-1 . The C-O stretch in the aryl ether group was observed at 1263 cm-1 , while C-O stretching in the phenol was observed To further confirm the incorporation of quercetin with PdNPs, we used FT-IR spectroscopy ( Figure 5). Our results indicated that quercetin showed characteristic transmittance bands. The OH group stretching was detectable at 3406 and 3283 cm −1 , while the OH bounded to the phenol group was detected at 1379 cm −1 . The aryl group was detectable at 1666 cm −1 , and the C=C from the aromatic ring was found at 1610, 1560, and 1510 cm −1. The C-H in aromatic hydrocarbon in the plane was detectable at 1317 cm −1, whereas the C-H out of the plane was observed at 933, 820, 679, and 600 cm −1 . The C-O stretch in the aryl ether group was observed at 1263 cm −1 , while C-O stretching in the phenol was observed at 1200 cm −1 , and the C-CO-C stretch in ketone was observed in 1165 cm −1 which has been reported as a band typical of quercetin [60]. at 1200 cm-1 , and the C-CO-C stretch in ketone was observed in 1165 cm-1, which has been reported as a band typical of quercetin [60]. The PVP characteristic bands in the 1655 cm-1 correspond to the C=O group. The bands stretching at 1492, 1460, and 1421 nm correspond to CH2 deformation, the 1372 cm-1 band corresponds to CH deformation and 646 N-C=O bands [61]. Quercetin used to load ZnO with PBA as a surfactant showed the successful load of quercetin into NPs [62]. Additionally, quercetin has been used to load silica NPs without the addition of surfactants, indicating the possibility of quercetin union without an intermediate [58]. The interaction of quercetin with the PVP could be mediated by hydrogen bonds between the OH from quercetin and the O and NH from PVP.
Nanoparticles Reactivity Characterization
PdNPs promises to be useful as catalysts in various reaction types as a Suzuki's reaction [63] or dye degradation [7]. This characteristic is important since it shows their reactivity in an environment that surrounds them. PdNPs works as an electron acceptor in the presence of H2 in a catalytic reaction [64]. These reaction mechanisms could be involved in the toxicity mechanisms with different cell types. It has been explained that a Fentontype reaction is involved in the toxicity mechanism of NPs. This catalytic reaction has also been used in organic pollutant degradation [65]. As a part of PdNPs-PVP-Q characterization, dye degradation was evaluated ( Figure 6). The PdNPs-PVP-Q showed catalytic activity in the MO and 4N (Figure 6a,b, respectively) but not in MB. The decolorization changes were observed immediately after Na2BH4 addition. The degradation of 4N has
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were ed to characterize NP size and morphology. The SEM micrographs showed a spherical noparticle with a size of approximately 5 nm without the surfactant and quercetin igure 2a). When the PVP and the quercetin were added to the reaction, an envelope was served (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown 4,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized ith PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, cause, in this experiment, a reduction method synthesis was used. The influence of ethod synthesis in size and morphology has been shown in previous works [47,48]. cle Characterization and transmission electron microscopy (SEM and TEM, respectively) were cterize NP size and morphology. The SEM micrographs showed a spherical ith a size of approximately 5 nm without the surfactant and quercetin hen the PVP and the quercetin were added to the reaction, an envelope was ure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown ver, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized hout agglomeration. Agglomeration could be attributed to PdNP synthesis, is experiment, a reduction method synthesis was used. The influence of esis in size and morphology has been shown in previous works [47,48]. tron microscopy (SEM and TEM, respectively) were rphology. The SEM micrographs showed a spherical ately 5 nm without the surfactant and quercetin uercetin were added to the reaction, an envelope was tion in PdNP synthesis with PVP has been shown howed a palladium cobalt nanoparticle synthesized gglomeration could be attributed to PdNP synthesis, ction method synthesis was used. The influence of ology has been shown in previous works [47,48]. PdNPs-PVP-Q.
The PVP characteristic bands in the 1655 cm −1 correspond to the C=O group. The bands stretching at 1492, 1460, and 1421 nm correspond to CH 2 deformation, the 1372 cm −1 band corresponds to CH deformation and 646 N-C=O bands [61]. Quercetin used to load ZnO with PBA as a surfactant showed the successful load of quercetin into NPs [62]. Additionally, quercetin has been used to load silica NPs without the addition of surfactants, indicating the possibility of quercetin union without an intermediate [58]. The interaction of quercetin with the PVP could be mediated by hydrogen bonds between the OH from quercetin and the O and NH from PVP.
Nanoparticles Reactivity Characterization
PdNPs promises to be useful as catalysts in various reaction types as a Suzuki's reaction [63] or dye degradation [7]. This characteristic is important since it shows their reactivity in an environment that surrounds them. PdNPs works as an electron acceptor in the presence of H 2 in a catalytic reaction [64]. These reaction mechanisms could be involved in the toxicity mechanisms with different cell types. It has been explained that a Fenton-type reaction is involved in the toxicity mechanism of NPs. This catalytic reaction has also been used in organic pollutant degradation [65]. As a part of PdNPs-PVP-Q characterization, dye degradation was evaluated ( Figure 6). The PdNPs-PVP-Q showed catalytic activity in the MO and 4N (Figure 6a,b, respectively) but not in MB. The decolorization changes were observed immediately after Na 2 BH 4 addition. The degradation of 4N has been reported by PdNPs synthesized using protein ferritin from Pyrococcus furios as a stabilizer [66]. The degradation of MB at a low rate due to a large kinetic energy barrier has been reported [52]. Many factors influence MB degradation, such as pH and the presence of amine groups [67]. Additionally, the photolytic activity degradation of MB by PdNPs has been demonstrated [7]. Dye water contamination is an important pollution problem [68], and NPs represent an alternative to a solution to this. Different NPs used for wastewater have been used as an alternative for dye degradation [68,69].
Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 18 been reported by PdNPs synthesized using protein ferritin from Pyrococcus furios as a stabilizer [66]. The degradation of MB at a low rate due to a large kinetic energy barrier has been reported [52]. Many factors influence MB degradation, such as pH and the presence of amine groups [67]. Additionally, the photolytic activity degradation of MB by PdNPs has been demonstrated [7]. Dye water contamination is an important pollution problem [68], and NPs represent an alternative to a solution to this. Different NPs used for wastewater have been used as an alternative for dye degradation [68,69].
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectiv used to characterize NP size and morphology. The SEM micrographs showed a nanoparticle with a size of approximately 5 nm without the surfactant and quer (Figure 2a). When the PVP and the quercetin were added to the reaction, an env observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has be [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle sy with PVP without agglomeration. Agglomeration could be attributed to PdNP because, in this experiment, a reduction method synthesis was used. The in
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) used to characterize NP size and morphology. The SEM micrographs showed a sph nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelop observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been sh [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthe with PVP without agglomeration. Agglomeration could be attributed to PdNP synt because, in this experiment, a reduction method synthesis was used. The influen
Nanoparticle Characterization
Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to characterize NP size and morphology. The SEM micrographs showed a spherical nanoparticle with a size of approximately 5 nm without the surfactant and quercetin (Figure 2a). When the PVP and the quercetin were added to the reaction, an envelope was observed (Figure 2b,c). Agglomeration in PdNP synthesis with PVP has been shown [44,45]. However, Çelik et al. [46] showed a palladium cobalt nanoparticle synthesized with PVP without agglomeration. Agglomeration could be attributed to PdNP synthesis, because, in this experiment, a reduction method synthesis was used. The influence of PdNPs-PVP-Q, Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 18 been reported by PdNPs synthesized using protein ferritin from Pyrococcus furios as a stabilizer [66]. The degradation of MB at a low rate due to a large kinetic energy barrier has been reported [52]. Many factors influence MB degradation, such as pH and the presence of amine groups [67]. Additionally, the photolytic activity degradation of MB by PdNPs has been demonstrated [7]. Dye water contamination is an important pollution problem [68], and NPs represent an alternative to a solution to this. Different NPs used for wastewater have been used as an alternative for dye degradation [68,69].
Quercetin Liberation
The capacity of nanoparticles as carriers has been widely reported [70,71]. In this work, we studied the ability of NPs to release quercetin at different pH levels (Figure 7). PdNPs-Q and PdNPs-PVP-Q were employed (orange and gray bars in Figure 7, respectively). Both nanoparticles were effective in quercetin liberation, but PdNPs-PVP were the most efficient.
pH was shown to affect this liberation. No influence of time was observed. The quercetin concentration was the same at 6 and 12 h ( Figure 8A,B). However, pH 7.5 allowed for the liberation of almost twice the concentration of quercetin than at pH 6.5 ( Figure 8B). pH influence in molecule liberation from NPs has been shown before [11,15]. Rhodamine B liberation of silica-alginate nanoparticles was more efficient at a pH of 7.5 than 2.5 [72]. Quercetin has been used as a load molecule for ZnONPs; however, in these nanoparticles, more efficient quercetin liberation was presented at a pH of 6.5 [62]. In contrast, Akal et al. [73] showed that in silica, NPs loaded with folic acid and quercetin, a pH of 4.4 was more efficient for quercetin liberation.
Effects of PdNPs-PVP-Q on the Cell Viability and Apoptosis of Cancer Colorectal Cells
A colorectal adenocarcinoma HCT-15 cell line was employed to evaluate the capacity of PdNPS-PVP-Q to inhibit cell proliferation. After 36 h of incubation, PdNPs-PVP and PdNPs-PVP-Q treatments resulted in a significant cell growth inhibition in comparison with the untreated control. Differences between both PdNPs-PVP and PdNPs-PVP-Q treatments were found (p < 0.05; Figure 8A). Afterward, the apoptotic percentage of cells exposed to PdNPs-PVP and PdNPs-PVP-Q was evaluated. The exposure of HCT-15 cells to PdNPs-PVP did not result in significant differences in cell death in comparison to the untreated control. In contrast, the incubation of cells with PdNPs-PVP-Q resulted in a slight but significant (p < 0.05) increase in apoptosis ( Figure 8B). As a control, we used quercetin (40 µM) alone and observed a considerable increase in cell death, as has been reported [74]. Additionally, as a positive control of cell death, we used cisplatin (50 µM), which induced extensive cell death, as expected. It has been proved that PdNPs generate oxidative stress, induce mitochondrial dysfunction, and induce apoptosis increasing caspase 3 in SKOV3 cells [75]. Additionally, porous palladium transferrin-conjugated nanoparticles have been reported to be effective in chemo-phototherapy with regard to MC-7 human breast adenocarcinoma cell lines [15]. Concave PdNPs have been used with ascorbate for the inhibition of colorectal adenocarcinoma cell viability. The addition of concave PdNPs to the ascorbate promoted dose-dependent PdNP toxicity and increased ascorbate activity [76]. Quercetin has been reported as a very potent antioxidant molecule [77] and as a chemosensitizer and cancer chemotherapy [78]. Quercetin triggers apoptotic mechanisms as a growth control of colorectal cancer [15]. Our data showed that PdNPs-PVP-Q induces a small increase (8%) in apoptosis relative to control. This slight effect may be due to (i) the low effective concentration of quercetin delivered in the cellular cytoplasm, (ii) the effect of temperature in the release of quercetin from PdNPs in the cellular microenvironment, and (iii) the potential agglomeration of PdNPs-PVP-Q in the culture media. Thus, further measurements of PdNPs-PVP-Q and quercetin in cellular compartments are required. Quercetin Liberation The capacity of nanoparticles as carriers has been widely reported [70,71]. In this work, we studied the ability of NPs to release quercetin at different pH levels (Figure 7). PdNPs-Q and PdNPs-PVP-Q were employed (orange and gray bars in Figure 7, respectively). Both nanoparticles were effective in quercetin liberation, but PdNPs-PVP were the most efficient. pH was shown to affect this liberation. No influence of time was observed. The quercetin concentration was the same at 6 and 12 h ( Figure 8A,B). However, pH 7.5 allowed for the liberation of almost twice the concentration of quercetin than at pH 6.5 ( Figure 8B). pH influence in molecule liberation from NPs has been shown before [11,15]. Rhodamine B liberation of silica-alginate nanoparticles was more efficient at a pH of 7.5 than 2.5 [72]. Quercetin has been used as a load molecule for ZnONPs; however, in these nanoparticles, more efficient quercetin liberation was presented at a pH of 6.5 [62]. In contrast, Akal et al. [73] showed that in silica, NPs loaded with folic acid and quercetin, a pH of 4.4 was more efficient for quercetin liberation. be due to (i) the low effective concentration of quercetin delivered in the cellular cytoplasm, (ii) the effect of temperature in the release of quercetin from PdNPs in the cellular microenvironment, and (iii) the potential agglomeration of PdNPs-PVP-Q in the culture media. Thus, further measurements of PdNPs-PVP-Q and quercetin in cellular compartments are required. . (B). Effect of PdNPs-PVP-Q on cell death. Graphs show the distribution of cell populations stained with Annexin-V and iodide propidium (IP) and sorted by flow cytometry. All assays were done in triplicate. Significance differences were indicated by * p < 0.05 and ** p < 0.005.
Conclusions
Here we report the successful development of novel PdNPs-PVP nanoparticles coupled to quercetin. The addition of quercetin to PdNPs-PVP was confirmed using UV/Vis and FT-IR spectrometry. Our data indicate that PdNPs-PVP-Q showed the smallest primary size, as well as the smallest hydrodynamic size and zeta potential than PdNPs or PdNPs-PVP. Furthermore, PdNPs-PVP-Q showed catalytic activity in the degradation of 4N and MO, but not for BM. In addition, we provide data indicating that quercetin liberation from PdNPs-PVP-Q was more efficient at a pH of 7.5, which is close to physiological pH (7.4), but no differences in quercetin liberation at 12 and 24 h were found. Additionally, significant differences in cell viability were observed in HCT-15 cells incubated with PdNPs-PVP or PdNPs-PVP-Q after 36 h. We also found that PdNPs-PVP-Q induced apoptosis of HCT-15 cells at 36 h relative to the control, suggesting a potential antitumoral effect in vitro. We are aware of the limitation of our study, as we observed that PdNPs-PVP-Q induced only a small increase (8%) in apoptosis of colorectal cancer cells relative to control, which may be due to the experimental conditions tested here which influenced the effective concentration of quercetin delivered in the cellular cytoplasm and the effect of temperature in the release of quercetin from PdNPs-PVP in the cellular microenvironment. Additionally, we did not discard the potential agglomeration of PdNPs-PVP-Q in the culture media and other unknown mechanisms involved in the cancer cell response to NDPs such as autophagy induction and cell cycle arrest. Therefore, further studies of PdNPs-PVP-Q and quercetin in cellular compartments are required to fully understand its utility in vitro and in vivo.
In conclusion, the result of this study reveals that PdNPs-PVP may be used as a carrier of molecules with biological activity such as quercetin. Further studies are needed to increase the quercetin liberation from PdNPs-PVP and to use these palladium-based nanoparticles as carriers for different therapeutic molecules in cancer and other human diseases. | v3-fos-license |
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} | pes2o/s2orc | Antimalarial Activity of Andrographis Paniculata Ness‘s N-hexane Extract and Its Major Compounds
Abstract Andrographis paniculata Ness is one of the plants that is under explored and could contain potentially active substances to serve as an antimalarial. Structure investigation of major compounds that have responsibility for the antimalarial activity of Andrographis paniculata Ness’s n-hexane extract is very important so that it is known whether the antimalarial activity is synergistic or from the major compounds. Menthol and dioctyladipate as major component from n-hexane extract of Andrographis paniculata Ness have been succesfully isolated using simple and inexpensive methods. The solvent system used are n-hexane : ethyl acetate (10:1) and n-hexane: chloroform (10:3) consecutively either for column or preparative thin layer chromatography. FT-IR, 1H-NMR and GC-MS were used to determine the structure of compounds. The activity of n-hexane extract, menthol and dioctyladipate related to the inhibition of heme polymerization have been done comprehensively. The inhibition of heme polymerization activity of isolate compounds and n-hexane extract are classified to a good level (dioctyl adipate IC50 = 1.15 ± 0.41 mg/mL, menthol IC50 = 0.31 ± 0,01 mg/mL, and n-hexane extract IC50 = 0.07 ± 0.03 mg/mL) and potentially as antimalarial. From the IC50, the antimalarial activity of Andrographis paniculata Ness’s n-hexane extract is working synergistically, not by the major compounds.
Introduction
Malaria continues to be a significant health problem and deadly parasitic disease across the world. During the last 100 years, the world has not given a clear contribution to the curing the disease [1]. The global commitment to the Millennium Development Goals (MDGs) puts malaria eradication into one of the common goals to be achieved. With the end of the MDGs by 2015, the commitment continues through Sustainable Development Goals (SDGs) whereby the specific goal of SDGs is to end the epidemic of neglected-tropical, tuberculosis and malaria diseases by 2030. The Indonesian Ministry of Health stated that malaria was one of the diseases, which was targeted to reduce the Annual Parasite Incidence (API) from 2 to 1 per 1000. Although nationally in 2011-2015 there was a decrease in the incidence of malaria, there are still areas with a high incidence of malaria especially in eastern Indonesia. Therefore effective efforts to address malaria problem are urgently required [2].
Drug development is important in malaria control due to the critical situation of drug resistance of P. Falciparum. Chloroquine, primaquine and pyrimethamine were developed and synthesized in Western countries in the 1940s and used as antimalarials. The resistance of chloroquine to Plasmodium falciparum was found and spread in the early 1960s [3]. During the Vietnamese War in the 1960s, there was an increase of incidence of chloroquine resistant. This situation encouraged many researchers to find and develop alternative antimalarials that were more effective to treat malaria than chloroquine.
In the late 1960s, Chinese scientists investigated some active compounds to solve the malaria problem from different medicinal herbs and their chemical structures were also determined and identified. It was a totally new agent: artemisinin. Therefore, the drug is not used alone as antimalarial but in combination with another antimalarial drug. The poor solubility and low curative rate of artemisinin stimulated the researchers synthesize artemisinin derivatives (artemether, artesunate, and dihydroartemisinin). Beside the isolation method, chemical synthesis was an important method for developing new antimalarials. The synthetic antimalarials (piperaquine, pyronaridine, lumefantrine and naphthoquine) were developed as new antimalarials between the 1960s and the 1980s [4].
The occurrence of parasitic resistance to malaria drugs (chloroquine and artemisinin) and the spread of resistance then encouraged researchers to find new antimalarial drugs which are more efficient and effective [5]. It is necessary to find new anti-malarials, especially from medicinal plants. Andrographis paniculata Ness is one of the plants that is under explored as a useful herbal medicine. Recent research suggests that the plant contains potentially active substances to serve as repellent, immunomodulators, hepatoprotectors, antibacterials, anti-inflammators, anticancer, and antimalarials [6], [7], [8]. Andrographolide is the main compound of andrographis paniculata and is active as an antimalarial [9]. However, it is necessary to search further for other potential compounds of Andrographis paniculata which also have potential as antimalarials.
The development of Andrographis paniculata's extract as antimalarials have been done very well, not only on in vitro wherein using both Plasmodium culture but also in vivo using experimental animals that have parasitic infection [10], [11]. Study of heme polymerization inhibition activity assay using Andrographis paniculata's extract has been conducted [12]. Heme polymerization inhibition activity assay is one method to determine the mechanism of action of a compound as an antimalarial. The mechanism that occurs is the interaction between the test material with the heme electrolyte system and or active groups in the test material which binds to the iron ion on heme [13]. Chloroquine as an antimalarial drug is also through inhibition of heme polymerization [14].
All of the Andrographis paniculata Ness's extracts with different polarity levels have the capability to inhibit heme polymerization. The IC 50 values are 2.19 ± 0.09 mg/ mL for n-hexane extract (nonpolar), 1.24 ± 0.01 mg/mL for ethyl acetate extract (semipolar) and 1.16 ± 0.02 mg/mL for ethanol extract (polar). All extracts are classified as active [12]. This provides an opportunity for the isolation process to find the main compound which is responsible for the activity of the extracts. If we can find the main compound responsibile for the antimalarial activity of the n-hexane extract, then in the future a synthesis approach can be made to save raw materials and costs. It is very important to know whether the antimalarial activity of this extract is synergistic or originates from its main compound. Therefore, this study conducted some isolation of major compounds from Andrographis paniculata Ness's n-hexane extract (origin from Yogyakarta) and continued with heme polymerization inhibition activity assay from the n-hexane extract and isolate.
Procedure
Andrographis paniculata Ness's n-hexane extract prepared from Andrographis paniculata Ness's plant by maceration method using n-hexane as solvent. The n-hexane extract was separated by using column chromatography with the best solvent system i.e. n-hexane : ethyl acetate (10:1) to produce fraction A and B with great separation (Figure 1).
All compounds were observed by using UV-lamp with 254 and 366 nm of wavelength. In the n-hexane extract and isolated compounds, phytochemical screening was performed i.e. alkaloids, flavonoids, terpenoids, and steroids screening. Isolated compounds were checked by using FT-IR, 1 H-NMR and GC-MS for determination of the compound. The conditions in the GC-MS analysis are column oven temperature 70°C, injection temperature 300°C with helium as carrier gas, and EI method on MS analysis. Analytical TLC was carried out using pre-coated silica gel plates (Merck TLC silica gel 60 F254). IR spectra were recorded on a Thermo Nicolet Avatar 360 using a NaCl cell; ṽ in cm -1 . 1 H-NMR spectra were recorded using a Agilent 500 MHz NMR spectrometer. Chemical shifts are reported in ppm relative to CHCl 3 (δ(H) 7.26) in CDCl 3 for 1 H-NMR. Splitting patterns are designated as s, d, t, q, and m, indicating singlet, doublet, triplet, quartet, and multiplet, respectively.
Heme polymerization inhibition activity assay were performed by In Vitro method using [13] a method which modified at levels of hematin solution and the level of test sample. Series of test sample levels were made in 10% DMSO with a concentration of 5; 2.5; 1.25; 0.625; and 0.3125 mg/ mL. Each level was made as much as 300 μL.
The testing process was carried out by placing 100 μL of 1 mM hematin solution in 0.1 M NaOH into Eppendorf tubes, then adding 50 μL of test material with various levels, namely 5; 2.5; 1.25; 0.625; and 0.3125 mg / mL. Replication is done 3 times for each level. To start the heme polymerization reaction, 50 μL of glacial acetic acid solution (pH 2.6) was added to the Eppendorf tube which contained hematin solution and samples, then incubated at 37°C for 24 hours. Positive control used was Mexaquin antimalarial drug, while the negative control was 10% DMSO. After the incubation ended, the Eppendorf tube was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the precipitate was washed 3 times with 200 μL of DMSO 100%. At each wash, centrifugation speed of 8000 rpm is carried out for 10 minutes. The obtained sediment was added 200 μL of 0.1 M NaOH. Every 100 μL of the solution obtained was put into a 96 microplate well and read the absorbance using Elisa Reader at a wavelength of 405 nm. Heme polymerization inhibitory activity is expressed by IC 50 values calculated by probit analysis using SPSS version 23. This assay was performed to n-hexane extract, isolated compounds and chloroquin in drug (Mexaquin Chloroquine).
Ethical approval: The conducted research is not related to either human or animal use.
Results and Discussion
This study was classified in explorative research. The results gave information about major compounds from Andrographis paniculata Ness's n-hexane extract and their antimalarial activity. In the first exploration, the class of compound was investigated by phytochemical screening. N-hexane extract collected in dark green oil with 0.4% of yield. Furthermore, there are two major compound which can be collected by chromatography process using n-hexane : ethyl acetate (10:1) and n-hexane: chloroform (10:3) as eluent. The first major compound is more nonpolar than another one with 7% of yield from n-hexane extract and isolated from Fraction A, called dioctyladipate .
The second major compound is more polar with 0.35% of yield from n-hexane extract and isolated from Fraction B, called menthol. The phytochemical screening also performed for isolate compounds. The results of phytochemical screening i.e. alkaloids, flavonoids, terpenoids, and steroids screening for all compounds are shown in Table 1. There are 3 group of compounds in n-hexane extract. This phenomenon is very unusual, but occurs in the extraction process where the alkaloid and flavonoid compounds can be extracted in n-hexane. Therefore, it can be predicted that in the process of separation and purification will continue to take a long time because it still has to separate the main compounds from 3 groups of compounds, alkaloids, flavonoids and terpenoids. This condition also makes the yield of the main compounds (first and second major compound) in the n-hexane extract very small.
In the second exploration, FT-IR, 1 H-NMR and GC-MS analysis were performed on both major compounds to determine the purity and structure of the compounds. Structure elucidation of first major compound, dioctyladipate was performed by means of FT-IR ( 4H, t). The spectrum displayed in Figure 3. By the 1 H-NMR spectrum results, it could be stated that dioctyladipate have been succesfully isolated as yellow oil. However, there are still many impurities.
The molecular formula was determined to be C 22 H 42 O 4 based on 1 H-NMR and GC-MS data (M + , calculated for being 370). The results of GC-MS analysis are shown in Table2 for dioctyladipate as first major compound. The chromatogram and mass spectra also presented in Figure 4.
From the result analysis of first the major compound, there are still many impurities (9 impurity compounds). However, the results concluded that the first major compound is dioctyladipate. This compound was classified as glyceride compound. When associated GC-MS analysis (Table 2 and Figure 4) with the results of phytochemical analysis (Table 1), there appears to be a mismatch between the results of phytochemical analysis with GC-MS analysis. This is because the main Figure 4: Chromatogram (a) and mass spectra (b) from first major compound, dioctyladipate.
compound, dioctyl adipate, is not including terpenoid group. However, when examined more deeply, the actual positive results of terpenoid on phytochemical analysis obtained from the content of impure compounds that still exist. From GC-MS analysis, it is clear that there are still 4 impurity compounds classified as terpenoids. From the MS analysis (Figure 4b), the spectra have 96% similarity index (SI) and it can be directly determined the name of the compound, as well as its structure.
In the third exploration, structure elucidation of second major compound, menthol was performed by means of FT-IR (Figure 2 Table 3 for menthol as second major compound. The chromatogram and mass spectra also presented in Figure 6. In contrast to the first major compound, GC-MS analysis of the second major compound ( Table 3 and Figure 6a) consisted of only 1 peak with 99% of purity. The results concluded that the second major compound is menthol and that compound was classified as terpenoid. This is in accordance with the results of phytochemical analysis ( Table 1) where in the analysis obtained that the compound is positive as terpenoid compounds. From the MS analysis (Figure 6b), the spectra have 90% similarity index (SI) and it can be directly determined the name of the compound, as well as its structure.
The M + from MS analysis of dioctyladipate and menthol were missing. This phenomena can be explain by fragmentation rules. Intensity of M + will decrease or invisible for branched and high molecular weight compound. Beside that, when ionization process, the molecules will be fragmented in the branches of their structure and will form more stable ions and these ions are seen in MS spectra. This is the reason why M + from dioctyladipate (370) and menthol (156) are invisible. After successfully in determining the structure, the exploration continued to HPIA assay. Hematin will polymerize into β-hematin crystals in acidic condition. Based on the antimalarial mechanism, the candidate will reduce β-hematin crystals formation. The IC 50 values of the isolate compound, negative and positive controls were written in Table 4. The results showed respectively that n-hexane extract, dioctyl adipate, menthol and positive control chloroquin in drug (Mexaquin Chloroquine) have IC 50 values 0.07 ± 0.03 mg/mL, 1.15 ± 0.41 mg/mL, 0.31 ± 0.01 mg/mL and 0.35 ± 0,13 mg/mL.
According to [13], a compound could be considered to have heme polymerization inhibitory activity if having IC 50 value less than chloroquine sulfate, i.e. 12 mg/mL. So it can be declared that the n-hexane extract of Andrographis paniculata Ness, dioctyl adipate and menthol have inhibition activity of heme polymerization, because their IC 50 values were lower than 12 mg/mL. However, the IC 50 of dioctyl adipate was higher than positive control chloroquin in drug (Mexaquin Chloroquine) but still lower than 12 mg/mL. This is allegedly because dioctyl adipate does not have hydroxyl groups that can interact with the heme's iron ion, while chloroquine and menthol have the group.
If the IC 50 value of n-hexane extract from Andrographis paniculata Ness, and the major compounds contained i.e. menthol and dioctyladipate compared to another antimalarials compound that have been found before, it can be concluded that menthol and dioctyladipate is quite good as antimalarials although not better than andrographolide which is the main compound of Andrographis paniculata Ness. However, n-hexane extract from Andrographis paniculata Ness has a better value and very potential to be used as an antimalarial wherein the preparation process is quite easy. The results of the toxicity test showed that n-hexane extracts have LC 50 values is 1,155 μg/mL [12]. Therefore all of the Andrographis paniculata Ness's extract is safe to use. Previous studies also reported that extracts of Andrographis paniculata Ness had no toxic effect on Artemia salina larvae [15].
In vivo testing to determine acute toxicity using animal testing of mice also showed that Andrographis paniculata Ness extract included in the safe category with an LD 50 value more than 5,000 mg/kg BW [16]. Thus all major compounds frome n-hexane extracts in this study are safe to use in humans. The activity of n-hexane extract of Andrographis paniculata is higher than their major compounds or main compounds because there are several compounds in the n-hexane extract wherein having synergistic activity. There are 3 class of compounds included in n-hexane extract (alkaloids, flavonoids and terpenoids). This condition has increased the activity of n-hexane extract to inhibit heme polymerization. The activity can be seen from IC 50 values of n-hexane extract, which is lower than both major compounds.
The presence of heme polymerization inhibition activity by n-hexane extract and menthol are suspected due to the interaction between hydroxyl groups contained in extracts or isolates with heme iron ions. This is based on research conducted by [13]. Terpenoid group compounds, phenols, and steroids have an inhibitory activity of heme polymerization due to the interaction between compounds with heme's electronic systems or hydroxyl group bonds with heme iron ions. Although there is no hydroxyl group on dioctyl adipate, this compound still has inhibition activity. This is possible because dioctyl adipate has an ester group in which the oxygen atoms of the ester group can also interact with iron even though the interaction is not as good as the hydroxyl group. The interaction of β-hematin with menthol and dioctyl adipate was described in Figure 5a and 5b.
Conclusion
In conclusion, we have presented the isolation of the major component from Andrographis paniculata Ness's n-hexane extract by using liquid chromatography method. Major compounds that can be isolated i.e. dioctyl adipate and menthol although there are still many impurities on the dioctyladipate. The inhibition of heme polymerization activity of both compounds are very good (dioctyl adipate IC 50 = 1.15 ± 0.41 mg/mL, menthol IC 50 = 0.31 ± 0.01 mg/mL, n-hexane extract IC 50 = 0.07 ± 0.03 mg/mL while n-hexane extract is more active than both of major compound. The antimalarial activity of Andrographis paniculata Ness's n-hexane extract is working synergistic, not by the major compounds. In case, menthol and n-hexane extract displayed better antimalarial activity than chloroquine (IC 50 =0.35 ± 0,13 mg/mL).
Conflict of interest:
Authors declare no conflict of interest. | v3-fos-license |
2020-12-03T09:07:09.996Z | 2020-11-30T00:00:00.000 | 227259267 | {
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} | pes2o/s2orc | pH-Sensitive Biomaterials for Drug Delivery
The development of precise and personalized medicine requires novel formulation strategies to deliver the therapeutic payloads to the pathological tissues, producing enhanced therapeutic outcome and reduced side effects. As many diseased tissues are feathered with acidic characteristics microenvironment, pH-sensitive biomaterials for drug delivery present great promise for the purpose, which could protect the therapeutic payloads from metabolism and degradation during in vivo circulation and exhibit responsive release of the therapeutics triggered by the acidic pathological tissues, especially for cancer treatment. In the past decades, many methodologies, such as acidic cleavage linkage, have been applied for fabrication of pH-responsive materials for both in vitro and in vivo applications. In this review, we will summarize some pH-sensitive drug delivery system for medical application, mainly focusing on the pH-sensitive linkage bonds and pH-sensitive biomaterials.
Introduction
Biomaterials have been widely developed as the carriers of various drugs for medical application [1][2][3][4]-of which pH-sensitive biomaterials represent a promising species, which can perform deformation or degradation after exposure to external acidic or alkaline environments. The pH-sensitive characteristics enable the changes in intramolecular or intermolecular forces of the formulations in external pH conditions, resulting in a trigger release of payloads [5][6][7]. There are basically two major different mechanisms to release the loading drugs in pH-sensitive drug delivery systems [8]: one is releasing the medicines generated from the changed hydrophobicity or a charge of carrier molecules by protonation/deprotonation induced by the external pH variation. The other is releasing the loading drugs based on pH-sensitive dynamic chemical bond cleavage [8].
Cancer is one of the leading causes of human death around the world [9,10]. In the past decades, nanomaterials based on the tumor microenvironment (TME) has been proposed to be a promising approach to improve the efficiency of cancer therapy [11,12]. TME constructs the external environment of tumor cells, which is closely related to the occurrence and metastasis of tumors [13,14]. The tumor microenvironment is composed of tumor cells, stromal cells, cytokines, and matrix. Due to the rapid growth and high volume expansion of tumor tissues, the tumor microenvironment is characterized by low oxygen [15][16][17] and mild acidity pH (6.5-6.9) [18]. With the better understanding of tumor cell biology, the tumor microenvironment is of great significance for the formation, development, metastasis, and other processes of tumors [19,20]. These abnormal microenvironments in return also provide the unique parameters for the designing of intelligent biomaterials for cancer therapy [21,22]. For control release of the therapeutics, many pH-responsive formulations have been designed based on the tumor acidic environment [23,24].
pH-sensitive biomaterials have been developed rapidly in the past decades and have made great achievements. As shown in Figure 1, we have summarized the number of publications about pH-sensitive biomaterials in the past 20 years (Figure 1), which increases every year. In this review, we focus on the pH-responsive linkage bonds and pH-sensitive nanomaterials for medical applications, especially for cancer therapy. Firstly, we summarize some common pH-sensitive biomaterials based on different types of pH-sensitive bonds. In the following, we summarize pH-sensitive nanomaterials for drug delivery purposes.
pH-Sensitive Bonds
The functionalization of biomaterials, which are covalently bonded to drug molecules, is the most common way to obtain intelligent biomaterials [25][26][27][28][29]. This type of carrier has high stability and can effectively prevent the premature release of drugs under physiological conditions [30]. However, in order to deliver drugs effectively, responsive biomaterials are showing great prospect [31-33]-of which, pH-responsive biomaterials equipped with acidic trigger release capacity have been widely used for the purpose. The pH-responsive biomaterials are expected to store the drugs in normal pH and release the drugs in a specific pH condition. To achieve this, pH-sensitive chemical bonds can be anchored within the drug carriers, and the configuration of the carriers can be deformation through the cleavage of the dynamic chemical bonds, so as to control release the payloaded drugs [8]. In this section, we will introduce the commonly used pH-sensitive chemical bonds (Table 1).
Imine Bonds
Imines are formed by condensation of primary amine with aldehydes or ketones. Imine bond can be hydrolyzed under weak acid conditions (pH~6.8, near the pH of solid tumors), but its stability in physiological pH environment can be improved [34-39].
Yang et al. synthesized a polymer poly(ethylene glycol)-cholic acid grafted poly-L-lysine (PEG-PLL-CA) anchored with benzoimide, and demonstrated that polycationic micelles formed by self-assembly of PEG-PLL-CA could function as the carriers of therapeutics for tumor therapy with pH-responsive characteristics [40]. The imine bond is liable to hydrolyze under acidic conditions which can also be used to modify the surface of the nanocarriers [41], and the acid-sensitive release of the drugs on the carrier can be realized triggered in the acidic environment. Xu et al. synthesized the pentaerythritol tetra(3-mercaptopropionate)-allylurea-poly(ethylene glycol) (PETMP-AU-PEG) anchored with imide bonds. They demonstrated that PETMP-AU-PEG loaded with DOX were highly stable in the neutral environment. While under the environment of weak acid, the formulation can be a responsive release of DOX for antitumor outcomes by cleavage of imide bond [42].
Hydrazone Bonds
Hydrazone is formed by condensation of aldehydes, ketones, or hydrazine. Similar to imine bonds, the hydrazone bonds are also a class of covalent bonds which are acidic responsive [43][44][45][46][47]. As hydrazone bonds are easily hydrolytic under acidic conditions, they have been widely utilized in the construction of acidic sensitive carriers for responsive formulations [48,49].
Rihova et al. applied N-(2-hydroxypropyl) methylacrylamide (HPMA) as the backbone to prepare HPMA-DOX formulations anchored with hydrazine bonds, and they reported that the formulations could increase the in vivo circulation time of DOX for optimal targeting accumulate in the tumor and afterwards responsive release triggered by the acidic tumor environment [50]. Song et al. first synthesized the polymer PEG-DiHyd-PLA containing the hydrazone bond by open-loop polymerization, and then the polymeric micelles were formed by self-assembly for loading DOX. They demonstrated that the formulations could release DOX rapidly and completely under the specific acidic conditions of tumor tissues [51].
Oxime Bonds
Oxime is formed by condensation of aldehydes, ketones, or hydroxylamine. Oxime can hydrolyze into aldehydes, ketones, and hydroxylamines under acidic conditions [52,53]. A potential advantage of using oxime bond for tumor-targeted nanocarriers is the tunability of its acid lability by facile variation of the substituents [54].
Zhu and colleagues described a polymeric drug carrier system by self-assembly of the triblock copolymer (PEG-OPCL-PEG) which were synthesized by conjugating of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic oxime-tethered polycaprolactone (OPCL), which was successfully prepared by reacting OPCL ligating with aldehyde-terminated PEG (PEG-CHO). During the self-assembly process, DOX as a drug model was loaded within the PEG-OPCL-PEG micelles. They demonstrated that the release rate of DOX in tumor tissue was significantly faster than in normal tissue, as a result of the acidic responsive of anchored oxime linkages within the micellers [53]. In another work, Zhu and colleagues described a flower micelle formulated by self-assembly of backbone-cleavable triblock copolymer polycaprolactone-oxime-poly(ethylene glycol)-oximepolycaprolactone (PCL-OPEG-PCL) in aqueous solution, during which the DOX were encapsulated within the micelles in high efficiency. In addition, the DOX loaded micelles had good stability in a neutral environment in vitro studies, but, under the condition of weak acid, DOX could be quickly released for the pH responsive characteristics and thus perform excellent antitumor outcomes [55].
Amide Bonds
Amide bond is formed by the substitution of the hydroxyl group in the carboxylic acid group by amino groups or hydrocarbon amino groups [53,56,57]. Amide bond can also be cleavage under acidic conditions [58][59][60][61][62], which are most widely used for designing pH-responsive formulations.
Gu et al. synthesized pH-sensitive polymeric micelle (mPEG-pH-PCL) through self-assembly of methoxy poly(ethylene glycol)-b-poly( -caprolactone) copolymer anchored with citraconic amide as a pH-sensitive bond, and DOX were efficiently encapsulated in the micelle during assembly. They demonstrated that the DOX released from mPEG-pH-PCL micelles in pH 5.5 solution was much faster than that of in pH 7.4, indicating a beneficial characteristic for drug targeting release in acidic tumors [63]. Forrest et al. used amide bonds to connect hyaluronic acid (HA) with DOX to form the HA-DOX conjugate. In the blood neutral environment, HA-DOX has good stability and biocompatibility during the circulation process, thus reducing dose-limiting cardiac toxicity and minimal toxicity observed in other normal tissues. However, once the therapeutic conjugation accumulated in the acidic environment of tumor tissue, the acidic hydrolysis of amide bond enabled DOX specific release, thus achieving better therapeutic effects [64].
Acetals
Acetals (including ketals) are promising acid-sensitive linkages for responsive formulations due to their sensitive hydrolysis with each unit of pH decrease [65,66]. Acetals are formed by condensation of aldehyde and alcohols. Under acidic conditions, acetal can be hydrolyzed into aldehyde and alcohols [67].
Zhong et al. conjugated acid biodegradable poly(acetal carbamate) (PAU) into a three block copolymer (PEG-PAU-PEG) and formulated to micelles by self-assembly for pH-triggered intracellular delivery of DOX. The three-block copolymer formed micelles had no cytotoxicity and performed good stability in the physiological pH. However, DOX could be triggered release in the acidic cytoplasm after uptake by endocytosis and induce apoptosis of cancer cells [68]. Pu and colleagues prepared tumor-pH-sensitive polymeric microspheres by using multi-block polyl-lactide (PLLA) with acetal bond as the backbone for efficient loading DOX. They reported better antitumor efficiency and prolonged life-span after intratumorally injection of the micelles compared to other control groups [69].
Orthoester
Orthoester is a functional group containing three alkoxy groups on the same carbon atom. Orthoester can be hydrolyzed into carboxylic acid and alcohol under acidic conditions [70].
Park et al. assembled micelles with copolymers bearing an acid-sensitive orthoester linkage, which composed of hydrophilic PEG and hydrophobic poly(γ-benzyl L-glutamate) (PBLG) and DOX. The DOX loaded polymeric micelles were stable under neutral conditions but could trigger the release of DOX for efficient therapy in the acidic tumor due to the acidic cleavage of orthoester [71].
pH-Sensitive Nanomaterials
In recent years, many administration routes have been widely developed for the medical application, and integrating the novel material engineering technologies with optimal administration route would enable their enhanced therapeutic efficiency and reduced side effects [72][73][74][75][76][77]. In terms of convenience, oral administration has great advantages. However, since drugs need to pass through the digestive tract during oral administration, and then arrive at the duodenum, jejunum, and ileum. The pH values in different parts of the gastrointestinal tract are different, that is, the stomach cavity is acidic, the pH range is 1-3, and the duodenum is alkaline [78]. When a drug enters the gastrointestinal tract by oral administration, the drug will be exposed to a series of acidic environments and biological enzymes, which may be degraded or inactivated. The tumor microenvironment also has features with acidic and other abnormal characteristics which may suppress the bioactivity of delivered drugs [79]. These required the novel drug carriers which can protect the drugs from degradation and metabolisms prior to their final acting places and triggered release of the therapeutic payloads in the abnormal pathological tissues according to the pH gradients or other conditions. Nanomaterials for drug delivery have broad application prospects [41, [80][81][82][83][84][85][86]. For example, the nanoparticles encapsulated with drug molecules can isolate drugs and environments that may degrade or inactivate drugs, providing a relative stable environment for drug molecules with expected bioavailability while passing through the complex in vivo environment. At the same time, since the nanoparticles allow them to penetrate the endothelium and capillary tubes, the use of nanoparticles to deliver drugs to target cells in inflammatory sites may be considered [87]. These carriers can release drugs in the extracellular environment, or within the cell through endocytosis. pH-responsive characteristics can be added into the nanoparticles which can be degradation or deformation under specific conditions, such as acidic conditions, thereby releasing the encapsulated drugs and acting on a specific location. After the changes of nanoparticles, the release of drugs from nanocarriers can be triggered based on the change of pH [88][89][90][91]. Compared with the ordinary nanoparticles, advantages of pH-responsive nanoparticles lie in applying acidic pH as a kind of external stimuli, resulting in the changes of drug release kinetics [92][93][94]. Herein, we present an overview of pH-sensitive nanomaterials [95] including hydrogel, liposomes, and polymer micelles.
Hydrogels
Hydrogels are composed of polymers that are crosslinked into a three-dimensional network [96][97][98]. Hydrogels can be made of synthetic or natural polymers and they are capable of imbibing large quantity of water or fluids [99,100]. There are a large number of hydrophilic groups on the polymer chain, such as -NH 2 , -OH, -COOH, and -SO 3 . With the increase of capillary action and osmotic pressure, hydrogels are relatively insoluble in the surrounding media due to the cross-linking between the polymer chains. The cross-linking in hydrogels is both physical (hydrogen bonding) and chemical (covalent bonding between functional groups) (Figure 2a). Hydrogel was first proposed in the 1960s by Wichterle and Lim using a hydrophilic network of poly(2-hydroxyethyl methacrylate) in contact lenses [101,102]. Hydrogels are valuable due to their easy fabrication process, small size, mouldability, immunity to electromagnetic radiation, and biocompatibility [100]. The physical characteristics of hydrogels can be tuned to provide specifications to match various applications in drug delivery systems or other aspects [103][104][105][106][107][108]. pH-responsive hydrogels been developed greatly over the past few years and shown to be very useful in biomedical applications, especially like targeted cancer treatment [109][110][111]. The use of pH-sensitive hydrogels can prolong drug release availability, and the synthesis of pH-sensitive hydrogels is also quick and cost-effective. In addition, pH-sensitive hydrogels have been investigated frequently for delivery of peptide drugs [112,113].
Currently, the use of pH-sensitive hydrogels for cancer therapy has been investigated as improving the efficiency of the released drugs [114][115][116][117][118]. Polymers commonly used to study pH-responsive hydrogels including polyacrylate, poly (N-isopropyl acrylamide), polyacrylate, n-vinyl caprolactam, sodium alginate, and carrageenan [119,120]. Some traditional hydrogels require the use of different substances, such as poly(N-isopropyl acrylamide) and poly(acrylic acid) attached to the PVF skeleton, fabricating hydrogels with dual temperature and pH response [119]. Alternatively, bivalent copper can be used in conjunction with alginate to form hydrogels by gel spheroidization. It is worth mentioning that the encapsulation system made by this method has been proved to maintain their structural integrity at pH 1.2, and the contents can only be released slowly at pH > 5, which suggests the stability of the encapsulation system under the conditions of simulated stomach, and release the contents slowly under the conditions of intestinal tract [123,124]. In addition to the traditional hydrogels, there are also novel hydrogels, such as gelation by crosslinking of poly (vinyl pyrrolidone) polymer and rotonic acid under γ-radiation [122]. The drug release rate from the gelation in acidic medium is much lower, while the release rate in neutral medium is faster. Therefore, similar to the former, it has the potential to act as a drug carrier to protect the release of drugs through the stomach in the intestinal tract. Some hydrogels also need to improve the stability of the pore wall to prevent swelling, such as the use of silica nanocomposites on top of polyn-isopropyl acrylamide [122]. Furthermore, the majority of the pH-responsive hydrogels [125] relies on the side chain by ionization, namely through the protonation of cationic groups or anionic groups, or the electric network with the surrounding environment, forming electrostatic repulsion [121] (Figure 2b,c).
In addition, dual temperature and pH-responsive hydrogels or dual magnetic and pH-responsive hydrogels were also expanding their medical application, allowing for more precise drug release behavior. Ren et al. synthesized the supramolecular hydrogels hybridized with magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs). These hybrid hydrogels which stimulated by temperature and pH showed reversible sol-gel transition [126] (Figure 3). There are also pH-responsive cellulose gels used as wound dressings based on hydrogels that could self-degrade on the mild acidic skin surfaces and have the ability to self-heal, which have broad application prospects. Amanda et al. showed that surface functionalization of cellulose nanocrystals (CNCs) with amine (CNC-NH 2 ) moieties enabled the CNCs with pH-responsive characteristics. The transition to hydrogels as observed at higher pH degraded into dispersion aqueous at low pH. This could be incorporated into a poly(vinyl acetate) matrix to yield mechanically adaptive pH-responsive nanocomposite films [127] (Figure 4).
Liposomes
In the 1960s, the first lipid-based vesicles for drug delivery was reported [128][129][130]. Liposomes [131] are a kind of biomimetic nanosome with a hollow structure consisting of phospholipid bilayers ( Figure 5). The ability of liposomes to encapsulate both hydrophilic and hydrophobic drugs, coupled with their biocompatibility and biodegradability, make liposomes attractive vehicles in the field of drug delivery [132][133][134][135]. Liposomes are frequently used for delivery of drugs, antigens, vaccines [136], DNA, and/or diagnostic units [137][138][139][140][141]. The liposomes could be internalized by cells through different endocytic pathways and effectively deliver the encapsulated drugs into the cell matrix [128,129,[142][143][144][145]. For their optimal in vivo application [146], the surface of liposomes can be functionalized with various hydrophilic polymers, such as PEG, to minimize reticuloendothelial system recognition and absorption [147,148]. The diameter of liposomes enables passive accumulation in tumor tissue for antitumor effect through the enhanced permeability and retention effect (EPR) [149][150][151][152]. The composition of liposomal systems can be easily modified to facilitate triggered release in response to environmental conditions. For example, pH-responsive liposomes are specifically designed to control the drug release in response to the acidic tumor microenvironment [153].
pH-responsive liposomes [154,155] represent a promising carrier for the loading of drugs for medical applications [153]. One common method to prepare pH-responsive liposomes is to use pH-sensitive components to fabricate liposomes. When these responsive units introduced into the liposomes, the liposome can be transformation or degradation in the acidic endosomes or environments of tumors, resulting in the trigger release of payload with pH-responsive behaviors [156]. In addition to the instability caused by pH-sensitive components, the stability of liposomes can also be affected by the integrated hydrophobic chains. These pH-liposomes have some significant advantages, such as low toxicity, simple preparation, and good biocompatibility as a result of the biocompatible degradable components [157,158]. pH-responsive liposomes [159] can function as the carrier of anti-tumor therapeutics for targeting delivery to the tumor and trigger release of the encapsulated drugs in the acidic environment. However, their use for in vivo application also presents great challenges. In general, the size of liposomes used for in vivo application is usually between 50 and 450 nm, and the liposomes within this diameter scale are easily removed by the RES system during blood circulation [157,160]. At the same time, although their structure and composition are similar to that of the cell membrane, liposomes are still easily recognizable as the antigens, and faced with the in vivo clearance by the immune system. To improve their in vivo fate for better therapeutic outcome, various surface modification strategies are developed such as introducing the PEG chains to the outside surface of liposomes, so as to prolong their in vivo circulation time [130,157,161,162] (Figure 6). In addition to the therapeutic purpose, pH-responsive liposomes are also becoming important tools for vaccine delivery. These liposomes are used to deliver small peptides or antibodies to produce an effective immune response and reduce their toxicity. Kim and colleagues successfully transferred the pH-sensitive liposomes loaded with cytotoxic T lymphocytes epitope peptides for tumor therapy, which resulted from the induced effective antigen-specific CTL responses after the responsive release of peptides in the acidic tumor [163].
Polymer Micelles
Polymer micelles are promising carriers for various kinds of drugs in medical application, thereby improving the efficacy of drugs and reducing side effects [164][165][166][167][168][169] (Figure 7). Micelles are usually formed by self-assembly of block polymers, which could be conjugated with different units such as polyethylene glycol and poly(amino acid) [169]. During the self-assembly of the polymers of micelles, therapeutic drugs such as DNA, RNA, proteins, and small molecular drugs with an abundant of functions could be encapsulated within the micelles [170][171][172][173][174]. Although polymer micelles have good stability in vivo, drugs released from the micelles usually in a very slow diffusion mode, which lead to a low concentration of free drugs in tumor cells and would suppress the therapeutic outcome. It is necessary to design polymer micelles to release drugs with desirable dynamic behavior in tumor sites. pH-responsive polymer micelles equipped with acidic trigger sections present an efficient drug delivery for cancer therapy [56], derived from the passive or active targeting potency and subsequent responsive release of payloads.
The design strategies of pH-sensitive polymer micelles mainly include: 1. Protonated groups are introduced into the polymer chain through the mechanism of protonation/deprotonation [175][176][177][178][179]. The common functional groups of protonation include the amine group, imidazolyl group, sulfonic acid group, and carboxyl group. When such polymer micelles reach acidic target sites, rapid targeted release of the drug contained in the micelles would be triggered as the unstable micelle structures due to acidic precipitation/aggregation, or depolymerization. 2.
Pun et al. recently developed pH-sensitive polymer micelles fabricated with self-assembly of lytic peptide modified diblock copolymers (PPCs). They demonstrated that significantly enhanced in vitro and in vivo transfection efficiency with melittin-like peptide polymer conjugate encapsulated in the micelles [191], indicating further in vivo gene therapy mediated by the peptide encapsulated micelles.
The surface modification of micelles, such as PEGylation, is important for their function [56]. The long chain of PEG may affect the uptake of micelles by cells for subsequent biological effects. Other modification strategies could be considered for improved uptake by cells, as a tactic folate acid and its variants can be used to modify the surface of micelles for improving cellular uptake, as folic acid receptors are usually highly expressed on many types of cancer cells. Xiong et al. successfully designed folate-conjugated crosslinked biodegradable micelles with poly(ethylene glycol)-b-poly(acryloyl carbonate)-b-poly(D,L-lactide) (PEG-PAC-PLA) and folate-PEG-PLA (FA-PEG-PLA) block copolymers for receptor-mediated delivery of paclitaxel (PTX) into KB cells for cancer therapy [192] (Figure 8).
Conclusions and Future Prospects
Over the last few decades, engineering biomaterials have strived to improve the therapeutic outcome with reduced effects, and great progress has been made in the application in drug delivery systems for medical applications, especially for cancer therapy. Several biomaterials with pH sensitivity have shown great potential for constructing effective drug delivery systems. In this review, we present an overview for pH-sensitive bonds and pH-sensitive nanomaterials for drug delivery in the past two decades. pH-sensitive biomaterials for drug delivery are a research hotspot in the fields of biochemistry and medicine, and great progress has also been made in recent years. At the same time, most of the research is still in the experimental stage, and there are still many problems that need to be solved for translational application, such as the biocompatibility of some pH-sensitive biomaterials, mass production with controllable quality, and potential toxicology of the carriers. Using pH-sensitive chemical bonds to design and prepare pH-sensitive biomedical materials based on acidic microenvironments of diseases would greatly improve the biocompatibilities and reduce potential toxicity of biomaterials. Microfluidics to screen the uniform size of biomaterials would greatly improve the consistency of synthesized samples with different batches. | v3-fos-license |
2018-04-03T00:05:39.989Z | 2017-10-25T00:00:00.000 | 8249953 | {
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} | pes2o/s2orc | Cellular splicing factor UAP56 stimulates trimeric NP formation for assembly of functional influenza viral ribonucleoprotein complexes
The influenza virus RNA genome exists as a ribonucleoprotein (RNP) complex by interacting with NP, one of virus-encoded RNA binding proteins. It is proposed that trimeric NP is a functional form, but it is not clear how trimeric NP is formed and transferred to RNA. UAP56, a cellular splicing factor, functions as a molecular chaperone for NP and is required for the replication-coupled RNP formation of newly synthesized viral genome, but the details of NP transfer to viral RNA by UAP56 is unclear. Here we found that UAP56 is complexed with trimeric NP, but not monomeric NP. Gel filtration analysis and atomic force microscopy analysis indicated that the complex consists of two trimeric NP connected by UAP56. We also found that UAP56 stimulates trimeric NP formation from monomeric NP even at physiological salt concentrations. Thus, UAP56 facilitates the transfer of NP to viral RNA since trimeric NP has higher RNA binding activity than monomeric NP. Further, UAP56 represses the binding of excess amount of NP to RNA possibly by transferring trimeric NP. Collectively, we propose that UAP56 stimulates viral RNP formation through promotion of the assembly of trimeric NP and is important for the structural integrity of NP-RNA complex.
The genome of influenza type A viruses is single-stranded RNAs of negative polarity. The viral genome (vRNA) exists as ribonucleoprotein (designated vRNP) complexes with heterotrimeric viral RNA-dependent RNA polymerases and nucleoprotein (NP) 1 . NP is one of the basic viral proteins and binds single-stranded RNA without sequence specificity. NP is essential to maintain the RNA template in an ordered conformation suitable for viral RNA syntheses [2][3][4][5][6] . Cryo-electron microscopy analysis revealed that the oligomerization of NP is important to form a double helical structure with anti-parallel strand of vRNP 7,8 . Oligomeric NP shows higher RNA binding activity than monomeric NP 9 . Although crystal structures of RNA-free NP showed that NP forms oligomers in the crystalline state, a broad size distribution was observed by gel filtration chromatography in solution at physiological salt concentrations [9][10][11][12] , suggesting that NP exists in an equilibrium between monomers and oligomers including trimers and tetramers 13 . Thus, an exact form of NP to be assembled into vRNP remains unknown.
For efficient viral transcription and replication, not only viral factors but also host factors are required. It has been reported that RAF-2p48/NPI-5/UAP56, Tat-SF1, and Prp18 function as molecular chaperones for NP to recruit NP to the viral RNAs 4,14,15 . NP chaperones are also required to suppress the aggregation of NP. UAP56 is a cellular splicing factor belonging to the DExD-box family of ATP-dependent RNA helicase 16 . It is reported that the newly synthesized viral genome is co-replicationally assembled into RNP complex by UAP56 3 . UAP56 binds to NP free of RNA but not NP-RNA complexes 15 . It is also reported that UAP56 interacts with N-terminal region of NP, and NP interacts with C-terminal region of UAP56 15 . However, the molecular mechanism of the transfer of NP to RNA by UAP56 is still unknown.
Here we examined the binding stoichiometry of NP-UAP56 complex using recombinant UAP56 and RNA-free NP. We found that UAP56 stably interacts with trimeric NP, and stimulates the assembly of trimers from monomeric NP. Gel filtration experiments showed that the molecular size of UAP56-NP complex is more than 440 kDa, suggesting that the complex is composed of 6 molecules of NP and 2 molecules of UAP56. By atomic force microscopy (AFM) analysis of NP-UAP56, we found a dumbbell-shaped complex which is considered as two trimeric NP connected by UAP56. We also revealed that UAP56 facilitates NP-RNA complex formation without excessively oligomerized NP-RNA complexes. Taken together, these results suggest that UAP56 stimulates the RNP formation through the assembly of trimeric NP and controls the amount of NP on RNA for the structural integrity of NP-RNA complex for efficient viral RNA synthesis.
Results and Discussion
Binding stoichiometry of NP and UAP56 complex. NP-His and GST-UAP56 were expressed in E. coli BL21(DE3) strain and purified by Ni-NTA resin and GST resin, respectively (Fig. 1a). The GST tag of UAP56 was digested with Prescission protease (Fig. 1a, lane 3). After buffer exchange to a buffer containing 25 mM HEPES-NaOH (pH 7.0), 150 mM NaCl, 1 mM DTT, and 5% glycerol, purified NP-His and UAP56 were incubated at 4 °C overnight. The NP-UAP56 complex was partially purified using Sephacryl S-200 gel filtration column, and then the NP-UAP56 complex was analyzed by Superose 6 gel filtration column (Fig. 1b). In the absence of NP, UAP56 was eluted as a homogenous single peak at elution volume of 1.6 ml, which corresponds to the molecular weight of about 100 kDa. Because theoretical molecular weight of UAP56 is 49.8 kDa, this result suggested that UAP56 forms a dimer. The elution peak of NP free of UAP56 was found at around 63 kDa, suggesting that NP mainly exists as a monomer (theoretical molecular weight is 57.9 kDa). We found that the NP-UAP56 complex was recovered as a single peak at elution volume of 1.4 ml, which corresponds to the molecular weight of more than 440 kDa. CBB staining of the peak fraction showed that the complex consists of NP and UAP56 at a 3:1 molar ratio, indicating that UAP56 interacts with trimeric NP (Fig. 1c, lanes 2-4). Similar results were obtained by an alternative staining method, called VisPRO ™ 5 Minutes Protein Stain Kit, which stains the gel but not the protein sample (Supplementary Figure S1). Since the observed molecular weight of NP-UAP56 complex on the gel filtration column was around 440 kDa, it is likely that the NP-UAP56 complex is composed of 6 molecules of NP and 2 molecules of UAP56 with the theoretical molecular weight of 447 kDa. To confirm the binding of NP to UAP56, aliquots of the peak fraction were applied to a 6% native PAGE (Fig. 1d). UAP56 is an acidic protein with isoelectric point of about 5.4 and can be migrated to the cathode side in native PAGE (Fig. 1d, lane 1), whereas NP is hardly migrated to the gel because NP is a highly basic protein with isoelectric point of about 9.6 ( Fig. 1d, lane 2). The NP-UAP56 complex showed a slower migration pattern than UAP56 free of NP, and there is no UAP56 free of NP (Fig. 1d, lane 3). These results suggest that UAP56 stably interacts with NP. UAP56 binds to trimeric NP but not monomeric NP. NP consists of head and body domains with a long tail loop, formed by residues 402-428, at the outlying part of RNA-binding groove. The interaction of the tail loop from one NP molecule with a neighboring subunit is important to form homo-oligomers. Next, to examine whether monomeric or oligomeric NP binds to UAP56, we purified a tail loop mutant, R416A, which is a monomeric NP mutant 9,12,17 . UAP56 was incubated with either wild type or R416A mutant at 4 °C overnight. The mixtures were fractionated through chromatography on a gel filtration column, and the fractions were analyzed by 10% SDS-PAGE (Fig. 2). We found that UAP56 was recovered with wt NP in the fractions around 440 kDa (Fig. 2a), but R416A mutant did not (Fig. 2b). This suggests that UAP56 specifically interacts with trimeric NP. It is noted that UAP56 recognizes the N-terminal 188 amino acid residues of NP 15 , suggesting that the tail loop is not the direct binding site for UAP56. It is reported that monomeric NP binds to RNA, and the association of additional monomers to the pre-existing monomeric NP-RNA complex leads to higher order oligomers 9 . This stepwise assembly of NP-RNA complex is thought to be a slow process 9 . In contrast, pre-formed trimeric NP interacts rapidly with RNA 9 . Although our purified NP mainly contains monomeric NP (Fig. 1b), a large part of NP interacting with UAP56 was recovered as trimeric NP as shown in Fig. 1b (Fig. 2a, lanes 6-8). Thus, it is quite likely that UAP56 promotes the trimer formation of NP, and thereby UAP56 facilitates the transfer of NP to RNA as a molecular chaperone.
Next, to demonstrate the protein composition of NP-UAP56 complex, we carried out AFM analyses (Fig. 3). In the absence of UAP56, NP was observed as globular proteins which are approximately either 3-4 nm or 12-13 nm in length ( Fig. 3a and b). According to the crystal structure of NP 12 , the size of monomeric NP and trimeric NP is thought to be approximately 5 nm and 15 nm, respectively. This suggests that the smaller particles are monomeric NP, while the bigger ones are trimeric NP. In contrast, NP-UAP56 complex was found as a dumbbell-shaped complex of approximately 40-50 nm in major axis ( Fig. 3c and d). Since the size of trimeric NP and UAP56 dimer are is approximately 15 nm and 6 nm 18 respectively, suggesting that the dumbbell-shaped complex seems to be two trimeric NP connected by UAP56 dimer.
UAP56-mediated NP-RNA complex formation.
To examine the chaperone activity of the purified NP-UAP56 complex, we performed EMSA with [ 32 P]-labeled 53 nt-long ( Fig. 4a and b) and 165 nt-long (Fig. 4c) model vRNAs, respectively. The formation of NP-RNA complex was stimulated in the presence of UAP56 (Fig. 4a, compare lanes 5-7 with lanes 8-10; Fig. 4c, compare lanes 1-3 with lanes 4-6). Further, the migration rate of NP-RNA complex in the presence of UAP56 was faster than that without UAP56 (Fig. 4a, compare lane 6 with lane 8; Fig. 4b). In the case of 165 nt-long model vRNA, excessively oligomerized NP-RNA complexes that cannot enter native PAGE were formed in the absence of UAP56 (Fig. 4c, lanes 1-3), whereas the NP-RNA complex assembled in the presence of UAP56 successfully migrated to the gel (Fig. 4c, lanes 4-6). It has been proposed that NP binds to a replicating RNA, and then additional NP molecules are subsequently recruited by the NP-NP oligomerization for the efficient encapsidation of nascent chains 5,17 . Thus, it is possible that UAP56 modulates the self-oligomerization of NP on viral RNAs to assemble the functional RNP complexes by recruiting trimeric NP to viral RNAs. To examine whether UAP56 interacts with NP-RNA complex or not, we carried out the western blotting using antibodies against UAP56 (Fig. 4d) and His-tag (Fig. 4e) after separating the NP-RNA complex by EMSA. UAP56 free of NP was incubated in the absence or presence of 53 nt-long model vRNA, and subjected to EMSA (Fig. 4d, lanes 1-6). The migration rate of UAP56 was not changed in the presence of RNA, suggesting that the negatively charged UAP56 does not bind to RNA as shown in Fig. 4a (lanes 2-4) and Fig. 4c (lanes 8-10). Further, by the addition of RNA to NP-UAP56 complex, UAP56 was not found in NP-RNA complex (compare Fig. 4d, lanes 13-15 with Fig. 4e, lanes 13-15) and migrated to the same position as UAP56 free of NP (compare Fig. 4d). This result suggests that NP-UAP56 complex was dissociated by the addition of RNA as previously proposed 15 .
In conclusion, UAP56 forms dimer in solution, and the binding stoichiometry of NP-UAP56 complex is a 1:3 molar ratio, suggesting that UAP56 interacts with trimeric NP. Thus, it is likely that dimerized UAP56 proteins bind to trimeric NP, respectively. We also found that UAP56 stimulates trimeric NP formation from monomeric NP even at physiological salt concentrations. Therefore, UAP56 facilitates the transfer of NP to viral RNA as a molecular chaperone since trimeric NP has higher RNA binding activity than monomeric NP 9 . In the absence of UAP56, monomeric NP first binds to RNA, and the additional monomers randomly bind to the monomeric NP on NP-RNA complex, slowly 9 (Fig. 5). In contrast, we found that UAP56 represses the binding of an excess amount of NP to RNA possibly by transferring trimeric NP (Fig. 5). This is in good agreement with previous reports that UAP56 provides functional NP-RNA complex competent for viral RNA synthesis 3,15 . Taken altogether, we propose that UAP56 simulates NP-RNA complex formation by recruiting trimeric NP and suppresses the formation of excessively oligomerized NP-RNA complex as shown in the latter model (Fig. 5). Our findings reveal the molecular mechanism of molecular chaperone-dependent ribonucleoprotein complex formation for viral RNA synthesis.
Preparation of recombinant proteins. Plasmids expressing NP-His, R416A NP-His, and GST-UAP56, were transformed into BL21(DE3) strain (Stratagene), respectively. The proteins were expressed in LB medium for 16 h at 18 °C after induction with 0.2 mM IPTG. For NP-His and R416A NP-His, the harvested cells were suspended in lysis buffer A (25 mM HEPES-NaOH [pH 7.9], 500 mM NaCl, 30 mM imidazole, and 5% glycerol) containing 0.1% Triton X-100 and 1 mM PMSF. After lysis by sonication, the lysates were centrifuged at 20,000 × g for 30 min. The supernatants were filtrated with a 0.45-μm filter and subjected to His-tag purification using Ni-NTA agarose beads (GE healthcare). After washing with five column volumes of buffer A, NP proteins were eluted in Figure 3. NP-UAP56 was observed to be a dumbbell-shaped complex by AFM. NP alone (panels a and b) and NP-UAP56 complex (panels C and D) were subjected to AFM analysis, respectively. Monomeric NP, trimeric NP, and NP-UAP56 particles are indicated by yellow, blue, and red circles, respectively (panels A and C). Scale bar, 100 nm. The size distribution of NP or NP-UAP56 particles in major axis is shown in panels B and D. Solid lines represent a Gaussian fit.
buffer A containing 300 mM imidazole. To remove the bacterial RNA bound to NP, the NP lysates were treated with RNase A before purification and washed with buffer A containing 1.5 M NaCl during purification. Purified NP with a ratio of the absorbance at 260 nm to that at 280 nm of between 0.57 and 0.59 was used for this study. For GST-UAP56, the cell pellet was resuspended in lysis buffer B (25 mM HEPES-NaOH [pH 7.3], 500 mM NaCl, 1 mM DTT, and 5% glycerol) containing 0.1% Triton X-100 and 1 mM PMSF. After sonication, the lysates were centrifuged at 20,000 × g for 30 min. The supernatants were filtrated with a 0.45-μm filter and subjected to GST-tag purification using glutathione Sepharose beads (GE healthcare). After washing with 5 column volumes of buffer B, GST-UAP56 immobilized on the resin was digested with an appropriate amount of PreScission protease for 12 h at 4 °C. UAP56 without GST tag was collected in the flow through fraction.
Gel filtration analysis. After buffer exchange of purified NP and UAP56 to buffer C (25 mM HEPES-NaOH [pH 7.0], 150 mM NaCl, 1 mM DTT, and 5% glycerol) using a desalting column (HiPreP 26/10 Desalting, GE Healthcare), purified NP and UAP56 were mixed at a 1:1 molar ratio, and were incubated at 4 °C for overnight. The NP-UAP56 complex was purified by a gel filtration chromatography using the HiPrep AFM analysis. NP alone and NP-UAP56 complex were subjected to AFM analysis in air at room temperature (Bruker Nanoscope VIII, Bruker). The system was operated in tapping mode with a 100-μm scanner. Probes made of a single silicon crystal with a cantilever length of 129 µm and spring constant of 33-62 N/m The samples were separated on 6% native PAGE in 0.5 × TBE buffer, and the gels were subjected to western blotting using anti-UAP56 (panel D) and anti-His-tag (panel E) antibodies, respectively.
Figure 5.
A proposed model. In the absence of UAP56, monomeric NP first binds to RNA, and the additional monomers randomly bind to the monomeric NP on NP-RNA complex, slowly. The dimerized UAP56 proteins bind to trimeric NP, respectively. UAP56 stimulates the transfer of NP to viral RNA by facilitating trimeric NP formation for the structural integrity of NP-RNA complex competent for viral RNA synthesis. | v3-fos-license |
2019-04-09T13:07:21.196Z | 2017-11-15T00:00:00.000 | 54860468 | {
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} | pes2o/s2orc | Determination of Microbial Activities and Biomass in Biofilm Associated with Treatment Wetlands Compartments to Investigate Active Pollutant Processing Site
Although Floating treatment wetlands (FTWs) provide immense advantages over other natural treatment facilities, there is no information about biofilm functioning and microbial-based processes in FTW. Therefore, this study was aimed to evaluate the magnitude of microbial-based processes in the root, bottom and water column zones of the FTW by employing of macrophytes. For this experiment, primary domestic wastewater effluent was used in two pairs of FTWs (I. psuedacorus and P. stratiotes) and a pair of control. Total microbial activity was estimated using FDA hydrolytic activity and specific microbial activities were examined as denitrification and nitrification activities, whilst viable microbial number and distribution in the FTW compartments were determined using ATP assay. The average nitrification rates in the FTWs were 0.55, 0.81 and 2.75 μg/ml of water, gravel and root surface per hour respectively; and denitrification rates were 0.022, 0.053 and 0.132 μg/ml of water, gravel and roots surface respectively. The mean fluorescein concentration for the FTWs were 9.2, 1.1 and 0.06 μg/ml of root, gravel and freewater respectively, indicating that the highest total microbial activity in the FTW occurs in the biofilm associated with the root system. Mean viable microbial community 3.85 × 108, 3.7 × 107 and 1.3 × 107 cells/ml of root surface, water and gravel surface. Therefore, all the result suggested that active pollutant removal in all FTW stakes place in the root zone.
Introduction
FTWs that incorporate common wetland plants growing in a hydroponic condition on floating rafts offer a potential solution to the major problems faced to ponds and conventional treatment wetlands [1]. FTW innovation can be practiced at all levels, with very low expense in all types of water body with ordinary engineering.
Despite the potential advantages of FTWs for the treatment of various wastewaters, there has been little information published to date about their design, construction and performance [2] and only few researchers assessed how the system functions [3,4].
Biofilms play the key roles in wastewater treatment systems including in conventional constructed wetlands and ponds. In FTWs, biofilm can effectively grow in the hanging roots, floating mat, sediment and in the free-water column between the sediment and the rhizosphere. Although, efficient removal of pollutants by FTW system is reported by few researchers, the location where the pollutants are actively processed and removed in the system has not been investigated yet.
Different zones of the system should be compared with respect to microbial parameters so that it gives clear information for the design and implementation of FTWs. Biofilm formations in the system need to be quantitatively explained. The active microbial cells distribution in the floating system should be also clearly identified so that the factors enhancing the microbial activity could be identified. This information is crucial in designing and implementing FTWs, which has no general design hitherto. Therefore, the objective of this study was to evaluate and compare microbial activities in different zones of the FTWs employing emergent macrophytes.
Experimental set up
Six mesocosms were prepared from six buckets and one hundred liter influent tank was placed higher in the laboratory (Figure 1). The bottoms of every bucket were covered with gravels measuring about 2 Liters. Two pairs of suspending racks were prepared from white floater and several small holes were made to suspend the plants.
Two species of emergent macrophytes, Iris pseudacorus (IP) and Phragmites australis (PA), were selected. The macrophytes were placed on the suspending floater in such a way that the roots could grow suspended down to the water column. A pair of buckets was used as a control (without plant and floating mat). All of the mesocosms were prepared in duplicate. Twenty I. pseudacorus, twenty P. australis macrophytes were placed. The influent tank was filled with primary wastewater (taken from primary sedimentation pond). The system was adjusted to provide five days retention time for the influent. The set up was run for several months before biofilm sampling was done.
Nitrification and denitrification activity tests
The microbial activity tests were done as suggested by Halsey [5]. Known volume of gravel and root containing biofilm were sampled from each FTWs. For biofilms in the water column, about 10 ml of water sample was taken from each FTW. All the samples were placed in 250 ml flask; and 180 ml of phosphate buffer and 0.4 ml of 25 g/L ammonium sulphate were added. The flasks were placed on a rotary shaker for 5 minutes with the speed of 150 rpm and allowed to stand for approximately 5 minutes after removing from the shaker. Approximately 10 ml aliquot was filtered and half of the sample was used to measure initial NO 3 --N concentration. The other half of the filtrated samples were placed in a refrigerator at 4°C to be used as reference. The remnant gravel, root and water sample in the flask was incubated for 72 hours at room temperature. 10 ml samples were taken at different time intervals and NO 3 --N concentration was measured.
Potential Denitrification Activity test was done similarly but the substrate was 2 ml of 9 g/L sodium nitrate and 2 ml of 12 g/L glucose were added and the test was done under anoxic conditions.
Measurement of total microbial activity in the biofilm using FDA assay
Total microbial activity in the three compartments of the FTWs was estimated by Fluorescein diacetate (3',6'-diacetylfluorescein) hydrolysis [6,7].
For biofilm in the free-water column, 50-100 ml of water was filtered using 0.2 μm pore size polycarbonate membrane filter and carefully removed and placed into falcon tubes. Known volume of root and gravel were taken and placed in 50 ml of falcon tube in duplicate and then, 35 ml of 20 mM of phosphate buffer was added. Two control falcon tubes were prepared one without sample (C 1 ) and the other without FDA (C 2 ). To all tubes except C 2 , 0.35 ml of 1 mg/ml FDA was added. All tubes were incubated for 30-60 minutes at 38°C. Immediately after incubation, drops of acetic acid was added into the tubes and gently shaken with hand. The entire falcon tubes were span at 5000 rpm for 5 minutes and 5 ml at the top (aqueous) was filtered with 0.2 μm filter GF-C filter. The fluorescence was measured using spectrophotometer at a wave length of 490 nm. First, the absorbance was adjusted for the "C 1 " and "C 2 " and then, converted to concentration of fluorescein using calibration curve determined from known concentration of disodium fluorescein stock solution. The amount of fluorescein produced was determined per ml of water, gravel and root biofilm.
Estimation of physiologically active microbial community using ATP assay
Root and gravel samples were placed in 10 ml Milli-Q water whereas 10 ml of water sample was for water column biofilm. The samples were prepared in duplicate and handled carefully to avoid ATP contamination and biofilm disturbances. The biofilm was separated from the gravel and roots by treating with Bransonic sonicator for 3 minutes and this sample was used for extracellular and total ATP determination. Total ATP was measured from ultrasonicated sample where as extracellular ATP was measured after removal of viable cells by high speed centrifugation followed by filtering through 0.1 µm pore size filter.
1 ml of the supernatant and Milli-Q water (for control) were placed in eppendorf tubes and preheated 50 μL of Bac-Titer Glo TM reagent / luciferase (Promega Co., WI, USA) added and shaken and incubated for 1 minute at 38°C. The photon produced was measured as relative light unit (RLU) by luminometer (GloMax 20/20 R , USA). RLU was converted to ATP using standard calibration curve of known ATP concentration. ATP concentration was then transformed into carbon equivalents using a conversion factor (i) 1 ng/ml ATP=250 ng/ml cell carbon [8] (ii) 2.95 × 10 -9 nmol ATP=1 μm 3 biovolume [9]. The number of the living cells was then calculated from the living biomass with the assumption that one cell contains 20 fgC [8,9].
Specific microbial activity in the FTWs
Nitrification activity: In all of the FTWs, nitrification by the root associated biofilm started immediately after incubation and increased rapidly ( Table 1). The concentration increased from 14.68 µg/ml at the beginning to 212.8 µg/ml of root surface within 72 hours of incubation. This shows that the root biofilm contains enough nitrifiers to start nitrification process immediately after incubation, unlike the other biofilms which was delayed by several hours to start. Nitrification occurs only under aerobic conditions at dissolved oxygen level of 1 mg/L or more; and below 0.5 mg/L of dissolved oxygen, growth of microbes is low [10] and hence, nitrification rate is slow. Therefore, the sharply increasing nitrate concentration associated was probably due to the presence of the plants as they provide oxygenated zone around the rhizosphere and this virtually favored nitrifying bacteria.
There were considerable variations in nitrification efficiency among FTWs, which can be related to several determinant factors such as oxygen production capacity and the quantity and quality of biofilm on the surface of the root. Oxygen evolution from roots depends on plant species, root type, location and age, light and temperature. For example, reports showed that highest oxygen release occurs in T. latifolia, P. australis and I. pseudacorus [11]. This fact is supported by Table 1: Nitrate production through nitrification processes in the biofilms.
The slow nitrification rate and lesser nitrate accumulation is an indication that physiologically active nitrifying bacterial number in the water column was not enough to start nitirification rapidly in all of the wetlands and could nitrify. The delayed nitrification process in the gravel biofilm is an indication that the gravel contains less physiologically active microbes than the other compartments.
Denitrification activity: Average denitrification rate in the root, gravel and water for the FTWs over 72 hours of incubation varied between 0.09 and 0.19; 0.03 and 0.1; 0.02 and 0.24 µg NO 3 --N/ml/hour respectively. The variation among the compartments was tested by One-way ANOVA (unstacked) with post hoc comparison. The analysis showed that denitrification associated with the root biofilm was significantly varied (P<0.05) from the other two compartments ( Table 2).
Nitrate concentration declined from 9.56 µg/ml to 0.05 µg/ml root surface within 72 hours of incubation ( Table 2). The high denitirification rate and nitrate removal in the root zone of the FTW scan be associated with plant attributes to denitrifiers. Although denitrification predominantly takes place in the sediments of wetlands [12], recent studies showed significant contributions of denitrification taking place on periphytic communities attached to submerged macrophytes [13]. Decaying parts of the macrophytes provide suitable condition for denitrifying bacterial growth [13]. Although macrophytes oxygen supply through the roots can be inhibitory, the net effect depends on plant species, growth rate and total biomass. Nitrate concentration declined from 8.75 µg/ml to 1.04 µg/ml gravel surface within 72 hours of incubation. The nitrate removal in gravel within 24 hours of incubation was very slow which was similar to denitrification in the free-water biofilm, but unlike the denitrification by free-water biofilm, the concentration rapidly declined and reached limiting concentration in 9 more hours of incubation. Compared to denitrification due to free-water biofilm, it was rapid and much better in terms of nitrate transformation capacity.
The low denitrification rate in the water column biofilm shows that physiologically active denitrifying bacteria suspended in the free-water column was low at the beginning and hence, until the denitrifiers multiply and become physiologically active, denitrification rate was low. This could be due to the influence of continuous aeration from the atmosphere into the water column, which may inhibit denitrifiers. The water column may not give diversified microhabitats like the rhizosphere so that the denitrification could be inhibited.
Total microbial activity
Total microbial activity in different zones of FTWs was measured as FDA hydrolysis. The mean hydrolysis product of the FDA, fluorescein concentration, varied from 7.7-11.3 μg/ml, 0.78-0.97 μg/ml and 7.5-12.7 μg/ml of root, gravel surfaces and water respectively ( Figure 2). One-way ANOVA with Tukey's post hoc test showed that the FDA hydrolysis in the root zone was significantly varied from the gravel and free-water compartments in all of the FTWs (P<0.05) but there were no statistically significant differences among the FTWs (P=0.36). The higher FDA hydrolysis associated with root biofilm was due to the fact that the roots of plants can create conducive microhabitat for a wide array of microbial communities by providing oxygen, optimized pH media and surface area for attachment. The variation in the microbial activity among different FTWs could be attributable to the specific plant species used because of their differences in the quality and quantity of metabolites they produce, capacity to release oxygen and other associated conditions. emergent macrophytes since the floating mat hampers light entrance and this enhances the growth of heterotrophic and anaerobic microbial-dominated biofilm community. There was higher fluorocein hydrolysis in the control than the FTWs and this may be due to the fact that light can reach to the surface so that it gives favorable conditions for diversified microbial community growth.
Microbial activity in the bulk water was low compared to the gravel and the root zone may be due to the rarity of suitable attachment site in the free-water zone.
The higher ATP molecule in the root zone (Table 3) is an indication of the presence of higher active bacterial biomass in the root biofilm. The dominant physiologically active microbial community associated with the root biofilm is possibly due to the fact that the root zone provides conducive microhabitats for biofilm formation, growth and its optimum activity. Plant species vary in the quality and quantity of organic substances they provide, surface area for microbial attachment and the capacity to oxygenate the medium [14].
The low number of active microbial community associated with gravel biofilm could be due to the influence of the surface cover by floating rack and the short time for the gravel biofilm to establish itself at the bottom. Active microbial community in the FTWs was much higher than the control, in fact this indicates the presence of vegetation, not only affect the root zone but also it influences the whole system in the FTWs.
In the present study, although the proportion of the root in each FTW was low (9%), it accounted more than half (59%) of the total physiologically active microbial cells in the system. Highest active microbial cell numbers (high intracellular ATP) is an indication of enhanced bacterial activity in the system.
Conclusion
• Evidences from the FDA hydrolysis for all of the FTWs showed that total microbial activity associated with the root biofilm was much higher than the other two compartments. • Denitrification and nitrification activity tests conducted for all of the FTWs also confirmed that the rate of nitrogen transformation in the root associated biofilm was higher than other compartments. • ATP assay confirmed that intracellular ATP concentration and estimated number of physiologically active bacterial cells was much higher in the root biofilm than the other compartments. Therefore, it is reasonable to conclude that in FTWs active pollutant transformation takes place primarily associated with the root biofilm. | v3-fos-license |
2020-11-19T09:17:54.974Z | 2020-11-01T00:00:00.000 | 227039422 | {
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} | pes2o/s2orc | A Molecular Perspective on Sirtuin Activity
The protein acetylation of either the α-amino groups of amino-terminal residues or of internal lysine or cysteine residues is one of the major posttranslational protein modifications that occur in the cell with repercussions at the protein as well as at the metabolome level. The lysine acetylation status is determined by the opposing activities of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), which add and remove acetyl groups from proteins, respectively. A special group of KDACs, named sirtuins, that require NAD+ as a substrate have received particular attention in recent years. They play critical roles in metabolism, and their abnormal activity has been implicated in several diseases. Conversely, the modulation of their activity has been associated with protection from age-related cardiovascular and metabolic diseases and with increased longevity. The benefits of either activating or inhibiting these enzymes have turned sirtuins into attractive therapeutic targets, and considerable effort has been directed toward developing specific sirtuin modulators. This review summarizes the protein acylation/deacylation processes with a special focus on the current developments in the sirtuin research field.
Introduction
Proteins are the structural and functional base of all living organisms. To date, the number of proteins that comprise the human proteome is still elusive. The analysis of the human genome shows the existence of approximately 25,000 protein-coding genes [1]. This would suggest the existence of the same number of proteins. Interestingly, to date, more than 90,000 different human proteins have been identified. This discrepancy has been attributed to three district mechanisms: the alternative splicing of precursor mRNAs, single amino acid polymorphisms (SAPs) and posttranslational modifications (PTMs) [2][3][4]. Together, these modifications raise the complexity of the proteomes by two to three orders of magnitude and help to explain the discrepancy between the complexity of vertebrate organisms and the sizes of their encoded genomes [2,5].
There are two general categories of protein PTM: the covalent cleavage of specific peptide bonds from a protein backbone, which can occur by autocatalytic cleavage or can be catalyzed by a group electrophilic fragment of a co-substrate, to a nucleophilic side-chain residue from the protein. The last reaction can be reversible or irreversible and can be enzymatically or non-enzymatically catalyzed.
The variability that these PTMs bring to proteins is further amplified by the existence of complex regulatory networks involving both positive and negative crosstalk between the different PTMs. This complex PTM crosstalk is the basis of a protein modification code and has significant importance in the regulation of cellular functions [5,[12][13][14].
The function of protein acylation reactions depends on the acyl group that is attached. It was proposed that the small acyl groups (formyl and acetyl) may function as recognition elements for protein-protein interactions, while long chains of fatty acids can target proteins to membranes and affect signal transduction [19].
The aim of this review is to provide a global overview of the mechanisms underlying both the acylation ( Figure 1) and deacylation ( Figure 2) of proteins, focusing on a special class of NAD +dependent protein deacylases named sirtuins.
The acyl group is generally attached to the side chain of an internal amino acid residue, but acylation can also occur in the protein N-terminus.
N-Terminal Acylation
The known examples of N-terminal acylation are the N-myristoylation of glycine, the Npalmitoylation of cysteine, and N-terminal acetylation [19]. Among those, the most common and best characterized is N-terminal acetylation [20].
N-Terminal Acetylation
In eukaryotes, the acetylation of an α-amino group of the N-terminus is an irreversible covalent modification catalyzed by N-terminal-acetyltransferases (NATs) that occurs co-translationally in more than 80% of all proteins [21,22]. N-terminal acetylation neutralizes the positive charge of the free amino group and blocks the N-terminus from further modifications. This modification influences several protein characteristics such as folding, lifetime and degradation, subcellular localization, interactions and complex formation [23,24]. Eukaryotes possess different NATs with different substrate specificities (NatA, NatB, NatC, NatD, NatE and NatF) [25]. Depending on the NAT isoform, the acetyl group may be transferred to the non-cleaved initial methionine residue of the nascent polypeptide chain, or the reaction may occur after the excision of the methionine residue by the ribosomally bound methionine aminopeptidases, and the acetyl group is added to the residue that is positioned immediately after the excised methionine.
The acyl group is generally attached to the side chain of an internal amino acid residue, but acylation can also occur in the protein N-terminus.
N-Terminal Acylation
The known examples of N-terminal acylation are the N-myristoylation of glycine, the N-palmitoylation of cysteine, and N-terminal acetylation [19]. Among those, the most common and best characterized is N-terminal acetylation [20].
N-Terminal Acetylation
In eukaryotes, the acetylation of an α-amino group of the N-terminus is an irreversible covalent modification catalyzed by N-terminal-acetyltransferases (NATs) that occurs co-translationally in more than 80% of all proteins [21,22]. N-terminal acetylation neutralizes the positive charge of the free amino group and blocks the N-terminus from further modifications. This modification influences several protein characteristics such as folding, lifetime and degradation, subcellular localization, interactions and complex formation [23,24]. Eukaryotes possess different NATs with different substrate specificities (NatA, NatB, NatC, NatD, NatE and NatF) [25]. Depending on the NAT isoform, the acetyl group may be transferred to the non-cleaved initial methionine residue of the nascent polypeptide chain, or the reaction may occur after the excision of the methionine residue by the ribosomally bound methionine aminopeptidases, and the acetyl group is added to the residue that is positioned immediately after the excised methionine.
Acylation of Internal Lysine Residues
The acylation of internal lysine residues is a reversible and highly regulated PTM that targets many proteins from different cellular compartments, such as the mitochondria, cytosol, nucleus and lumen of the endoplasmic reticulum (ER) [26][27][28]. The target proteins are involved in distinct cellular processes, such as the cell cycle, nuclear transport, chromatin remodeling, mRNA splicing, actin nucleation, signal transduction [26], protein homeostasis and autophagy [27,28].
Although protein acylation can be an activating or inhibitory modification, recent metabolic studies have suggested that, in mitochondria, acylation has a consistently inhibitory role [29,30] Protein acylation may occur by two distinct mechanisms: it may be catalyzed by acyl-specific transferases that transfer acyl groups from acyl-coenzyme A (acyl-CoA) to the amino acid residues [24,[31][32][33], or it may occur non-enzymatically due to the intrinsic reactivity of acyl-CoA thioesters [29,[33][34][35].
The first evidence of acylation as a PTM came in 1964 when lysine acetylation was discovered as a PTM of histones [36]. The evidence for the addition of other acyl groups as a PTM was only recently discovered, and therefore, the function and mechanisms behind them are still elusive [7].
Acetylation of Internal Lysine Residues
Similar to terminal acetylation, the addition of an acetyl group to an internal lysine residue increases the size of its side chain and neutralizes the positive charge of its amino group. This modification will raise the protein hydrophobicity and consequently induce significant conformational changes that will ultimately affect several protein properties, including transcriptional activity, DNA-protein interactions, subcellular localization, enzymatic activity, folding, peptide-receptor recognition and protein stability [15].
Enzymatic Nε-lysine acetylation has been deeply studied and requires an acetyl group donor (acetyl-CoA), an acetyl group acceptor (the ε-amino group of an internal lysine residue from a polypeptide chain) and an acetyl-CoA:lysine acetyltransferase (lysine acetyltransferase (KAT)) to catalyze the acetyl exchange. The mechanism behind the enzymatic addition of other acyl groups to Nε-lysine requires further studies [29].
The particular biochemical properties of the mitochondria, namely, its pH and metabolite concentrations, are proposed to favor the occurrence of other non-enzymatic acylation reactions, namely, succinylation, malonylation and glutarylation [29].
S-Palmitoylation of Cysteine Residues
The reversible attachment of palmitic acid to an internal cysteine residue (S-palmitoylation) is the most common type of protein fatty acylation in eukaryotic cells [43][44][45]. The attachment of the palmitic acid to the protein is catalyzed by palmitoyl transferases [46,47], and its removal is mediated by palmitoyl thioesterases.
The palmitoyl transferases belong to the zinc-finger DHHC domain-containing protein family that is characterized by the presence of a conserved aspartate-histidine-histidine-cysteine (DHHC) cysteine-rich domain [46,47]. They are integral membrane proteins, with four to six transmembrane domains and the N and C termini present in the cytosol [48], a characteristic that has made difficult their purification and consequently their identification.
In spite of the recent advances in the field, there are still many questions regarding the functioning of DHHC enzymes. To date, their selectivity, their forms of substate selection and the identification of palmitoylation sites are still elusive [44].
The Influence of Protein Acylation in Human Health and Disease
The attachment of acyl groups to positively charged lysine residues neutralizes the positive charge and consequently affects the protein's physicochemical properties, ultimately affecting its stability, function, catalytic activity, protein-protein and protein-DNA interactions, degradation and subcellular location [43,44,49].
Many of these post-translationally acylated proteins have relevant roles in vital physiological processes including gene transcription, cell division, cytoskeleton organization, DNA damage repair, DNA replication, signal transduction, protein folding, autophagy, apoptosis, lipid storage and breakdown, mitochondrial fission and fusion, protein synthesis, ion transport, redox and metabolism regulation. [49] Additionally, protein acylation has also been related to the protein aggregation process, which is implicated in several neurological pathologies such as amyotrophic lateral sclerosis [50] and Alzheimer's disease [51].
Therefore, the correct functioning and regulation of the enzymes involved in protein acylation (and deacylation) are essential for human health, and their misregulation has been associated with several pathologies.
In spite of its evident therapeutic potential, the inhibition of KATs has not been widely explored. A recent study described the development of A-485, a potent and selective catalytic inhibitor of p300 and CREB-binding protein (CBP) that competes with the substrate acetyl-CoA. The compound selectively inhibited cell proliferation in lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer [59].
Another study reported the development of two highly potent and selective inhibitors of KAT6A and KAT6B named WM-8014 and WM-1119, respectively, that are reversible competitors of acetyl-CoA. The inhibition of KAT6A and KAT6B inhibited the growth of lymphoma in mice [60].
Detailed knowledge about the catalytic activity of the different KATs would be important for identifying potent and selective inhibitors for other KATs that would enable obtaining deep knowledge about their biological functions and exploring their therapeutic potential.
Reversible S-palmitoylation is a dynamic process that has several important functions in subcellular protein trafficking, in protein stability (by the prevention of ubiquitination and subsequent degradation) and in the modulation of protein interactions (adhesion and signaling), but its most studied function is its capability to increase the affinity of soluble proteins for lipophilic membranes [61].
The alteration of DHHC expression and the consequent palmitoylation impairment have been associated with several cancers (for example, leukemia, colorectal, hepatocellular and non-small-cell lung cancers) [44,62,63] and with several other disease states resulting from organ-specific processes. One of the most studied cases is the relation between palmitoylation and neuronal functions. There are many reports relating defects in palmitoylation regulation or in the enzymes responsible for palmitoylation and depalmitoylation processes with several neurological disorders such as Alzheimer's, Parkinson's or Huntington's disease, schizophrenia and intellectual disability [44,64].
Protein Acylation and "Carbon Stress"
It has been recently suggested that an increase in the concentration of reactive carbon metabolites, resulting from physiological or pathological situations, can culminate in the abnormal occurrence of non-enzymatic protein acylation reactions [29]. This situation can have a detrimental effect on protein function and ultimately disrupt cellular homeostasis. In these scenarios, the non-enzymatic protein acylations are considered a form of "carbon stress" [29].
Recently, it has been found that under carbon stress situations that lead to an increase in protein acylation, a particular group of deacylases, named sirtuins (Sirts), are called to intervene, and, in some conditions, their expression can be upregulated. The sirtuins are part of a protein quality control response that is vital for reducing protein acylation, ensuring protein quality control and consequently reducing carbon stress. An increase in carbon stress and/or the impairment of the carbon stress response will reduce protein quality control and ultimately reduce protein function, leading to several age-related diseases such as neurodegeneration, diabetes, cardiovascular disease or cancer [18,[65][66][67][68][69][70].
The hydrolysis of acyl groups from acyl-lysine residues is catalyzed by a group of enzymes named lysine deacetylases (KDACs).
The human genome encodes a total of 18 KDACs that can be grouped into two categories or superfamilies: the Zn 2+ -dependent KDACs or histone deacetylases (HDACs) and the NAD + -dependent sirtuin deacetylases ( Figure 2).
It should be noted that although grouped in the same family, some KDACs have different acyl selectivities [80][81][82][83], and others are thought to have poor or no deacetylase activity [84].
Zn 2+ -Dependent KDACs
The Zn 2+ -dependent KDACs (or HDACs) are usually called the classic KDACs and account for 11 of the 18 KDACs. They share a highly conserved deacetylase domain and, based on their phylogenetic conservation and in-sequence similarities, can be divided into four classes, named classes I, IIa, IIb and IV ( Figure 2). They differ in their enzymatic function, structure, expression patterns and subcellular localization. Classes I and IV are nuclear, class IIb is cytoplasmic, and class IIa is primarily nuclear but upon the activation of signaling is exported to the cytoplasm [84]. Some in vitro experiments suggest that vertebrate class IIa KDACs show poor catalytic activity, which may be related with the replacement of a conserved tyrosine by a histidine in the catalytic pocket [85].
These enzymes exist in multiprotein complexes that determine their substrate specificity, and their catalysis usually generates deacetylated lysine and acetate [49,84,86].
NAD + -Dependent Sirtuin Deacetylases
The remaining seven KDACs are named sirtuins and unlike the classic KDACs, they require the cofactor NAD + as a co-substrate [87].
The name sirtuin came from the family's founding member, the silent information regulator 2 (Sir2) from Saccharomyces cerevisiae [88].
They comprise the class III family of KDACs and have seven members (Sirt1-Sirt7). Sirtuins are evolutionarily conserved in all domains of life, and, based on their sequence similarity, they are classified into five classes (I-IV and U) (Figure 2). The seven mammalian sirtuin genes are included in classes I to IV: Sirt1, Sirt2 and Sirt3 are in class I, Sirt4 in class II, Sirt5 in class III, and Sirt6 and Sirt7 in class IV (Figure 2) [89]. Sirt1 has the highest sequence homology to Sir2 in yeast, and it is the most studied sirtuin. The different sirtuins are distinguished by their subcellular localization, acyl lysine substrate specificity, enzymatic activity, and biochemical and metabolic functions. Their impairment is related to different metabolic and health issues [90].
Although deacylation is the main activity catalyzed by the sirtuin enzymes, there is evidence that some sirtuins (Sirt1, 4 and 6) can also ADP-ribosylate protein substrates [91].
Sirtuin Structure
Sirtuin proteins contain a conserved catalytic core domain composed of approximately 275 amino acid residues flanked by N-and C-terminal regions with a variable sequence and length [78,92]. The catalytic core domain shows a high degree of structural superposition among the different sirtuins. It adopts an elongated shape containing a classical open α/β Rossmann-fold structure that is characteristic of NAD + /NADH-binding proteins, and a smaller globular domain composed of two insertions in the Rossmann fold. One of these insertions binds a structural zinc ion that is coordinated with four conserved cysteine residues [93]; the other insertion is a helical module (Figure 3a).
Between the two domains exists a deep cleft where the enzyme active site is located and where both NAD + and acetyl-lysine substrates bind [90]. It was proposed that when the peptide containing the acylated lysine binds to the enzyme's cleft, its main chain establishes β sheet-like interactions with two flanking strands. One of those strands is positioned in the Rossmann fold. The other one is located in a loop that contains a highly conserved FGExL motif, and is positioned between the Rossmann fold and the Zn 2+ -binding module [94] (Figure 3b). The formation of this so-called "β staple" interaction, in which the substrate links the Rossmann fold and the Zn 2+ -binding module, inserts the acetyl-lysine side chain into a conserved, mainly hydrophobic tunnel and consequently changes the enzyme´s conformation from an open to a closed conformation [93,95]. The closed conformation of the enzyme facilitates the correct binding of NAD + inside a conserved hydrophobic C pocket that is adjacent to the acyl-lysine-binding tunnel. This binding order is important because the occupation of the acetyl-lysine-binding tunnel seems to restrict the binding conformation of NAD + and force it to adopt a "productive conformation" in which its adenine ring forms extensive hydrogen bonds and van der Waals interactions with the Rossmann-fold domain, and its nicotinamide ring is inserted into the C pocket ( Figure 3c) structure that is characteristic of NAD + /NADH-binding proteins, and a smaller globular domain composed of two insertions in the Rossmann fold. One of these insertions binds a structural zinc ion that is coordinated with four conserved cysteine residues [93]; the other insertion is a helical module (Figure 3a). Between the two domains exists a deep cleft where the enzyme active site is located and where both NAD + and acetyl-lysine substrates bind [90]. It was proposed that when the peptide containing the acylated lysine binds to the enzyme's cleft, its main chain establishes β sheet-like interactions with two flanking strands. One of those strands is positioned in the Rossmann fold. The other one is located in a loop that contains a highly conserved FGExL motif, and is positioned between the Rossmann fold and the Zn 2+ -binding module [94] (Figure 3b). The formation of this so-called "β staple" interaction, in which the substrate links the Rossmann fold and the Zn 2+ -binding module, inserts the acetyl-lysine side chain into a conserved, mainly hydrophobic tunnel and consequently changes the enzyme´s conformation from an open to a closed conformation [93,95]. The closed conformation of the enzyme facilitates the correct binding of NAD + inside a conserved hydrophobic C pocket that is adjacent to the acyl-lysine-binding tunnel. This binding order is important because the occupation of the acetyl-lysine-binding tunnel seems to restrict the binding conformation of NAD + and force it to adopt a "productive conformation" in which its adenine ring forms extensive hydrogen bonds and van der Waals interactions with the Rossmann-fold domain, and its nicotinamide ring is inserted into the C pocket (Figure 3c) [95,96].
Substrate Specificity of Sirtuins
A few studies demonstrate that sirtuins show a high level of substrate specificity for certain acetylation sites in specific substrates [98][99][100]. Accordingly, substrate recognition by sirtuins is affected by differences in the sirtuins' binding clefts, by the subcellular localization and by some particular characteristics of the substrate, namely, the acylated residue, the attached acyl group, the three-dimensional structure of the substrate, the substrate sequence and the in vivo interactions of the substrate.
The Influence of Sirtuin Structure
The sirtuin's active site is positioned in a cleft composed of the Rossmann-fold domain, the Zn 2+ -binding domain and the four loops that connect the two domains. This cleft is the region of the enzyme that contains the highest sequence conservation within the different sirtuins [98].
The non-Zn 2+ -binding module from the small Zn 2+ -binding domain is the area that shows more variability, either in the primary sequence or in the secondary and tertiary structures among the different Sir2 homologues. This observation suggests that this domain may be involved in the sirtuin's substrate specificity. Additionally, it has also been observed that the small Zn 2+ -binding domains of the archaeal and bacterial sirtuins have a similar overall topology, while, in eukaryotic sirtuins, they show a higher secondary structure variability [90]. This difference may reflect the higher number of sirtuins that are expressed in eukaryotes and that are required to distinguish a greater number of substrates [101].
Other regions distanced from the active site may also be involved in the sirtuin's discrimination between different substrates [90].
The Influence of the Subcellular Localization of Sirtuins
The different cellular compartments have different proteins; therefore, the distinct subcellular localizations of the seven sirtuins play an important role in their substrate specificities.
Sirt1 is localized predominantly in the nucleus but was also found in the cytoplasm [102]. Sirt2 is mainly cytosolic but was also found in the nucleus [103]. Sirt3 is predominantly localized in the mitochondrial matrix [104], but there is evidence that it moves from the nucleus to mitochondria during cellular stress [105]. Sirt4 was only detected in the mitochondrial matrix. Sirt5 is predominately mitochondrial but is also active in the cytosol [106]. Currently, Sirt6 and Sirt7 are thought to be nuclear.
The Influence of the Acylated Residue
The existing data suggest that sirtuins only have deacylation activity on acylated lysine residues [107]. However, since our knowledge of their activity is still very limited, the possibility that they can also deacylate other residues should not be excluded.
The Influence of the Acyl Group
In vitro studies showed that only class I sirtuins (Sirt1, 2 and 3) have robust deacetylase activity, although they could also remove long-chain fatty acyl groups. Sirtuins 4-7 have preferences for longer acyl chains [108,109].
Many of these studies were performed in vitro and, therefore, require in vivo validation. The recent development of new methodologies for exploring protein acylation in vivo may bring a new breath of life to this field [19,112].
The Influence of Protein Structure
Previous studies suggested that sirtuins deacetylate lysine residues located in regions without a defined secondary structure or in loop regions of the substrate proteins [113]. Recent studies demonstrated that sirtuins are also able to deacetylate lysine residues located in structured or rigid regions of a protein [114]. One of the major limitations in this field is the difficulty of obtaining the co-crystallized structures of sirtuins with natively folded proteins. Indeed, most studies use small peptides containing acetylated lysine residues to study the enzyme-substrate interaction, an approach that may preclude some important information or even skew the conclusions. To get a full picture of the enzyme-substrate interaction, it would be necessary to increase the number of studies using a structural and functional approach in a context of site-specifically acetylated full-length and natively folded substrate proteins.
The Influence of Protein Sequence
Structural data show that during catalysis, the side chains of the residues preceding and proceeding the acyl lysine interact with both the Rossmann fold and Zn 2+ -binding module in a "β staple" interaction, as described in the previous section. In order for this enzyme-substrate interaction to be possible and energetically favorable, the side chains of the substrate residues that interact with the enzyme must be chemically and geometrically compatible with the residues that compose the binding cleft of each sirtuin. Because the binding cleft from each sirtuin presents some specific features that distinguish it, the existence of some sequence similarities among the substrates preferentially catalyzed by a given sirtuin would be expected [98,115]. Although some studies have suggested that some sirtuins have a preference for certain amino acid residues in determined sequence positions [98][99][100]114], the results between different studies are not always convergent in their conclusions. A study that performed an analysis of sequences of biochemically confirmed substrates for Sirt2 or Sirt3 concluded that there was no clear consensus in their sequence [93]. On the other hand, some data suggest that the deacetylase activity of Sirt6 is sequence-dependent [110]. In a recent review, it was suggested that this chemical interaction between the enzyme-binding pocket and the substrate residues flanking the acyl lysine could be more important for substrates whose acyl groups show weaker binding affinities for the sirtuin. For acyl groups that bind more tightly to the enzyme´s catalytic pocket, the chemical contribution of those substrate residues would be less important [93].
The Influence of Protein Interactions
Under physiological conditions, sirtuins can interact with other proteins or with DNA, and those interactions influence the sirtuin's substrate specificity. For example, Sirt1 and Sirt3 bind so tightly to their substrate proteins p53 and AceCS2, respectively, that they coimmunoprecipitate [116,117]. In other cases, the recruitment of a sirtuin for a given substrate or vice versa may be mediated by the interaction with other proteins (e.g., transcription factors). For example, Sirt6 and Sirt7 bind to certain transcription factors, which recruit them to different chromatin regions, where they catalyze the deacetylation of a specific histone at specific target genes [118][119][120].
Catalytic Mechanism of Sirtuin Deacylation
According to the existing data, sirtuins are catalytically active only when the peptide containing the acylated lysine is correctly positioned inside the binding tunnel, the enzyme is in the closed conformation, and NAD + has its nicotinamide ring inside the hydrophobic C pocket and the α face of its N-ribose ring exposed to the acetyl lysine carbonyl group [95,96].
Although it is well established that the general sirtuin catalytic mechanism proceeds in two consecutive stages (Figure 4), the details about the chemistry involved in the generation of each reaction intermediate are still not consensual [121,122]. In Stage I occurs the ADP-ribosylation of acetyl lysine, which involves the cleavage of the nicotinamide moiety of NAD + and the nucleophilic attack of the side chain of the acetylated lysine from the protein substrate to form a positively charged ADP-ribosyl-peptidylimidate (or C1′-Oalkylamidate) intermediate and nicotinamide. It was proposed that this reaction occurs through a highly dissociative and concerted displacement mechanism [121]. This reaction is reversible, so NAD + can be resynthesized when nicotinamide concentrations are elevated in solution.
Computational and experimental evidence has shown that Stage II starts with the deprotonation of the 2′-OH group by a conserved histidine residue that acts as a general base (Step 1 from Figure 4). This facilitates the intra-molecular nucleophilic attack of the 2′ hydroxyl onto the positively charged iminium carbon and culminates in the formation of a bicyclic intermediate. In the second step, which is the reaction-limiting step, occurs the collapse of the bicyclic intermediate, in the presence of a water In Stage I occurs the ADP-ribosylation of acetyl lysine, which involves the cleavage of the nicotinamide moiety of NAD + and the nucleophilic attack of the side chain of the acetylated lysine from the protein substrate to form a positively charged ADP-ribosyl-peptidylimidate (or C1 -O-alkylamidate) intermediate and nicotinamide. It was proposed that this reaction occurs through a highly dissociative and concerted displacement mechanism [121]. This reaction is reversible, so NAD + can be resynthesized when nicotinamide concentrations are elevated in solution.
Computational and experimental evidence has shown that Stage II starts with the deprotonation of the 2 -OH group by a conserved histidine residue that acts as a general base (Step 1 from Figure 4). This facilitates the intra-molecular nucleophilic attack of the 2 hydroxyl onto the positively charged iminium carbon and culminates in the formation of a bicyclic intermediate. In the second step, which is the reaction-limiting step, occurs the collapse of the bicyclic intermediate, in the presence of a water molecule, generating a tetrahedral intermediate (Steps 2a and 2b in Figure 4). In the third step, there is a proton transfer from the positively charged histidine to the amino group of the tetrahedral intermediate (Step 3 in Figure 4). In the fourth step occurs the breakdown of the tetrahedral intermediate into the reaction products: the deacylated lysine and the 2 -O-acetyl-ADP-ribose (2 -AADPR) (Step 4 in Figure 4) [122,123], which can be non-enzymatically isomerized to 3 -O-acetyl-ADP-ribose.
The reaction products are a mixture of 2 -and 3 -O-acetyl ADP-ribose, nicotinamide and the deacetylated peptide [92,124,125].
The Influence of Sirtuins in Human Health and Disease
The sirtuins are a family of enzymes that target different proteins, including histones, transcription factors or proteins involved in DNA repair. Their variable subcellular distribution and the variability of the substrates allows them to control several vital molecular pathways that are involved in cell survival, neuronal signaling, energy metabolism, tissue regeneration, DNA repair, inflammation or circadian rhythms [94,103,118,119,[126][127][128][129] Caloric restriction is the only effective way to naturally extend lifespan and eventually health span in several organisms including humans [130].
Several studies suggest that caloric restriction increases the expression levels of sirtuins, with the exception of Sirt4 [131][132][133] This relation between sirtuin activation and increased lifespan has suggested that sirtuins may have a role in the beneficial effects elicited by a caloric restriction diet [134]. This assumption has boosted the search for potent sirtuin-activating compounds (STACs) [135].
The abnormal activity and/or expression of several sirtuins has been correlated with several cancer types. Several studies suggest that Sirt1 can act both as a tumor promoter and as a tumor suppressor [136,137]. It regulates many tumor suppressors and DNA repair genes, and its upregulation was corelated with a higher chance of being resistant to chemotherapy [138].
Sirt2, similarly to Sirt1, also has a regulatory function, and it has been suggested that it can act both as a tumor promoter and as a tumor suppressor [139].
Sirt3 was shown to act as a tumor suppressor by inhibiting glycolysis metabolism through the deacetylation and consequent activation of pyruvate dehydrogenase [140], but it is also possible that it can act as a tumor promoter in some situations.
Sirt4 was correlated with the inhibition of the progression of colorectal cancer, and its underexpression was correlated with a worse prognosis [141].
The overexpression of Sirt5 in non-small-cell lung cancer tissues was found to be a marker of low survival [142].
Sirt6 showed a controversial role in several cancers. A reduction in Sirt6 expression was correlated with tumor progression with a poor clinical outcome. Its overexpression was shown to promote oncogenic activity in solid and in hematologic tumors [143].
The overexpression of Sirt7 has been associated with aggressive cancers and low survival, whereas its depletion has been associated with a less aggressive phenotype [120].
Sirt1 is the most studied sirtuin. Alterations of the level of Sirt1 expression were associated with the outcomes of several metabolic and neurodegenerative diseases, cancer and aging. A reduction in Sirt1 expression has been related to cardiovascular and neurodegenerative diseases such as Alzheimer's and Parkinson's, and with some metabolic diseases such as obesity and diabetes [144][145][146][147][148]. It has been proposed that the downregulation of Sirt1 along with the disease progression may result from the concomitant increase in oxidative stress and inflammation [146,149].
Some age-related diseases and endocrine system dysfunctions are associated with an increase in Sirt1 expression, albeit with a decrease in its activity. It has been hypothesized that in these cases, the increase in Sirt1 expression is a way to compensate for the decline in Sirt1 activity [144].
The current knowledge about the relationships between sirtuins and certain health and disease conditions strongly suggests that the development of molecules capable of selectively activating each human sirtuin variant may bring health benefits through the stimulation of its anti-inflammatory, cardio-protective, neuroprotective and anti-tumor activities. On the other hand, the relationship between the overexpression of some sirtuin variants and the proliferation of certain cancer cells and the development of some metabolic disorders also suggests that their selective inhibition would also be beneficial in certain disease conditions. Therefore, several activators of Sirt1 and inhibitors of Sirt1 and Sirt2 have been developed and are actually in clinical trials. This issue has been deeply explored in a recent review [150].
Conclusions
Although it is now evident that protein acylation is a complex PTM that embraces the addition of a wide range of different acyl groups to specific protein residues, both the acylation and the deacylation processes require deeper investigation. The major limitation of previous studies is the lack of efficient methodologies that are capable of providing an unbiased identification of the acylated proteins, their acylation modification sites and the specific acyl modifications in vivo. As a significant portion of the current knowledge about these mechanisms arose from in vitro studies, it is important to validate those results in vivo both to prove their biological occurrence and to correlate those modifications with their function in the cell and ultimately in the organism.
The correct functioning and regulation of the enzymes that catalyze the addition and the removal of the acyl groups are of major importance for a variety of metabolic processes, and their impairment has already been related to several human pathologies, including neurodegeneration [64,151,152], cancer [62,63,153,154] and cardiovascular diseases [155,156].
The seminal discovery that the upregulation of the Sir2 gene was able to increase the replicative lifespan of yeast [79,157] has sparked great interest in sirtuin biology. From there on, several studies have shown that sirtuins play critical roles in epigenetics, cell death and lifespan regulation [58,127,128,158] and that their abnormal activity is implicated in several diseases, such as cancer, neurodegenerative disorders, obesity and diabetes [129,159].
Recently, it was found that increasing their activity was associated with the delay of some age-related cardiometabolic diseases [160] and could even increase longevity [161][162][163][164]. These findings have turned sirtuins into attractive therapeutic targets, and considerable effort has been directed toward developing specific sirtuin activators and inhibitors. A deep knowledge of sirtuin enzymatic activity and allosteric regulation is imperative for the development of highly specific mechanism-based sirtuin modulators.
The determination of the catalytic mechanisms of acylation and deacylation by each enzyme, and the identification of the specificities that distinguish them among the members of the same families would be of great importance for improving the current knowledge about these enzymes and the associated enzymatic processes. The knowledge of the transition state structures of the rate-limiting steps of each reaction, with atomistic detail, would provide a promising approach for the design of potent and specific molecules with activating or inhibitory characteristics.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. | v3-fos-license |
2020-12-10T09:03:21.993Z | 2020-12-09T00:00:00.000 | 229174606 | {
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} | pes2o/s2orc | Circ_0005198 enhances temozolomide resistance of glioma cells through miR-198/TRIM14 axis
Circular RNAs (circRNAs) are associated with chemoresistance in many cancers. However, the function of circ_0005198 in the temozolomide (TMZ) resistance of glioma has not been well elucidated. Here, we demonstrated that circ_0005198 was considerably up-regulated in glioma tissues, serum samples and TMZ-resistant glioma cells. Silencing of circ_0005198 restrained TMZ resistance, restricted the proliferation and facilitated the apoptosis of TMZ-resistant glioma cells. MiR-198 could be sponged by circ_0005198, and we demonstrated that the effect of circ_0005198 on the progression of TMZ-resistant glioma cells was attributed to the inhibition of miR-198 activity. Moreover, TRIM14 was a target of miR-198 and silencing of TRIM14 hindered TMZ resistance and suppressed the progression of TMZ-resistant glioma cells, while TRIM14 over-expression rescued the inhibiting effect of miR-198 over-expression. We conclude that circ_0005198-miR-198-TRIM14 regulatory pathway is critical to TMZ resistance of glioma.
INTRODUCTION
Glioma originates from the neuroglial stem or progenitor cells and is a malignancy with strong aggressiveness [1]. With the advancement of surgeryrelated technologies and neoadjuvant chemical therapy, the mortality rate of glioma has considerably decreased, and the prognosis has improved [2]. Unfortunately, prognosis remains poor for patients enhancing resistance to chemotherapeutic drugs. Increasing evidence has shown that glioma stem cells (GSCs) account for tumor proliferation even after chemotherapy [3]. For this reason, delving into the molecular pathogenesis of glioma and the mechanism of chemoresistance acquisition will facilitate the advancement of personalized medicine as well as targeted therapies. Circular RNAs (circRNAs) refer to one type of endogenous non-coding RNAs and present in the form of closed loops which cannot code protein [4]. Existing researches reported that circRNAs participate in a wide variety of physiological and pathophysiological procedures (e.g., concocting modulating alternative splicing, sponging microRNA (miRNA), as well as regulating the interactions and genetic expression of protein-RNA) [5][6][7]. Moreover, abnormally expressed circRNAs are implicated with the progression of various tumors [8,9]. For instance, circ_001680 could facilitate colorectal carcinoma proliferation and AGING migration and mediate its chemoresistance [10]. Moreover, circRBM33 could regulate IL-6 to facilitate gastric cancer progression [11]. Circ_0005198 was mapped to chr13:25072253-25077915 with a spliced length of 592 nt and involved in the regulation of invading, migrating, apoptotic, and proliferating processes in glioma [12]. However, its effect in the chemoresistance of glioma is still worth further exploration.
Recent data has also demonstrated the key effect of circRNAs in the development of cancers by sponging microRNAs and regulating RNA binding proteins [13,14]. We have predicted that miR-198 has putative binding sites of circ_0005198, based on our search in Circular RNA Interactome (https://circinteractome. nia.nih.gov/index.html). MiR-198 was verified involved in the progression of malignant melanoma [15]. Also, Liu et al. reported that miR-198 could inhibit the development of papillary thyroid carcinoma [16]. Nevertheless, the role of miR-198 in regulating the biological processes of glioma is largely unexplored.
Tripartite motif-containing 14 (TRIM14), consisting of a B-box, a coiled-coil region and a C-terminal PRYSPRY region, involves in cell apoptosis, cell cycle regulation and viral response [17]. We speculated that TRIM14 is the putative target of miR-198 based on the website database TargetScan (http://www.targetscan. org/). Previous studies demonstrated that TRIM14 could be targeted by miR-124-3p to impact tongue cancer development [18]. Besides, TRIM14 facilitated epithelial-mesenchymal transition and chemoresistance of glioma [19,20]. Hence, TRIM14 was expected to be a good target for glioma treatment.
The aim of our study here was to investigate the role of circ_0005198 in TMZ resistance of glioma cells and dissect its mechanism via bioinformatics prediction and experimental verification. The discovery of the circ_0005198-miR-198-TRIM14 axis is promising to provide a novel therapeutic insight for glioma chemoresistance.
Circ_0005198 expression was increased in glioma
Firstly, we tested the expression of circ_0005198 in glioma. In clinical samples, the expression level of circ_0005198 was higher in glioma tissues than normal tissues and its expression displayed positive correlations with tumor grades ( Figure 1A). We then examined the expression of circ_0005198 in serum samples. As shown in Figure 1B, circ_0005198 expression was gradually increased in glioma compared to the control, and its expression also displayed positive correlations with tumor grades. Besides, we showed that the expression of circ_0005198 was up-regulated in all glioma cell lines in vitro, especially in U138 and LN18 cells, compared to normal human astrocytes ( Figure 1C). Furthermore, circ_0005198 expression in U138/TMZ and LN18/TMZ cells was higher than that in U138 and LN18 cells ( Figure 1D), demonstrating that circ_0005198 might be related to TMZ resistance of glioma.
Circ_0005198 knockdown inhibited the progression of TMZ-resistant glioma cells
Circ_0005198 expression was knocked down in U138/TMZ and LN18/TMZ cells to investigate the biological function of circ_0005198 in glioma cells ( Figure 2A). Cell viability assay showed that the cell proliferation of U138/TMZ and LN18/TMZ cells was significantly inhibited by circ_0005198 knockdown ( Figure 2B). Likewise, colony formation assays showed that the clone numbers of U138/TMZ and LN18/TMZ cells were significantly reduced with circ_0005198 down-regulation as well ( Figure 2C). The apoptotic rate of U138/TMZ and LN18/TMZ cells transfected with si-circ_0005198-2 was increased ( Figure 2D) compared to the scrambled group. The IC50 value of cells was significantly decreased after circ_0005198 knockdown, indicating that TMZ resistances of U138/TMZ and LN18/TMZ cells were suppressed ( Figure 2E). As a result, we verified that circ_0005198 played an active effect in TMZ resistance of glioma cells.
Circ _0005198 directly interacted with miR-198
Subsequently, we attempted to delve into the possible action mechanism of circ_0005198 in glioma cells. Predominantly cytoplasmic enrichment of circ_0005198 was identified ( Figure 3A), demonstrating that circ_0005198 might hinder target mRNA repression as the molecular sponges for miRNAs. Based on Circular RNA Interactome, miR-198 binding sequence was shared by that of circ_0005198 at 3′-UTR ( Figure 3B). We transfected miR-198 mimic into U138/TMZ and LN18/TMZ cells respectively ( Figure 3C). Over-expression of miR-198 inhibited luciferase activity in U138/TMZ and LN18/TMZ cells transfected with circ_0005198-WT but not in cells transfected with circ_0005198-MT ( Figure 3D). In particular, miR-198 expression level was elevated considerably with circ_0005198 knockdown ( Figure 3E). Furthermore, RIP assay proved the specific interaction of circ_0005198 and miR-198 in U138/TMZ and LN18/TMZ cells ( Figure 3F). Taken together, circ_0005198 could act as a sponge for miR-198.
Over-expression of miR-198 inhibited the progression of TMZ-resistant glioma cells
Next, the impacts exerted by miR-198 on TMZ resistance of glioma were explored. MiR-198 was down-regulated in glioma tissues and TMZ-resistant glioma cells ( Figure 4A, 4B). We further elevated the expression of miR-198 in TMZ-resistant glioma cells by transfecting miR-198 mimic. As revealed from the results of CCK-8 and colony formation assays, miR-198 over-expression restricted U138/TMZ and LN18/TMZ cells proliferation ( Figure 4C, 4D). The apoptotic rates of U138/TMZ and LN18/TMZ cells transfected with miR-198 mimic were increased ( Figure 4E). The IC50 value of the cells evidently declined after miR-198 over-expression, indicating that TMZ resistance was suppressed ( Figure 4F). Given the mentioned data, miR-198 was critical to the TMZ resistance of glioma.
Circ_0005198 was mediated by the negative regulation of miR-198
We then inhibit miR-198 level by transfecting miR-198 inhibitor into U138/TMZ and LN18/TMZ cells ( Figure 5A). MiR-198 expression was enriched by si-circ_0005198-2 and partially reversed via cotransfection with miR-198 inhibitor ( Figure 5B). As indicated from the results of CCK-8 and colony formation assays, the cell proliferation inhibition mediated by si-circ_0005198-2 could receive partial reversion by co-transfection with miR-198 inhibitor ( Figure 5C, 5D), demonstrating that circ_0005198 facilitated cell proliferation by restricting miR-198 expression. Additionally, flow cytometry assay showed that the apoptotic rate of U138/TMZ and LN18/TMZ cells was increased by si-circ_0005198-2, and partially reversed through co-transfection with miR-198 inhibitor AGING ( Figure 5E). Likewise, knockdown of circ_0005198 decreased the IC50 value of cells, and this effect was partially abolished by the silencing of miR-198 ( Figure 5F). These results suggested that circ_0005198 regulated TMZ resistance in glioma via negatively regulating miR-198 expression in U138/TMZ and LN18/TMZ cells.
TRIM14 was a target gene of miR-198
To investigate the molecular mechanism of circ_0005198 in TMZ-resistant glioma cells, the website database TargetScan was adopted for predicting the target gene of miR-198. A putative miR-198 binding site was identified in the 3'UTR of TRIM14 ( Figure 6A). As indicated from the results of dual-luciferase reporter assay, miR-198 over-expression considerably suppressed the luciferase activity of TRIM14-WT in U138/TMZ and LN18/TMZ cells, whereas TRIM14-MT was not affected ( Figure 6B). By ascertaining the mRNA and protein expression, we reported that overexpression of miR-198 hindered TRIM14 expression in U138/TMZ and LN18/TMZ cells ( Figure 6C, 6D). These results indicated that TRIM14 was the putative target of miR-198.
Silencing of TRIM14 inhibited the progression of TMZ-resistant glioma cells
We investigated the impacts exerted by TRIM14 on TMZ resistance of glioma. TRIM14 was highly expressed in glioma tissues and TMZ-resistant glioma AGING cells ( Figure 7A, 7B). Then we knocked down TRIM14 expression in U138/TMZ and LN18/TMZ cells ( Figure 7C). CCK-8 and colony formation assays revealed that inhibition of TRIM14 reduced U138/TMZ and LN18/TMZ cells proliferation ( Figure 7D, 7E). The apoptotic rate of U138/TMZ and LN18/TMZ cells transfected with si-TRIM14-1 was increased ( Figure 7F). The IC50 value of cells was significantly decreased after TRIM14 knockdown, indicating that TMZ resistance was suppressed ( Figure 7G). All the above data determined that TRIM14 expression was crucial to the occurrence of TMZ resistance of glioma cells.
MiR-198 mediated the regulatory effects of TRIM14 on the progression of TMZ-resistant glioma cells
To determine the regulatory relationship between TRIM14 and miR-198, we measured the protein expression of TRIM14 in U138/TMZ and LN18/TMZ cells transfected miR-198 mimic and TRIM14 overexpression plasmid (pc-TRIM14). The results indicated that miR-198 over-expression inhibited the expression of TRIM14, while the over-expression of TRIM14 could increase its expression ( Figure 8A). CCK-8 and colony formation assays showed that the proliferation inhibition mediated by miR-198 mimic can be partially reversed by co-transfection with TRIM14 over-expression plasmid ( Figure 8B, 8C), demonstrating that miR-198 inhibited cell proliferation by restricting TRIM14 expression. Moreover, flow cytometry assay indicated that the apoptotic rate was up-regulated by miR-198 mimic and partially reversed through co-transfection with pc-TRIM14 ( Figure 8D). Likewise, miR-198 mimic lowered the IC50 value of cells; such effect was partially abolished by overexpressing TRIM14 via the AGING transfection of pc-TRIM14 ( Figure 8E). Our results suggested that TRIM14 expression was altered by miR-198 on the progression of TMZ-resistant glioma cells. Taken together, our study revealed that circ_0005198 exerted its function as a ceRNA through sponging miR-198 to regulate TRIM14 expression, and therefore contributed to TMZ resistance in glioma (Figure 9).
DISCUSSION
Glioma refers to an aggressive tumor arising from glial cells of the central nervous system. Despite the development of new treatment options, the survival rate of glioma remains low. In particular, glioblastomas patients have a median survival of 15 months [21]. Surgery and the following radiotherapy assisted with temozolomide (TMZ) or cisplatin adjuvant chemotherapy is the normal strategy to treat glioma; however, the prognosis remains poor as there is growing resistance to the chemotherapeutic drugs [22,23]. For this reason, researches stressing the molecular and cellular mechanisms regarding the glioma chemoresistance would prompt the development of more effective treatments on the malignant glioma.
Currently, proteins, non-coding RNAs and many other molecules have been identified as the potential biomarkers to diagnose or prognostically predict glioma [24][25][26][27]. Growing evidence has proved the crucial function of circRNAs to tumor occurrences and progressions [28,29]. Owing to its naturally closed loop structure, circRNA can be stable in the tissues and serum [7]. So far, there have been many studies focusing on circRNAs regulating the progression of glioma. For instance, Chao Yi et al. showed that upregulation of circ_0034642 demonstrated a poor glioma prognosis and facilitated the proliferation and invasion via the miR-1205/BATF3 axis [30]. Fei Shi et al. reported that circ_0014359 contributed to the glioma progression via sponging miR-153 and regulating PI3K signal [31]. In existing studies, circ_0005198 was correlated with glioma development [12]. In the present study, we showed that circ_0005198 expression was up-regulated in glioma tissues, serum and TMZ-resistant glioma cells. Silencing of circ_ 0005198 suppressed the resistance of glioma cells to TMZ, inhibited the proliferation and enhanced the apoptosis of TMZ-resistant glioma cells. These results confirmed the positive effect of circ_0005198 on glioma resistance and provided a theoretical basis for circ_0005198 to become an ideal biomarker for glioma resistance diagnosis and treatment.
Numerous studies have shown that miRNA regulatory networks played an essential effect on the progression and chemoresistance of cancer [32,33]. Liu et al. showed that miR-497-5p enhanced apoptosis in OC cells [34]. Besides, Ueda et al. suggested that miR-27a ameliorated chemoresistance of breast cancer cells by AGING disruption of reactive oxygen species homeostasis [35]. Based on the findings here, circ_0005198, as "miRNA sponge", interacted with the miR-198 and suppressed the miR-198 activity. As reported in this study, miR-198 could hinder the TMZ resistance and suppress the progression of TMZ-resistant glioma cells, complying with the conclusion of Nie et al. [36]. Furthermore, we found that the effect of circ_0005198 on the progression of TMZ-resistant glioma cells could be achieved by silencing of miR-198. For this reason, the mentioned results uncovered that circ_0005198 regulated glioma resistance through the inhibitory effect on miR-198. Meanwhile, we showed that TRIM14 as a target of miR-198 and contributed to TMZ resistance.
Investigations have been reported on the involvement of TRIM14 in glioma chemoresistance. Tan et al. found that TRIM14 promoted chemoresistance in gliomas by activating Wnt/β-catenin signaling pathway [20]. Our study verified that TRIM14 was highly expressed in glioma and its silencing inhibited the progression of TMZ-resistant glioma cells. We showed that TRIM14 expression was required to maintain TMZ resistance in glioma cells. Furthermore, TRIM14 over-expression could invert the inhibiting effect of miR-198 on the progression of TMZ-resistant glioma cells, thereby confirming that TRIM14 functioned as a target gene of miR-198.
Although there are considerable reports on circRNAs, more potential functions of circRNAs in cancer require further investigation. This study demonstrated that circ_0005198 induced TMZ resistance of glioma by regulating the miR-198-TRIM14 axis, and this novel molecular mechanism added a new respective to the oncogenic function of circ_0005198 in glioma.
Clinical samples
Twenty glioma samples were collected from patients receiving surgical resection and 15 normal brain tissues were acquired from individuals died in traffic accidents from January 2016 to December 2019 in the Department of Neurosurgery at the Third Xiangya Hospital of Central South University. Glioma specimens were diagnosed pathologically and split to low-grade group (stage I-II, n=10) and high-grade group (stage III-IV, n=10) abiding by the WHO classification system by at least 2 advanced clinical pathologists. Prior to surgery, none of these patients was treated by chemotherapy or radiotherapy. Besides, we collected blood samples from these patients. The serum extracted from the blood samples were collected using standard procedures. The study was conducted in accordance with the Declaration of Helsinki. Written informed consents were collected from patients or their relatives regarding the utilization of their specimens and disease information. The project was approved by the Third Xiangya Hospital of Central South University. All samples were stored in the liquid nitrogen following resection till use.
Cell culture
The human glioma cell lines U87, U251, U138 and LN18, as well as normal human astrocytes (NHA) were provided by ATCC. U138 and LN18 were administrated with rising concentrations of TMZ to form TMZresistant glioma cells (U138/TMZ and LN18/TMZ). All cells were kept and incubated in Dulbecco's Modified Eagle Medium (DMEM; Hyclone, South-Logan, UT, USA) added with 10% fetal bovine serum (FBS; Hyclone) and without antibiotics. Cells received the incubating process in wet atmosphere containing 5% CO2 at 37° C.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were harvested from tissues, serum or cells with the use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions of manufacturer. For circRNA and mRNA, PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China) helped to generate the cDNA; TB Green™Premix Ex Taq™ II (TaKaRa) helped to perform the real-time PCR; GAPDH was applied as the endogenous control. In terms of miRNA, miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN) was adopted for generating cDNA; miRcute Plus miRNA qPCR Kit (SYBR Green) was adopted to perform real-time PCR to measure the comparative expression of circ_0005198, miR-198 and TRIM14 with 2 −ΔΔCt methods. U6 was considered internal controls. Sangon Biotech (Shanghai, China) contributed to synthesizing primers. Primer sequences were: circ_0005198, forward: 5'-GGTTGCACTAGC GTTATTC-3' and reverse:
Cell transfection
Circ_0005198 small interfering RNA (si-circ_0005198) or their negative controls (si-NC), TRIM14 small interfering RNA and over-expression plasmid (si-TRIM14 and pc-TRIM14) or their negative controls (si-NC and pc-NC) were provided by RiboBio (Guangzhou, China). Genepharma Company (Shanghai, China) contributed to synthesizing miR-198 mimic and inhibitor or their negative controls (miR-NC and anti-miR-NC). Lipofectamine TM 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) was employed for building cells transfecting process.
Cell TMZ resistance and proliferation assays
For determining the TMZ resistance and proliferation of cells, CCK-8 reagent (Dojindo, Japan) was used to detect cell viability. U138/TMZ and LN18/TMZ cells underwent the seeding process into 96-well plates. The cells were then treated with TMZ at a range of levels (0 μM, 200 μM, 400 μM, 600 μM, 800 μM, 1000 μM and 1200 μM) for 48 h. CCK-8 reagent was added to respective well and then incubated in the incubator containing 5% CO2 at 37° C for 2 h. For evaluating the TMZ resistance of cells, we measured the absorbance at 490 nm and then calculated the half-maximal inhibitory concentration (IC50). Furthermore, U138/TMZ and LN18/TMZ cells were administrated with CCK-8 at the specified time point after transfection. Then, we examined the absorbance at 490 nm for assessing cell proliferation.
Colony formation assay
We seeded cells undergone transfection in six-well plates with culture medium containing 10% FBS and incubated throughout the night. Fourteen days later, methanol was adopted for fixing cells followed by staining treatment with 0.1% Crystal Violet. Under a light microscope, we counted the colonies.
Flow cytometry
U138/TMZ and LN18/TMZ cells were gained after they were transfected for 48 h and stained with the Annexin V-fluorescein isothiocyanate (FITC)/ Propidium iodide (PI) Apoptosis Detection Kit (Yeasen, Shanghai, China). For gaining fluorescence signals and ascertaining the apoptosis rate of U138/TMZ and LN18/TMZ cells, Flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was employed.
RNAs isolation from nucleus and cytoplasmic fractions
The PARIS™ Kit (Invitrogen) was applied to isolate the cytoplasmic fraction and nuclear fraction following the manufacturer's directives. Briefly, we first harvested cells, followed by using cell fractionation buffer to lyse the harvested cells, and then separated cytoplasmic fraction and nuclear fraction with centrifugation. We also harvested supernatant containing cytoplasmic fraction and then transferred them to a fresh tube without RNase. The Cell Disruption Buffer was employed for lysing nuclear pellet. We mixed the nuclear lysate and cytoplasmic fraction with the 2X Lysis/Binding Solution and subsequently mixed them with 100% ethanol. A Filter Cartridge contributed to drawing sample mixture. Besides, Washing Solution was adopted for sample washing. The Elution Solution was employed for eluting the RNAs of cytoplasmic fraction and nuclear fraction. U6 snRNA and GAPDH acted as the positive control for the nuclear fraction and the cytoplasmic fraction, respectively.
RNA immunoprecipitation (RIP) assay
A Magna RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) contributed to implementing an RNA immunoprecipitation. In brief, a RIP buffer covering magnetic beads was employed for incubating entire-cell lysate, displaying conjugation with human anti-Ago2 antibody (1:50, Millipore) or normal mouse IgG (Millipore), classified to be a negative control. Proteinase K buffer helped to incubate samples. The qRT-PCR contributed to extracting and analyzing immunoprecipitated RNA to reveal the presence of circ_0005198 and miR-198 expression.
Western blot (WB) analysis
BCA protein assay kit (Sigma, USA) was applied to extracting the total proteins derived from glioma cells and detect protein concentration. Proteins were resolved with 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE; Sigma). After separation, the proteins were transferred to the PVDF membrane (Bio-Rad, USA). The membranes were incubated with TRIM14 antibody (Cell Signaling Technology, USA), AGING and GAPDH (Santa Cruz Biotechnology, USA) at 4° C overnight. Afterwards, blotted membranes underwent 2h of incubation with HRP-conjugated secondary antibody at ambient temperature. ECL Substrates (Millipore) contributed to visualizing the signals.
Statistical analysis
SPSS 21.0 software was applied in statistical analysis. The experimental processes in the study were carried out in triplicate, and Mean ± SD represents the results. The two-tail Student's t-test or one-way ANOVA was applied for assessing the difference between the two groups. Correlations were analyzed by Spearman rank correlation. It was set that the difference was of statistical significance with P value < 0.05.
AUTHOR CONTRIBUTIONS
XM, YD, HZ conceived and designed the experiment. XM, YD, LX performed most of the experiments. CL analyzed the statistical analyses. XM wrote the manuscript. XM, YD, HZ reviewed the manuscript. All authors have approved the manuscript. | v3-fos-license |
2017-10-23T13:38:44.235Z | 2017-10-01T00:00:00.000 | 26291897 | {
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} | pes2o/s2orc | Solvent-Free Addition of Indole to Aldehydes: Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl) Alkyl]-1H-Indoles and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells
New 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,3′- and 1,3′-diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated.
Introduction
Indole is one of the most versatile heterocyclic nuclei, identified as a pharmacophore in a large number of natural and synthetic biologically active molecules [1]. Due to its electron-rich character, indole promptly reacts with hard and soft electrophiles; thus, it is fair to consider it a privileged structure, quaintly referred to as the 'lord of the rings' [2].
Interestingly, several natural [24] and synthetic 3,3′-BIMs have shown important anti-cancer properties and demonstrated the capacity to sensitize cancer cells to apoptosis by signaling various proapoptotic genes and proteins [25][26][27]. In this regard, we present a preliminarily evaluation of the effect of these novel 3,3′-and 1,3′-BIM derivatives on the growth of the hepatoma cell line FaO.
Results and Discussion
In the quest to develop a simple and eco-friendly protocol to 3,3′-BIM derivatives, we unexpectedly observed, from the very beginning of our study, that, when the reaction was carried out with formaldehyde or aliphatic aldehydes, both 3,3′-and 1,3′-bisindolyl methane (3,3′-and 1,3′-BIM) derivatives were obtained. Conversely, aromatic aldehydes gave only classical 3,3′-BIMs. To understand these unusual results, the reaction between indole 1, and formaldehyde 2a, derived from paraformaldehyde, was first investigated (Scheme 2).
Scheme 2. Reaction between indole 1 and formaldehyde 2a.
Surprisingly, apart from the expected 3,3′-diindolyl methane 3a, we observed, as shown in Scheme 2, the formation of indole-1-carbinol 5 [28] and 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a, which was fully characterized via mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy experiments (spectra available in Supplementary Materials). The best results were achieved at 100 °C, and also observed an improvement in the yields when an excess of calcium oxide (CaO) was used. The reaction did not take place at all at room temperature, both with or without CaO. Similarly, when the operative temperature was fixed at 60 °C, traces of both isomers were detectable. Thus, it was speculated that CaO, which has recently been shown to catalyse some Mannich reactions for the preparation of lariat ethers [29], might affect our experiments. Hence, the role of CaO was explored, noticing that, when it was used in stoichiometric amounts or in large excess, the reaction seemed to proceed smoothly. Use of CaO in a catalytic amount (10 mol %), did not seem to produce any effect. Reasonably, the temperature and CaO might have a synergic effect in the reaction activation. We can plausibly assume that, in these conditions, the paraformaldehyde is rapidly converted to free formaldehyde and the traces of formic acid produced during prolonged heating are rapidly neutralized by CaO. Following the experiment by means of gas chromatography-mass spectrometry (GC-MS) analysis (Figure 1), we also observed, from the very beginning of the reaction and only in the presence of CaO, the formation of the indole-1-carbinol 5, which is usually obtained in a strong basic condition [30,31]. Surprisingly, apart from the expected 3,3 -diindolyl methane 3a, we observed, as shown in Scheme 2, the formation of indole-1-carbinol 5 [28] and 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a, which was fully characterized via mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy experiments (spectra available in Supplementary Materials). The best results were achieved at 100 • C, and also observed an improvement in the yields when an excess of calcium oxide (CaO) was used. The reaction did not take place at all at room temperature, both with or without CaO. Similarly, when the operative temperature was fixed at 60 • C, traces of both isomers were detectable. Thus, it was speculated that CaO, which has recently been shown to catalyse some Mannich reactions for the preparation of lariat ethers [29], might affect our experiments. Hence, the role of CaO was explored, noticing that, when it was used in stoichiometric amounts or in large excess, the reaction seemed to proceed smoothly. Use of CaO in a catalytic amount (10 mol %), did not seem to produce any effect. Reasonably, the temperature and CaO might have a synergic effect in the reaction activation. We can plausibly assume that, in these conditions, the paraformaldehyde is rapidly converted to free formaldehyde and the traces of formic acid produced during prolonged heating are rapidly neutralized by CaO. Following the experiment by means of gas chromatography-mass spectrometry (GC-MS) analysis (Figure 1), we also observed, from the very beginning of the reaction and only in the presence of CaO, the formation of the indole-1-carbinol 5, which is usually obtained in a strong basic condition [30,31]. We postulate that, in our experimental conditions, CaO induces a partial N-H proton abstraction and, despite the modest ionic character of the N-Ca bond, formaldehyde is so reactive as to undergo a nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used as polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as a major product in a high regioselective manner.
In addition, an interesting yield improvement, especially for isomer 4a, was noticed when KOH was used instead of CaO. A possible explanation, apart from the stronger electropositivity of K + , might be that indole-1-carbinol 5, when heated in the presence of KOH, decomposes to regenerate indole and HCHO [30] that react again to afford both 3,3′-and 1,3′-BIM isomers (Table 1). To gain a better comprehension of the reaction mechanism, we examined the possible role of compound 5. It did not demonstrate being a real intermediate in the formation of 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a. In fact, when 5 could react with indole 1 and CaO, only traces of the two isomers were detected (Scheme 3a). Interestingly, the classical bis (indolyl) methane 3a, could derive from a thermal-induced isomerisation of 4a (Scheme 3b) [20]. We postulate that, in our experimental conditions, CaO induces a partial N-H proton abstraction and, despite the modest ionic character of the N-Ca bond, formaldehyde is so reactive as to undergo a nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used as polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as a major product in a high regioselective manner.
In addition, an interesting yield improvement, especially for isomer 4a, was noticed when KOH was used instead of CaO. A possible explanation, apart from the stronger electropositivity of K + , might be that indole-1-carbinol 5, when heated in the presence of KOH, decomposes to regenerate indole and HCHO [30] that react again to afford both 3,3 -and 1,3 -BIM isomers (Table 1). To gain a better comprehension of the reaction mechanism, we examined the possible role of compound 5. It did not demonstrate being a real intermediate in the formation of 1-[1-(1H-indol-3-yl)methyl]-1H-indole 4a. In fact, when 5 could react with indole 1 and CaO, only traces of the two isomers were detected (Scheme 3a). Interestingly, the classical bis (indolyl) methane 3a, could derive from a thermal-induced isomerisation of 4a (Scheme 3b) [20]. We postulate that, in our experimental conditions, CaO induces a partial N-H proton abstraction and, despite the modest ionic character of the N-Ca bond, formaldehyde is so reactive as to undergo a nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used as polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as a major product in a high regioselective manner.
In addition, an interesting yield improvement, especially for isomer 4a, was noticed when KOH was used instead of CaO. A possible explanation, apart from the stronger electropositivity of K + , might be that indole-1-carbinol 5, when heated in the presence of KOH, decomposes to regenerate indole and HCHO [30] that react again to afford both 3,3′-and 1,3′-BIM isomers (Table 1). To gain a better comprehension of the reaction mechanism, we examined the possible role of compound 5. It did not demonstrate being a real intermediate in the formation of 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a. In fact, when 5 could react with indole 1 and CaO, only traces of the two isomers were detected (Scheme 3a). Interestingly, the classical bis (indolyl) methane 3a, could derive from a thermal-induced isomerisation of 4a (Scheme 3b) [20]. With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3 -diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). With the perception that the reaction, if successful with formaldehyde, would allow similar 1,3′-diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions with diverse ketones and aldehydes were carried out ( Table 2). It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)-2-propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3′-and 1,3′-isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3′-and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)-2-propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3′-and 1,3′-isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3′-and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)-2-propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3′-and 1,3′-isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3′-and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)-2-propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3′-and 1,3′-isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3′-and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4 -methylacetophenone and 1-(p-methoxyphenyl)-2propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3 -and 1,3 -isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH 3 CN, CHCl 3 , THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3 -and 1,3 -BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. It was immediately evident that the reaction was highly chemoselective for aldehydes; in fact, ketones such as 2-octanone, 2-hexanone, 4′-methylacetophenone and 1-(p-methoxyphenyl)-2-propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,3′-and 1,3′-isomers, while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly, the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the formation of 3,3′-and 1,3′-BIMs isomers. Both routes share the same well-known 2-azafulvene intermediate [20][21][22][23]36,37] which may add to nucleophilic species. Specifically, if the path a could be merely assumed as a Friedel-Crafts alkylation, the path b is presumably a Mannich-type N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems plausible given their intrinsically diverse steric and electronic features, and associated with the modest nucleophilicity of the indole nitrogen. Electron-impact (EI) mass spectra allowed us to characterize and differentiate both isomers easily ( Figure 2). diindolyl isomers when different carbonyl substrates were used, some other exploratory tions with diverse ketones and aldehydes were carried out ( Table 2). In fact, in the mass spectra of all 1,3 -diindolyl alkane isomers, the formation of the [M-116] + ion, presumably due to N-C bond cleavage, is the most favored fragmentation process (RA % 100), while in the case of 3,3 -isomers, the formation of [M-116] + ion (RA % 5), related to the rupture of the more stable C-C bond, is suppressed in favor of m/z 245 ion (RA % 100), generated by the radical loss of R substituent. MS/MS experiments are now in progress to investigate the most characteristic fragmentation pathways. NMR analysis included 1 H, 13 C, COSY, gHSQC, gHMQC and ROESY (spectra available in Supplementary Materials), and confirmed the structure of 1,3 -diindolyl alkane isomers.
Recent studies have reported on the pleiotropic protective properties on the chronic liver injuries steatohepatitis [38] and hepatocarcinoma [39,40], and on the multiple anti-tumour activities, including the apoptotic, anti-proliferative and anti-angiogenetic effects [24], of 3,3 -bisindolylmethane, the parent compound of 3,3 -BIMs and one of the most abundant dietary compounds derived from Brassica-genus vegetables. Hence, we decided to evaluate the effect of our novel 1,3 and 3,3 -BIMs on cell proliferation of the rat hepatoma cell line FaO by comparing their effects to those induced by 3,3 -bisindolylmethane 3a and indole-3-carbinol (I3C), the natural active precursor of 3,3 -bisindolylmethane. In experiments, carried out on a selection of compounds, FaO cells were treated with increasing concentrations of 3b, 4a and 4b as well as 400 µM of I3C for 24 h. As shown in Figure 3, the hepatoma cells were highly susceptible to the anti-proliferative effect of these BIMs. In particular, compounds 4a, 3b and 4b exhibited a concentration-dependent growth inhibitory effect in FaO cells similar to that observed after treatment with the well-characterized BIM 3a [38,39].
Electron-impact (EI) mass spectra allowed us to characterize and differentiate both isomers easily ( Figure 2). In fact, in the mass spectra of all 1,3′-diindolyl alkane isomers, the formation of the [M-116]+ ion, presumably due to N-C bond cleavage, is the most favored fragmentation process (RA % 100), while in the case of 3,3′-isomers, the formation of [M-116] + ion (RA % 5), related to the rupture of the more stable C-C bond, is suppressed in favor of m/z 245 ion (RA % 100), generated by the radical loss of R substituent. MS/MS experiments are now in progress to investigate the most characteristic fragmentation pathways. NMR analysis included 1 H, 13 C, COSY, gHSQC, gHMQC and ROESY (spectra available in Supplementary Materials), and confirmed the structure of 1,3′-diindolyl alkane isomers.
Recent studies have reported on the pleiotropic protective properties on the chronic liver injuries steatohepatitis [38] and hepatocarcinoma [39,40], and on the multiple anti-tumour activities, including the apoptotic, anti-proliferative and anti-angiogenetic effects [24], of 3,3′-bisindolylmethane, the parent compound of 3,3′-BIMs and one of the most abundant dietary compounds derived from Brassica-genus vegetables. Hence, we decided to evaluate the effect of our novel 1,3′ and 3,3′-BIMs on cell proliferation of the rat hepatoma cell line FaO by comparing their effects to those induced by 3,3′-bisindolylmethane 3a and indole-3-carbinol (I3C), the natural active precursor of 3,3′-bisindolylmethane. In experiments, carried out on a selection of compounds, FaO cells were treated with increasing concentrations of 3b, 4a and 4b as well as 400 µM of I3C for 24 h. As shown in Figure 3, the hepatoma cells were highly susceptible to the anti-proliferative effect of these BIMs. In particular, compounds 4a, 3b and 4b exhibited a concentration-dependent growth inhibitory effect in FaO cells similar to that observed after treatment with the well-characterized BIM 3a [38,39]. More importantly, our novel derivatives, as well as 3a, were 20-to 4-fold more potent than I3C in suppressing the viability of FaO cells with an IC50 value ranging from 50-100 µM after treatment with 4a and from 20-100 µM and 25-100 µM after treatment with 3b and 4b, respectively. It was noteworthy that all the tested BIMs induced a significant inhibition of growth of hepatoma cells at More importantly, our novel derivatives, as well as 3a, were 20-to 4-fold more potent than I3C in suppressing the viability of FaO cells with an IC 50 value ranging from 50-100 µM after treatment with 4a and from 20-100 µM and 25-100 µM after treatment with 3b and 4b, respectively. It was noteworthy that all the tested BIMs induced a significant inhibition of growth of hepatoma cells at lower concentrations than I3C. Furthermore, 3b was more effective in inducing loss of viability at very low doses.
Glutamax I, Invitrogen S.r.l., Milano, Italy) supplemented with penicillin, streptomycin and 10% heat-inactivated fetal calf-serum (FCS) (Invitrogen) in a humidified atmosphere of 5% CO 2 /95% air, at 37 • C. Indole 3-carbinol (I3C) and compound 3a, 3b, 4a and 4b were dissolved in DMSO and were added to the culture media to the final concentrations specified in the text. Control cells were treated with an equivalent amount of the solvent alone.
Cell Viability
Cell viability was determined by the uptake of neutral red by lysosome of viable cells. Determination of viability of adherent cells by NRU assay was performed according to Borefreund and Puerner [40]. The value obtained for treated cells was expressed as percentage of the value obtained in control cells. All experiments were performed in triplicate.
Statistical Analysis
Instant software (GraphpAd Prism 5, GraphPad Software Inc., San Diego, CA, USA) was used to analyse data. One-way analysis of variance (ANOVA) with post hoc analysis using Tukey's multiple comparison test was used for parametric data. The results of multiple observations were presented as the means ± S.D. of three experiments. A p value of <0.05 was considered statistically significant. | v3-fos-license |
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} | pes2o/s2orc | Assessing Raman Spectroscopy as a Prescreening Tool for the Selection of Archaeological Bone for Stable Isotopic Analysis
Stable isotope analyses for paleodiet investigations require good preservation of bone protein, the collagen, to obtain reliable stable isotope values. Burial environments cause diagenetic alterations to collagen, especially in the leaching of the organic bone content. The survival of bone protein may be assessed by the weight % collagen, % carbon and % nitrogen yields, but these values are achieved only after destructive chemical processing. A non-destructive method of determining whether bone is suitably preserved would be desirable, as it would be less costly than chemical processing, and would also preserve skeletal collections. Raman analysis is one such potential non-destructive screening method. In previous applications, Raman spectroscopy has been used to test both the alteration of the mineral portion of bone, as well as to indicate the relative amount of organic material within the bone structure. However, there has been no research to test the relationship between the Raman spectroscopic results and the survival of bone protein. We use a set of 41 bone samples from the prehistoric archaeological site of Ban Non Wat, Northeast Thailand, to assess if Raman spectroscopy analysis of the organic-phosphate ratio has a significant correlation with the weight % collagen, and carbon and nitrogen yields obtained by isotopic analysis. The correlation coefficients are highly statistically significant in all cases (r = 0.716 for collagen, r = 0.630 for carbon and r = 0.706 for nitrogen, p≤0.001 for all) with approximately or close to half of the variation in each explained by variation in the organic-phosphate ratio (51.2% for collagen, 39.6% for carbon, and 49.8% for nitrogen). Although the Raman screening method cannot directly quantify the extent of collagen survival, it could be of use in the selection of bone most likely to have viable protein required for reliable results from stable isotope analysis.
Introduction
Stable isotope analysis of bone protein (collagen) and bone mineral (apatite) have been used extensively to determine the diet and movement of past people [1][2][3][4][5][6]. Experimental data have indicated that different bone tissues reflect different components of the diet [7], [8]. Stable isotope values in bone collagen are mainly influenced by the protein portion of the diet, while the bone and tooth enamel mineral component (carbonated-hydroxyapatite or apatite), is reflective of a mixture of dietary protein, carbohydrates and fats. Quantitative estimates of dietary influences from various sources (e.g. terrestrial or marine, and plant or animal protein components) can also be made from analysis of bone collagen and apatite. Bone collagen and bone apatite are remodelled during life and thus their isotopic composition reflects dietary averages over a certain time period [6], [9], [10].
The isotopic signatures of bone collagen and apatite can be compromised by poor preservation. Among the types of burial environments that most severely alter bone protein and bone apatite are conditions found in the monsoonal tropics, where heat and alternating wet and dry seasons leach bone protein and recrystallise bone apatite. The preservation state of collagen is particularly important in the application of stable isotope analysis for dietary studies. The isotopic values of carbon and nitrogen in bone protein are derived from the metabolism of food sources, but diagenetic changes to bone protein from the burial environment can alter the original diet-derived isotopic signature [2], [11]. These diagenetic changes include the breakdown of the hydrogen bonds in bone collagen from excessively hot temperatures and extremes of aridity or moisture [12] or the intrusion of exogenous carbon from humic and fulvic acids (fulvic acids are humic acids of lower molecular weight and higher oxygen content) in burial soils. As collagen deteriorates there is a reduction in % nitrogen, in part due to the denaturing of the collagen helix and cleaving-off of amino acids, which are more susceptible to degradation [13]. As each amino acid which constitutes the whole collagen protein has a d 13 C and d 15 N signature arising from its source and metabolic generation [14], [15], the loss of certain amino acids during protein degradation may alter the overall stable isotope value of the collagen of interest for dietary analysis [10], [16][17][18] with a possible shift of d 13 C to depleted values and an enrichment of d 15 N signals [13]. The isotopic abundance of a sample is preceded by the notational symbol d, which is called the delta ratio. The delta ratio of a sample is calculated by the formula: [19]. Where d expresses the abundance of isotope A of element X in a sample relative to the abundance of that same isotope in an isotopic standard, which is analysed along with the unknown samples. Because of this potential collagen degradation, paleodietary studies firstly assess collagen integrity by one or more screening methods. A suite of indicators can be used to assess collagen survival and presence of exogenous carbon, including techniques such as % collagen yield (weight % collagen derived from undemineralised bone), collagen amino acid composition, weight % carbon and nitrogen [2], [6], [11], [20][21][22], and carbon to nitrogen (CN) ratios, indicative of preserved bone protein in the 2.9-3.6 range [23]. Each of these screening methods requires bone to be destroyed during chemical preparation to isolate the collagen. It would be useful if a screening method was available to select the best preserved bone for stable isotope analyses from often precious collections. Archaeological research in tropical areas, including Asia, have long faced a limited availability of sound skeletal material for isotopic analysis. Recent research by King at al. [24] on the bone of 40 adults from the prehistoric archaeological site of Ban Non Wat in Northeast Thailand used Raman spectroscopy to qualitatively assess the extent of diagenesis. The research investigated chemical changes in bone such as fluoridation, carbonate substitution in the apatite, and the presence or absence of collagen [24]. In King et al.'s [24] study, Raman results were interpreted as indicating a total leaching of bone protein from all samples considered. However, % collagen, and % carbon or % nitrogen yield derived from stable isotope analysis were not produced to compare with the qualitative measures of Raman spectroscopy. The aim of the present paper is to analyse how Raman organic-phosphate ratios correlate with actual percentages of collagen, carbon and nitrogen retrieved from bone samples to assess the usefulness of Raman spectroscopy as a tool for detecting the protein component of bone. There was a statistically significant correlation between the Raman organic-phosphate ratio and % collagen, % carbon and % nitrogen, which indicates that Raman spectroscopy could be used in the future as a pre-screening tool for preservation of the organic component of bone for stable isotope analyses.
Raman spectroscopy
Raman spectroscopy involves a sample being irradiated with a laser, with some of the resulting scattered photons containing information on the vibrational energy levels of the sample. These data may be used to quantify the constituents of a sample [25].
Uses of Raman spectroscopy include the examination of bone quality and health in patients by assessing organic-phosphate ratios [26], lamellar bone orientation and bone composition [27], and mechanical stress on collagen fibrils in bone [28]. Raman spectroscopy can also be used in forensic contexts to determine the post-mortem interval of death by monitoring organic material loss in bones [29]. In addition, Raman spectroscopy is used to detect exogenous metal ions incorporated into the apatite lattice of bone, which reveals if a specimen's apatite has undergone recrystalisation, raising the possibility that exposed collagen might have also undergone diagenesis (e.g. [24,30]). This can be investigated because the band energy and width corresponding to the symmetric PO 4 3vibration in the Raman spectrum is sensitive to changes in the apatite unit cell size due to foreign ion incorporation. [30], [31].
Raman spectroscopy has a fast scan-time and is non-destructive [32]. The technique is also insensitive to the presence of water, and it can be coupled to a light microscope to give sub-micron spatial resolution for dense surface sampling [33]. Raman spectroscopy can be used quantitatively, but to do so requires homogenous calibration standards with known analyte concentrations. When calibration standards are not available, it can be used to provide relative quantification [34][35][36].
The aim of this study is to evaluate how well % collagen yield, % carbon yield and % nitrogen yield as indicators of protein survival, correlate with the Raman spectroscopy evidence for the presence of protein (organic-phosphate ratios). If Raman spectroscopy produced a reasonably reliable ''pre-screen'' for archaeological bone being considered for stable isotope analysis, it would be a relatively inexpensive, fast and non-destructive test to select bone with the most viable collagen content prior to destructive preparation work and running of mass spectroscopy for dietary stable isotopes from archaeological samples.
Ban Non Wat
The site of Ban Non Wat is situated in the Upper Mun River Valley on the Khorat Plateau, Northeast Thailand ( Fig. 1). This site has yielded 630 inhumation burials within an occupation sequence from 3800-1500 BP based on 14 C AMS dates [37]. The main excavation of this site was carried out under The Origins of Angkor Archaeological Project directed by Professor Charles Higham (University of Otago), Dr Rachanie Thosarat and Dr Amphan Kijngam (Thai Fine Arts Department) over 7 seasons from 2002-2008 [38]. All of the human skeletal remains are stored at the Bone Storage Facility at the 12 th Regional Office at Phimai, Northeast Thailand. Full permission from the National Research Council of Thailand (NRCT) and the Thai Fine Arts Department has been granted for this research (NRCT permit number 0002.3/ 8713). Specimen numbers are designated with burial numbers and Raman IDs, with details given in the results section. During part of a larger study assessing weaning and diet of all subadults excavated from this site (N = 197), bone from 41 of those infants and children were submitted for Raman spectroscopy analysis prior to stable isotope analysis for carbon and nitrogen.
These samples were chosen to represent the different chronological phases of the site, qualified as Neolithic (n = 5), Bronze (n = 23) and Iron Age (n = 13) [38]. Prior to selection for nondestructive Raman and destructive stable isotope analysis all osteological data including age, bone growth, growth disruption and any skeletal and dental pathological information was collected using standard methods outlined in Halcrow [39].
Raman spectroscopy analysis
For each skeleton, a rib bone was the preferred skeletal element. In cases of poor preservation of ribs, skeletal elements such as segments of long bones were selected. Each bone was scraped with a scalpel to remove any dirt to minimise fluorescence effects, which obscure peaks in the Raman spectrum. The scalpel was soaked briefly in 10% HCl between different bone samples to remove any residues before being rinsed extensively with distilled water and wiped.
Spectra were obtained with a Fourier Transform (FT) Raman spectrometer Equinox 55 Interferometer (Bruker Optics, Ettlingen, Germany) equipped with a FRA-106 Raman accessory and a D418-T liquid nitrogen Ge detector. The 1064 nm excitation laser with a power of 300 mW was used to minimise fluorescence without destroying the organic content of the bone (Nd:YAG laser, Coherent Inc. Santa Clara, USA). A downward-looking objective was used with a spot size of 1 mm to obtain representative spectra. Three replicate spectra (0-3200 cm 21 ) were recorded on different regions of each sample's compact bone. A resolution of 1 cm 21 was used to concurrently analyse diagenesis in the mineral phase (results not presented). Each spectrum was the result of the coaddition of 300 scans to produce an acceptable signal-noise ratio. If a strong emission signal was observed before a measurement, that region was not used for data collection. Each Raman spectrum was integrated at 3060-2800 cm 21 (C-H symmetric and asymmetric vibrations), 983-930 cm 21 (symmetric PO 4 32 vibration) and 566-300 cm 21 to obtain band areas.
Stable Isotope analyses (d 15 N, d 13 C, %C, %N) For the stable isotope determinations, the surfaces of bone samples were mechanically cleaned with a small drill or pared with a scalpel to remove degraded surface material and any adhering soil. The bone was then broken into 2-3 cm fragments and placed in clean plastic 50 ml vials for chemical demineralisation using a modified Longin [40] method with 0.5 M HCl. The bone sample was demineralised for at least 12 hours at room temperature, then each sample was centrifuged, the spent supernatant decanted, and fresh 0.5 M HCl added. This step was repeated until each sample was completely demineralised. The insoluble collagen was isolated by centrifuging and decanting of the supernatant acid, then rinsed to neutral by topping each vial with MilliQ deionised water, centrifuging, and decanting supernatant in repeated washes. The insoluble collagen was gelatinised with 0.01 M HCl at 60uC for 24 h, then double-filtered through Whatman GF/C and 0.45 mm Acrodisc filters. The filtered gelatine was lyophilised to obtain gelatine yields relative to starting weight of physically cleaned bone.
Stable isotope analysis for d 13 C, d 15 N, %C and %N was performed at the University of Otago's Isotope Ratio Mass Spectrometry Unit on a Europa Hydra coupled to a Carlo Erba NC 2500, with an average of 0.8 mg of bone gelatine in duplicate samples. All reference materials and internal standards are calibrated and traceable to the international standards V-PDB for 13 C [41], [42] and AIR for 15 N [43]. The calibration standard EDTA accompanied each analytical run. Measurements of an
FT-Raman analysis
All spectra of the bones contained four bands corresponding to the vibrations of the tetrahedral PO 4 32 within the apatite lattice. The vibration at ,960 cm 21 was the most intense and assigned as PO 4 32 stretching symmetrically (v 1 ) with all four oxygens vibrating in phase (Fig. 2). Carbonate incorporated into the apatite lattice had a symmetric stretch (v 1 ) present at ,1071 cm 21 . Only six bones showed prominent organic vibrational bands, which indicated definite proteinaceous content in the specimens (Raman IDs 22,24,28,29,33 and 41). The C-H stretch region, composed of symmetric and asymmetric stretching, had three bands in the range of 3060-2800 cm 21 , which were the primary indicator of organic content. There was also a broad C-H bending vibration (d C-H) at ,1425 cm 21 . The remaining samples (n = 12) showed poorly-defined C-H bands with a lesser intensity or no indication of organic content whatsoever (n = 23). Amide I manifested as a broad band at ,1667 cm 21 which is primarily due to the vibrations of carbonyls within the polypeptide and is indicative of the a-helical secondary structure of collagen [28]. Most bones showed this weak, ill-defined amide I feature. In addition to the organic and mineral components of the bone, secondary mineralisation products (calcite and barite) were also observed. This suggests that the apatite portion of the bone within each sample had been exposed to crystallisation processes from ground fluids. The ratio of organic material to apatite was examined using the band areas of the C-H region (3060-2800 cm 21 ) with those of the PO 4 32 (n 1 and n 2 ). The broad band centred at 431 cm 21 corresponds to O-P-O bending vibrations (v 2 ). Figure 3 shows the Raman spectra of a modern bone, a well-preserved archaeological sheep bone and the Ban Non Wat sample from Figure 2. These spectra demonstrate the poor preservation of the Ban Non Wat samples. In addition to the intense C-H stretching region, the modern bone contains amide III and phenylalanine bands not seen in any Ban Non Wat specimens. The lack of these features is a reflection of the loss of protein in the Ban Non Wat bone.
Previous Raman studies of bone have shown that measurements on two orthogonal planes of orientation affect certain band intensities [27]. The use of a large spot size (diameter 1 mm) was used to mitigate these effects as the sampling area is much larger than domains of orientated collagen fibrils.
Stable isotope
In Table 1, the % yield of collagen in the BNW bone samples is overall quite low, with a mean of 1.07% (minimum 0.14%; maximum of 6%). Collagen yields of less than 1-2% are usually suspect for adequate stable isotope results. However, due to the nodes of exogenous minerals from burial conditions within the bone structure, starting raw bone weights may not be representative of the bone weight itself, leading to calculation of lower % collagen yield.
Seven burials had nitrogen of 7 mg or less, and produced only carbon delta values and weight percents. An eighth sample, BNW 479, also had a nitrogen value of less than 7 mg did not produce a carbon delta result (Table 2). Figure 4 shows % collagen versus the organic-phosphate ratio (v 1 ), Figure 5 shows % carbon in the same way, and Figure 6 shows % nitrogen similarly (n = 31 in all graphs). The correlation coefficients are highly statistically significant in all cases (r = 0.716 for collagen, r = 0.630 for carbon, and r = 0.706 for nitrogen, p# 0.001 for all) with approximately or close to half of the variation in each explained by variation in the organic-phosphate ratio (51.2% for collagen, 39.6% for carbon, and 49.8% for nitrogen). Removing the one clear outlier and two leverage points to the right of the graph from the % collagen scatter plot would reduce the coefficient of determination to 17.8% (p = 0.025) and removing the one leverage point to the right of the carbon graph would only slightly reduce R 2 to 36.1%. Removing the one leverage point to the right of the graph from the % nitrogen scatter plot would reduce R 2 to 43.8% (p,0.001). There was no evidence before or after removing these points that the association between the organic-phosphate ratio and % carbon was stronger than with % collagen (difference in correlation coefficients p = 0.555 before and p = 0.379 after removing the unusual points), or for % nitrogen than with % collagen (difference in correlation coefficients p = 0.942 before and p = 0.214 after removing the unusual points), or for % carbon than with % nitrogen (difference in correlation coefficients p = 0.606 before and p = 0.712 after removing the unusual points). Table 2. Weight % nitrogen (%N), weight % carbon (%C), % yield of gelatin relative to undemineralised bone starting weight (% collagen), and Raman organic-phosphate ratio [C-H/PO 4 32 (n 1 )] for eight samples for which stable isotope analysis indicates weight % nitrogen at extremely low levels (7 mg or less), including one sample with extremely low weight % carbon.
Discussion
In this experiment FT-Raman spectroscopy organic-phosphate ratio results are compared with % collagen yield from the whole bone sample, and % carbon and % nitrogen from isotopic analysis. Because statistically significant correlations between these variables were found, it is feasible that Raman spectroscopy could be useful in the selection of bone for subsequent destructive chemical treatment in the course of stable isotope analysis. There was no evidence (whether or not the odd data points are kept or removed) that there is a stronger association between the organic-phosphate ratio with % collagen, % carbon or % nitrogen. All samples with greater than 1% collagen yields showed C-H stretches from visual examination of the Raman spectra (n = 12). Considering bones that have % collagen yields below 1-2% are undesirable for isotope analysis, visual inspection of the spectra allowed viable bones to be identified. The Raman spectra allowed qualitative presence or absence of protein to be determined which can save time, expense and preserve sample integrity before other techniques are applied. The relationship between the Raman spectroscopy qualitative organic scores and % collagen and nitrogen preservation found in this study could be further It has been shown by previous studies (e.g. [44]) that collagen content is not lost uniformly, and exogenous ions are not incorporated uniformly through the bone. Some of the variability in the correlation between the Raman collagen and actual collagen yield from isotopic preparation may be attributable to subsampling. This is because Raman scattering primarily occurs on the surface of samples with penetration depth of a few hundred micrometres for a 1064 nm laser [45], [46]. In contrast, the weight percent of carbon and nitrogen by mass spectrometry was determined from collagen samples derived from the whole bone, which may have variable preservation. However, homogeneity on the surface of the bone as indicated in the triplicate spectra appeared reasonable with the exception of three bones. In addition, as noted in the methods, in order to record a representative composition, each spectrum gathered data from a spot 1 mm in diameter. Further research should be conducted into the extent of variable collagen preservation within bone specimens especially those which are poorly preserved.
These results are consistent with previous research, which showed that there is a relationship between bone recrystalisation and collagen preservation [47]. King's [25] study of diagenesis at Ban Non Wat identified secondary mineralisation products of calcite and barite in their Raman analysis of adult bones from BNW, which was also supported with tentative identification of calcite and barite crystals using SEM. The present study's results showed that secondary mineralisation products (calcite and barite) were also present, suggesting that the apatite portion of the bone within each sample had been exposed to recrystallisation processes from ground water. | v3-fos-license |
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} | pes2o/s2orc | Effects of solvation shells and cluster size on the reaction of aluminum clusters with water
the oxidation of aluminum nanoparticle: Multimillion-atom reactive molecular dynamics Reaction of aluminum clusters, Al n ( n = 16, 17 and 18), with liquid water is investigated using quantum molecular dynamics simulations, which show rapid production of hydrogen molecules assisted by proton transfer along a chain of hydrogen bonds (H-bonds) between water molecules, i.e . Grotthuss mechanism. The simulation results provide answers to two unsolved questions: (1) What is the role of a solvation shell formed by non-reacting H-bonds surrounding the H-bond chain; and (2) whether the high size-selectivity observed in gas-phase Al n -water reaction persists in liquid phase? First, the solvation shell is found to play a crucial role in facilitating proton transfer and hence H 2 production. Namely, it greatly modifies the energy barrier, generally to much lower values ( < 0.1 eV). Second, we find that H 2 production by Al n in liquid water does not depend strongly on the cluster size, in contrast to the existence of magic numbers in gas-phase reaction. This paper elucidates atom-istic mechanisms underlying these observations. Copyright 2011 Author(s). This article is distributed under a Creative Commons Attribution 3.0 Unported License .
I. INTRODUCTION
Reaction of water with metal to produce hydrogen gas has been widely studied, with aluminumwater reaction being most intensely investigated. [1][2][3][4][5][6][7] Although the reaction, 2Al + 6H 2 O → 2Al(OH) 3 + 3H 2 , is exothermic, formation of an aluminum-oxide layer on the aluminum surface prevents continuous reaction. 8 In order to overcome this bottleneck, continual removal of the oxide layer has been attempted using various promoters such as hydroxides, 5 oxides, 9 and salts. 10 Unfortunately, none of these techniques has achieved a sufficiently fast rate of H 2 production for commercialization. 8 Nanotechnology has opened new avenues toward solving this problem. For example, the surfacearea to volume ratio is drastically increased, 11 quantum effects on the atomic scale often lead to novel catalytic activities 12 and enhanced reaction rates, 1,7,[13][14][15][16] and surfaces can be tailored at the nanometer scale to prevent oxidation. 17 Metal clusters have been found to react chemically in unusual ways. A remarkable example is an Al superatom, i.e., a cluster consisting of a magic number of Al atoms. 18,19 Hydrogen molecules were produced through gas-phase reaction of Al superatoms with water molecules, where Al 12 and Al 17 showed particularly higher reactivity compared with other cluster sizes. 20,21 The size selectivity of superatoms for gas-phase reaction with water molecules has been explained through geometric factors. 20,21 First-principles calculations show the existence of sites with Lewis base character and those with Lewis acid character on superatom surfaces. In the proposed H 2 -production mechanism (hereafter referred to as R-mechanism), water molecules are first split into protons and hydroxyl anions, which respectively bond to Lewis base and acid sites. Subsequently, a hydrogen molecule is produced from two surface protons that are bonded to two proximate Lewis base sites, with the energy barrier of about 1 eV. Our previous quantum molecular dynamics (MD) study investigated H 2 production of the magic-number Al n (n = 12, 17) in liquid water. 22,23 It is found that proton transfer along a chain of hydrogen bonds (H-bonds) between water molecules, i.e. Grotthuss mechanism, [24][25][26][27][28] plays an important role not only in splitting water into surface hydrogen and hydroxyl groups, but also in recombining hydrogen atoms into hydrogen molecules. However, the H-bond chain is surrounded by non-reacting H-bonds that form a solvation shell, and the role of the solvation shell for H 2 production has not been addressed in the previous study. Thus, the central questions are: (1) What is the role of a solvation shell formed by non-reacting H-bonds surrounding the H-bond chain; and (2) whether the high size-selectivity observed in gas-phase Al n -water reaction persists in liquid phase?
To answer these questions, we investigate the reaction of superatoms, Al n (n = 16, 17, and 18), with liquid water using quantum MD simulations. Here, size-selectivity is studied by comparing highly reactive Al 17 with less reactive Al 16 and Al 18 , as observed in gas-phase reaction. 20,21 The observed fast reaction rates and calculated low reaction energy barriers are explained by the same reaction mechanisms as in our previous study. 22,23 In addition, we find that the formation and breaking up of branching H-bonds, which connect the H-bond chain with the solvation shell, are crucial to facilitate proton transfer. This explains why the energy barrier varies strongly upon the rearrangement of surrounding water molecules, generally resulting in extremely low values (< 0.1 eV) in the presence of branching H-bonds. Thermal deformation of superatoms also modifies the arrangement of Lewis base and acid sites, driving some of the surface hydrogen atoms to diffuse around neighbor aluminum sites and lowering the energy barrier. The Al 18 system undergoes further oxidation, from Al n -OH 2 to Al-OH-Al to Al-O-Al, revealing the mechanism of the formation of aluminum-oxide layers coating aluminum particles. Due to the thermal deformation of Al n , as well as the delocalized nature of the reaction assisted by H-bond chains, Al n -water reaction in liquid water is less sensitive to the local geometrical arrangement of Lewis acid and bases sites on the superatom surface, and accordingly the size-selectivity is less profound.
II. METHOD OF CALCULATION
Our quantum-mechanical molecular dynamics (MD) simulation is based on density function theory (DFT) within the generalized gradient approximation (GGA). 29 The projector-augmentedwave (PAW) method 30, 31 is used to calculate the electronic states, where the plane-wave cutoff energies are 30 and 250 Ry, respectively, for the electronic pseudo-wave functions and the pseudocharge density. A preconditioned conjugate-gradient method 32, 33 is used to minimize the energy functional iteratively. The Brillouin zone sampling is performed at point. The projector functions are generated for O (2s, 2p), H (1s), and Al (3s, 3p, 3d) states. Nosè-Hoover thermostat 34, 35 is used for MD simulation in the canonical ensemble, where the equations of motion are numerically integrated by an explicit reversible integrator 36 with a time step of 13 a.u. (∼0.314 fs).
With periodic boundary conditions, in a cell of 12.58×12.58×18.87 Å 3 , four systems are studied in our MD simulations. They are superatoms Al n (n = 16, 17, and 18) immersed in 84 H 2 O molecules, as well as a reference system consisting only of 96 H 2 O molecules. A snapshot of the Al 16 simulation cell is shown in Fig. 1(a). The MD simulations are performed at a temperature of 773 K to accelerate reactions, so that as many events as possible are sampled during 20 ps of simulation for each system. Once all reaction mechanisms in the MD simulations are identified, the corresponding reaction energy barriers are calculated with the nudged elastic band (NEB) method, 37 which is capable of finding the minimum-energy path between given initial and final states. With the help of transition state theory, 38 the reaction rates at room temperature are then estimated from the energy barriers.
III. RESULTS AND DISCUSSION
To study the hydrogen-production process, the time evolution of the numbers of H-H and Al-O bonds are plotted in Fig. 1(b). We consider two atoms to be bonded when their distance is less than a cutoff distance R c during a prescribed bond lifetime of 12 fs, where R c for Al-O, Al-H, and H-H are 2.4, 2.0, and 1.0 Å, respectively. The number of H-H bonds shows that, within 20 ps, three H 2 molecules are produced each in the Al 16 and Al 17 systems and that six H 2 molecules are produced in the Al 18 system. In contrast, no H 2 molecule is produced in the pure water system. The production of a larger number of H 2 molecules in the Al 18 system is attributed to the increasing number of Al-(OH)-Al bridging bonds, which will be discussed later in this paper. We do not observe any stable bridging bonds in Al 16 and Al 17 systems. After the first H 2 is produced in the Al 17 system, we also observe that one aluminum atom of the Al 17 -superatom is detached due to the high temperature. The detached Al atom forms H H > Al < O H O H 2 , which has two Al-H bonds. The three Al-H bonds in the final Al 17 system include two from the detached Al atom. Thus the final Al 17 superatom has only one Al-H bond corresponding to a surface H atom, which is the same number as in the Al 16 and Al 18 systems.
Before we investigate the H 2 production mechanisms in details, it is worth summarizing the mechanisms found in our previous study. 22 Figure 2 is a schematic of these mechanisms. An H-bond chain provides a channel for proton transfer (known as the Grotthuss mechanism) not only in the initial water splitting but also in H 2 production. The proton-donating side of the H-bond chain is a water molecule adsorbed at a Lewis-acid site (labeled A in where Al and Al respectively denote aluminum atoms at Lewis base and acid sites. In Eq. (1), the reacting H-bond chain length l is defined as the number of oxygen atoms directly involved in the proton transfer. Reaction A → D is the water splitting process: Reaction A → B is proton exchange between Al n -bonded water and hydroxyl, where Al is another Lewis base site. In Eq. (3), the reaction could occur in both directions. Our previous study has revealed that the H-bond chain can substantially reduce the energy barrier of the reaction in Eq. (2) from 0.42 eV to 0.20 eV, 22 providing an example of H-bond related catalytic properties of water molecules. 41 To study the effect of H-bond chain length, we calculate the energy barrier for the H 2 -production reaction in Eq. (1) involving Al n (n = 16, 17, 18) with various chain length l = 1∼4. We start with a superatom in the minimum-energy state. The initial state of the reaction is prepared by first adding a water molecule to one of the strongest Lewis acid sites (i.e. the most negative site according to Mulliken population analysis) 13,42 of Al n to form Al-OH 2 , and adding a H atom to the nearby Lewis base site of Al n to form Al -H (see Fig. 3(a) for the case on Al 18 ). We then insert l-1 water molecules chained by the H-bonds to connect the Al-OH 2 and Al -H. The final state is prepared by proton transfer along the H-bond chain as shown in Fig. 3(a). Both initial and final states are relaxed to local minimum-energy configurations by the conjugate gradient method. Given the initial and final states, the minimum energy reaction path between them is obtained by NEB calculation. The difference between the maximum energy along the reaction path and the initial-state energy is the activation barrier of the reaction. Figure 3(b) shows the calculated energy barriers for Al n (n = 16, 17, 18) with various chain length l = 1∼4. The increasing rate of energy barrier versus length is about 0.20 eV for all three systems, which may be interpreted as the energy required for transferring a proton from a hydrogen donor to an acceptor. For l >1, we have obtained different energy barriers (within discrepancy ∼0.1 eV) even for the same superatom with the same H-bond chain length, depending on the initial arrangement of the water molecules along the H-bond chain.
Besides the proton transfer along the H-bond chain, the existence of massive H-bonds between the reacting H-bond chain and surrounding water molecules in liquid water (which we call the branching H-bonds) could play an important role in these reactions. To study how the H 2 -production mechanism is influenced by the branching H-bonds, Fig. 4 shows different stages of the H 2 production mechanism in the Al 16 system during MD simulation, for which the H-bond chain length l = 2. Figure 5 plots the distance R ij and bond-overlap population O ij between several atomic pairs i and j involved in this reaction. Here, the bond-overlap is calculated by population analysis 13,42 to clarify the change in the bonding properties. By expanding the electronic wave functions in an atomic-orbital basis set, we obtained the overlap population O ij between the i th and j th atoms as well as the gross charge Q i for the i th atom. The Al atom labeled Al1 in Figs. 4 and 5 is the proton-accepting Lewis base site, while Al2 is the proton-donating Lewis acid site. The reaction begins with the configuration in Fig. 4(a), where the O3· · ·H3-O1 H-bond has formed. Here, · · · denotes a hydrogen bond, and the H-bonds represented by red dashed lines in Fig. 4 are defined by the criteria: R oo < 3.5 Å and θ HOO < 30 • . The existence of those H-bonds has been confirmed by calculating the bond-overlap population O ij between the proton and the oxygen, as will be shown in the analyses below. The proton H4 transfers from O2 to O1 during (a)∼(b). As shown in Fig. 5(a), the bond-overlap between O3 and H3 increases, indicating the strengthening of the O3· · ·H3-O1 H-bond. At the same time, R O3-H3 decreases and R O1-H3 increases as shown in Fig. 5(b), indicating that H3 moves away from O1 towards O3. As H3 moves away from O1 and the H3-O1 bond weakens, it becomes for O1 to receive another hydrogen labeled H4. During (b)∼(c), breakage of the O3· · ·H3-O1 H-bond is indicated by the decrease of the bond-overlap population O O3-H3 in Fig. 5(a). Together with the decrease of O1-H3 and O1-H4 bond lengths, the breakage of the O3· · ·H3-O1 bond promotes breakage of the O1-H2 bond (R O1-H3 increases to ∼ 2 Å as shown in Fig. 5(b)) and pushes H2 into H1's binding region (R H1-H2 decreases to below 1 Å as shown in Fig. 5(b)). The O3· · ·H3-O1 bond breaks at 15.81 fs by way of water reorientation, which causes O3, H3, and O1 to misalign as shown in Fig. 4(c). This bond breakage is accompanied by the formation of another H-bond, O4· · ·H6-O3 in Fig. 4(c). Between (c) and (d), O1-H2 and H1-H2 bonds change until O1-H2 is broken and H1-H2 is bonded. From (d) to (e) is further structural change of the surface H 2 (H1-H2), and this H 2 molecule is released from the superatom surface during (e)∼(f). A notable observation during (c) and (f) is that the H 2 molecule is formed by the approach of H2 toward the Al1 atom while the H2-H1 bond is formed, instead of H1 being released to bond to H2 away from the superatom surface. We also observe that the O2-H5 bond within a surface-bonded hydroxyl group has higher kinetic energy (manifested by large-amplitude vibration of R O2-H5 ) than the O1-H3 and O1-H4 bonds in the free water molecule (see Fig. 5(b)). As demonstrated in the above example, branching H-bonds formed with surrounding water molecules (e.g. O3-H3 in Fig. 4) are actively involved in the proton transfer along the H-bond chain.
To quantify the effect of the surrounding water molecules (i.e. solvation shell) on the energy barrier for H 2 production, we consider the same H 2 -production mechanism as in Fig. 3(a) (with l = 1) by adding a surrounding water molecule; see H4-O2-H5 in Fig. 6(a). The extra water H4-O2-H5 serves as a proton donor to form a branching H-bond O2-H4· · ·O1. In order to study the dependence of the reaction energy barrier on the H4-O1 distance, we perform NEB calculation to obtain the H 2 -production reaction path, where R O1-H4 between H4 and O1 is constrained to a fixed value. The range of the constraint R O1-H4 is chosen in reference to the equilibrium O-H distance (1.75 Å) of H-bonds, which is the second peak position of the radial distribution function g(r) in our Al 18 MD simulation (see in Fig. 6(b)). (The first peak of g(r) signifies the covalent O-H bond length within water molecules.) By varying R O1-H4 = 1.75, 1.9, 2.0, and 2.1 Å, we obtain the energy barrier as a function of R O1-H4 as shown in Fig. 6(c). The energy barrier is reduced to below 0.02 eV (the blue dashed line) when R O1-H4 is 1.75∼2.0 Å, and the barrier increases to 0.08 eV for R O1-H4 = 2.1 Å. In the limit, R O1-H4 → ∞, we recover the case considered in Fig. 3 (i.e., in the absence of surrounding water molecules), where the energy barrier is 0.14 eV as indicated by the red dashed line in Fig. 6(c). This result indicates that branching H-bonds can greatly reduce the energy barrier of hydrogen production hence can increase the reaction rate.
To study the energy barrier with a larger solvation shell, we next perform NEB calculations using configurations from MD simulations, where the positions of the surrounding atoms (i.e., those outside the superatom and the reacting H-bond chain) are fixed. (A similar procedure of holding the surrounding water atoms to calculate the minimum energy path was used in Ref. 43.) Figures 7(a) and 7(b) respectively show the reactant configuration (corresponding to Fig. 4(a)) and the product configuration ( Fig. 4(f)) of the H 2 -production reaction in the MD simulation of the Al 16 system. For each configuration, we calculate the energy barrier of the same H 2 -production reaction involving Al 16 with the H-bond chain of length l = 2 using NEB, where the surrounding-atom positions are fixed. To prepare the initial state for the reactant configuration, we minimize the energy by relaxing the positions of the reacting H and O atoms (i.e. those in the reacting H-bond chain, which are colored in Fig. 7(a)) with all the other atoms fixed. To prepare the final state for the same reactant configuration, we move the reacting H and O atoms along a reaction path to form a product H 2 . The positions of the reacting H and O atoms are then relaxed to minimize the energy and obtain the final state for the reactant configuration. 44 Given the initial and final states, the reaction path with the surrounding atoms in the reactant configuration is calculated by NEB (see the red curve in Fig. 7(c)). The calculated final-state energy for the reactant configuration is 0.48 eV higher than the initial-state energy and is not stable. The initial and final states for the product configuration are prepared in a similar manner, and the calculated energy profile along the reaction path is shown by the red curve in Fig. 7(d). The reaction energy barrier for the product configuration is calculated to be 0.23 eV. The time difference between the reactant and product configurations in the MD simulation is ∼ 50 fs, which does not allow the superatom to deform noticeably. Therefore, the difference of Al 16 geometry between the reactant and product configurations is ignorable, and the difference in the energy barrier should arise from the different surrounding H-bond networks. The large energybarrier difference (from 0.48 to 0.23 eV) indicates the significance of concerted rearrangement of surrounding atoms to promote H 2 -production reaction. Since hydrogen molecules are not physically adsorbed to superatoms, holding non-reacting atoms fixed makes the H 2 confined closer to the superatom. Accordingly the final-state energy is higher than the initial-state energy (by 0.05 eV for the red curve in Fig. 7(d)).
To confirm the barrier reduction due to surrounding water in other systems, we also calculate H 2 production in the Al 18 system with the H-bond chain length l = 1 and 2. The corresponding energies along the reaction paths are respectively given by the blue and black curves in Figs. 7(c) (for the reactant configuration) and 7(d) (for the product configuration). For the reactant configurations, the energy barriers are typically less than 0.70 eV, while the product configurations are characterized by much lower energy barriers ( ∼ 0.05 eV). This again demonstrates the significant role of surrounding-atom configurations in lowering the energy barrier. The H 2 products in these two configurations are not tightly confined to the superatoms, and accordingly the calculated final-state energy is high (> 0.8 eV).
During our MD simulations, the Al 17 superatom splits into an Al 16 superatom plus one free Al atom, and the Al 18 superatom undergoes great deformation. These observations suggest that the geometry of the superatom plays an important role in the reactions. To investigate the effect of superatom deformation on H 2 production, Fig. 8 compares the energy barrier of H 2 production (see Eq. (4)) for Al n in the minimum-energy configuration and that for a deformed Al n taken from MD simulation with the H-bond chain length l = 1.
(4) Figure 8(a) is the initial and final states for the system involving the minimum-energy Al 18 . They are prepared in the same way as in Fig. 3(a). Figure 8(b) is the initial and final states for the system with a deformed Al 18 , of which atomic positions are taken from MD simulation and then O and H atom positions are relaxed to the local energy minimum. Figure 8(c) shows that the energy barrier is reduced from 0.14 eV (corresponding to Fig. 8(a)) to 0.004 eV (corresponding to Fig. 8(b)) due to the deformation of Al 18 . This is because the deformation of the superatom changes the effective charge of each site, in particular modifying the strength of the Lewis base site. This is reflected in the length of the Al-H bonds. For the minimum-energy Al 18 in Fig. 8(a), the Al-H bond length marked by the red circle is R(Al sym. -H) = 1.611 Å, which is shorter than R(Al def. -H) = 1.718 Å of the deformed Al 18 in Fig. 8(b). The same deformation effect is also observed for Al n (n = 16,17) as shown in Fig. 8(c). Besides the reduction of the energy barrier, deformation enhances the diffusion of surface H atoms on the superatom surface. Since a surface H atom bonded to the strongest base sites has the lowest energy, deformation drives the surface H atom to diffuse around several Lewis base sites, as the strength distribution of the Lewis base sites changes due to geometric deformation. This explains why we see migration of surface H atoms, reflected in spikes on the Al-H distance curve in Fig. 1(b). It is also worth noting that the Lewis-base character is quite sensitive to radicals (-OH etc.) bonded to the superatom. 20 Besides revealing plausible mechanisms of H 2 production, our MD simulation of the Al 18 system reveals the oxidization mechanism of Al atoms in water. The Al 18 -superatom undergoes higher geometric deformation, and hence the Al-(OH)-Al bridge bond is more favorable in an Al 18 system. Such bridge bonds seen in the Al 18 system are formed almost linearly depending on the oxidation level, i.e. the number of H 2 molecules produced, while on the contrary we did not observe stable bridge bonds in the Al 16 and Al 17 systems. Figure 9(a) through 9(c) shows the formation of bridge bond Al1-(O1H1)-Al2 in about 45 fs. The H-bond O1-H2· · ·O2 exerts force to drive O1 toward Al2, and the transfer of H2 from O1 to O2 occurs during the formation of the bridge bond. Subsequently, the O1-H1 bond in bridge bonds breaks to form Al1-O1-Al2 during Fig. 9(d) through 9(f). Al-O-Al bonds are basic building blocks of aluminum oxide, and the observed reaction from Al n (H 2 O) m to Al-(OH)-Al to Al-O-Al provides such an oxidation mechanism. This describes the formation of an aluminum-oxide layer, which covers aluminum particles to prevent the thermodynamically favorable reaction, 2Al + 6H 2 O → 2Al(OH) 3 + 3H 2 . According to the above oxidation mechanism, if the oxidation of Al-superatoms should be limited, the intermediate state, Al-(OH)-Al, should be avoided. The facts that Al 16 and Al 17 systems do not form Al-(OH)-Al bridge bonds and temperature-assisted geometric deformation facilitates such reactions provides a guideline for choosing the proper superatom size and reaction temperature for efficient H 2 production.
IV. SUMMARY
Our quantum MD simulation reveals H-bond assisted mechanisms for hydrogen production by the reaction of aluminum superatoms with water. Proton transfer along a chain of H-bonded water molecules is involved not only in the production of water but also in the splitting of water. Simulation results reveal the important role of solvation shell in accelerating the reactions. The NEB method is used to calculate various reaction paths, showing that H-bond chains as well as surrounding H-bonds in the solvation shell can substantially reduce the reaction energy barrier, thereby enhancing the reaction rate drastically. The character of Lewis acid and base sites is strongly dependent on the geometry of superatoms. However, thermal deformation of superatoms, as well as the delocalized nature of the H-bond-chain assisted reaction, makes the size-selectivity of the reaction less profound in liquid water compared with gas-phase reaction. Furthermore, the formation mechanism of aluminum-oxide layers coating aluminum in water is proposed. | v3-fos-license |
2018-04-03T00:35:58.150Z | 2016-11-22T00:00:00.000 | 13369882 | {
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} | pes2o/s2orc | Comparison of antioxidant activities among four kinds of Japanese traditional fermented tea
Abstract Antioxidant activities of four kinds of Japanese traditional fermented tea, Gishi‐cha, Ishizuchi‐kurocha, Awa‐bancha, and Batabatacha, were compared. Antioxidant activity was evaluated by three parameters: copper ion reduction ability, radical trapping ability, and oxygen consumption rate. Processes of fermentation of these fermented teas are different. Goichi‐cha and Ishizuchi‐kurocha are produced by a two‐stage fermentation process, aerobic fermentation and subsequent anaerobic fermentation. Awa‐bancha is produced by anaerobic fermentation. And batabata‐cha is produced by aerobic fermentation. Additionally, unfermented green tea was also employed as control. These tea leaves were extracted by boiling water and measured antioxidant activities. And concentrations of caffeine and catechins were measured in green tea and in the four kinds of fermented tea: Ishizuchi‐kurocha, Goishi‐cha, Awa‐Bancha, and Batabata‐cha. Concentrations of caffeine and catechins were lower in the fermented teas than in green tea. Among the fermented teas, epigallocatechin content was the highest in Ishizuchi‐kurocha, whereas Batabata‐cha hardly contained any epigallocatechin. Goichi‐cha, Ishizuchi‐kurocha, and Awa‐bancha showed antioxidative activity regardless of measurement method. Batabatacha had hardly any antioxidative activity. Among the fermented teas, Ishizuchi‐kurocha had the strongest antioxidant activity. The antioxidative activities of green tea and the four kinds of fermented tea were significantly different among each other (p < .01). Implication of this study is as follows: although contents of catechins were lower than that of green tea, three kinds of fermented tea showed antioxidative activity comparable to green tea. The results suggest that anaerobic fermentation process is beneficial at least for antioxidative activity.
postfermented tea is made by fermentation of tea leaves by microorganisms. Simply stated, the postfermented tea is a pickle of tea leaves.
In many cases, the characteristic flavor of fermented tea is produced by fungi and lactic acid bacteria. The most famous postfermented tea is Pu-erh tea. Traditional fermented tea is made in only two areas in the world. One is the area of Yunnan (China), Myanmar, and northern Thailand. Postfermented teas in this area are Pu-erh (Yunnan), Lahpetso (Myanmar), and Miang (northern Thailand). The other area is Japan. There are four kinds of traditional postfermented tea in Japan: Ishizuchi-Kurocha in Ehime, Goishi-cha in Kochi, Awa-bancha in Tokushima, and Batabata-cha in Toyama. Among these postfermented teas, three varieties are made on Shikoku Island. The processes of fermentation and production of these four kinds of postfermented tea are different, respectively (Murakami, 1990;Nakagawa, 1979;Ukeda, 2013) (Figure 1). Ishizuchi-Kurocha and Goishi-cha are produced by a two-stage fermentation process, aerobic fermentation mainly by fungi, and subsequent anaerobic fermentation mostly by lactic acid bacteria. In the process of production of Ishizuchi-kurocha, tea leaves are kneaded after aerobic fermentation; this is not the case for Goishicha. Awa-bancha is produced only by anaerobic fermentation by lactic acid bacteria. Batabata-cha is produced only by aerobic fermentation by fungi. For these reasons, Japanese postfermented teas each have a unique flavor. Recently, postfermented tea attracted attention not only as a favorite beverage but also as a functional beverage (Noguchi, Hamauzu, & Yasui, 2008;Shimamura, Matsuura, Moriyama, Takeda, & Ukeda, 2008;Yoshioka et al., 2013). Two factors can contribute to the functional effect of the postfermented tea. One is the effect mediated by the microorganisms such as lactic acid bacteria. However, association of the fermentation-related microorganisms to the beneficial effects of the teas remains unclear. The other is the effect of functional compounds present in the tea extract for drinking. Ingredients of postfermented tea are different from those of green tea because of fungal and/or bacterial metabolic activities (Kato et al., 1993(Kato et al., , 1995. Therefore, fungi and bacteria involved in the fermentation are important for functional properties of postfermented tea. In this study, we focused on antioxidant activity of tea. Antioxidant activity of postfermented tea has been studied previously in Goishitea. Goishi-tea shows superoxide anion scavenging activity of the same magnitude as green tea does (Shimamura et al., 2008). It is also reported that Awa-bancha has a strong antioxidant activity (Masuda et al., 2013). Nevertheless, there are no studies on the comparison of antioxidant activities among postfermented teas by the same method.
Generally, strong antioxidant activity of green tea is derived from catechins, which are some of the polyphenols present in tea leave (Matsuzaki & Hara, 1985). Epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) are known as the catechins of green tea. Among these catechins, EGCg has the strongest antioxidant activity (Nanjo, 1998). Catechins are converted into theaflavin, proanthocyanidin, and theasinensin by polymerization during production of black tea (Nanjo, 2000). Although antioxidant activities have been studied in green tea and black tea varieties, details of antioxidant activity of postfermented tea remain unclear. Chemical changes in catechins in tea leaves during microbial fermentation are poorly understood, and the association of these molecules with the antioxidant activity of postfermented tea remains unknown. Therefore, in this study, we measured concentrations of catechins in postfermented teas and compared the antioxidant activities.
Antioxidant activity of an antioxidant compound depends on the radical-scavenging ability (Niki & Noguchi, 2000). The simplest method for evaluation of antioxidant activity is DPPH radical-scavenging activity. Because the DPPH method evaluates the scavenging ability toward artificial radicals per unit of time, the measured antioxidant activity depends on time. This is a demerit of the DPPH method.
Although the method of oxygen radical absorbance capacity (ORAC) is a widespread approach to evaluation of antioxidant activity of foods (Cao, Alessio, & Cutler, 1993), this method cannot distinguish whether the cause of the antioxidant activity is the concentration or reactivity of the relevant compounds (Niki, 2010). In this study, the antioxidant activities were measured by two kinds of probes, which have a different rate of reaction with radicals (Takashima et al., 2012). This is an improved method, free of the drawback of the ORAC method.
Antioxidant activity in terms of lipid peroxidation was measured by oxygen consumption. Additionally, the metal ion-reducing ability was evaluated because metal ions generate radicals in the human body.
We evaluated all the results on these antioxidative properties and estimated total antioxidant activity of postfermented tea.
| Tea materials
All tea leaves were purchased in 2014. Common green tea leaves that were produced in Kagawa, Japan, in 2014 as "Takase-cha" (Nishimorien, Takamatsu, Japan) were also purchased as a control.
| Analysis of caffeine and polyphenols
Caffeine and polyphenols in tea extracts were determined by highperformance liquid chromatography (HPLC) based on the method described by Huang et al. (Huang, Inoue, Li, Tanaka, & lshimaru, 2004).
One hundred milliliters of boiling water was added to 1.5 g of dry tea leaves with incubation for 5 min. Then, the extract was passed through No. 2 filter paper (Advantec Toyo Kaisha, Ltd., Tokyo, Japan) and a 0.45-μm membrane filter (Advantec Toyo Kaisha). The filtered extract was applied to a Shimadzu LC-10A HPLC system (LC-10AD pump, SPD-M10AVP PDA detector, and SPD-10AV UV-Vis detector; Shimadzu Corporation, Kyoto, Japan). Analysis was performed on a Inertsil ODS-3 column (4.6 × 150 mm; GL Sciences Inc., Tokyo, Japan). The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) (buffer A) and acetonitrile (buffer B) under the following linear gradient conditions: 10-50% B at 0-30 min, 10% B at 33 min, and 10% B at 40 min. The flow rate was 1.0 ml min −1 and column temperature 30°C.
Elution was monitored by absorbance at 280 nm. The standard was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
| Evaluation of copper ion-reducing activity
It is known that metal ions generate radicals. Thus, the metal ionreducing activity of postfermented tea is involved in its antioxidant activity. For evaluation of copper ion-reducing activity, 100 ml of boiling water was added to 1.5 g of dry tea leaves with incubation for 5 min.
The extract and twofold dilution of the extract with distilled water were subjected to the measurements. Reducing activity of the tea extracts toward Cu(II) was measured with the TAC Assay Kit (Metallogenics Co., Ltd., Chiba, Japan) as total antioxidant capacity (TAC) (Campos, Guzmán, López-Fernández, & Casado, 2009). Cu(II) was reduced to Cu(I) by the antioxidants in the tea extract, and the Cu(I) formed a complex with bathocuproine. The absorbance of the Cu(I)-bathocuproine complex (490 nm) was measured on an Infinite F200 PRO microplate reader (Tecan Group, Ltd., Männedorf, Switzerland).
| Evaluation of the radical-scavenging ability
For evaluation of this ability, 100 ml of boiling water was added to 0.3 g of dry tea leaves with incubation for 5 min. The extract was diluted to an appropriate concentration with distilled water. Details of the method have been described previously (Takashima et al., 2012).
The ability of the tea extract to scavenge radicals was assessed on the basis of the effects on the rate of decay of fluorescein (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and pyrogallol red (PGR, Aldrich Chemical Company, Inc., Milwaukee, WI, USA) in a reaction with free radicals generated by 2,2′-azo-bis(2-methylpropionamidine) dihydrochloride (AAPH; Wako Pure Chemical Industries). Next, the reaction rates of fluorescein and PGR with free radicals in phosphatebuffered saline (PBS) were determined by measuring probe decay at 494 and 540 nm by means of a UV-VIS spectrophotometer (Shinadzu UV1800, Shimadzu Corporation, Kyoto, Japan) equipped with a thermostated cell maintained at 37°C for 2 hr.
The number of radicals (n) that were trapped by one molecule of an antioxidant was estimated using the following formula: where t, [IH], and R i are lag phase time (s), antioxidant concentration (mol\L), and the rate of radical flux from the azo initiator (mol [l·s] −1 ), respectively. In this analysis, R i was estimated by measurement of Trolox (Cayman Chemical, Ann Arbor, MI, USA) because the tea extract contains a mixture of several antioxidants.
| Evaluation of antioxidant activity by oxygen consumption
Here, we assessed the antioxidant activity of tea extracts toward oxidation of methyl linolate in micelles of cholic acid (pH 7.4) (Escobar, Salvador, Contreras, & Escamilla, 1990;Umeno et al., 2015). One hundred milliliters of boiling water was added to 0.3 g of dry tea leaves with F I G U R E 1 Manufacturing processes of fermented tea. Awa-bancha is produced by only anaerobic fermentation. Batabata-cha is produced by only aerobic fermentation. Goishi-cha and Ishizuchi-kurocha are produced by two-step fermentation: aerobic and subsequently anaerobic fermentation. The tea leaves including branch and nut are harvested in early summer. The tea leaves are boiled or steamed for enzyme inactivation and sterilization. In process of Awa-bancha and Ishizuchi-kuroha, tea leaves are kneaded for development of anaerobic fermentation. The tea leaves are dried by sunlight after fermentation. We referred several reports for manufacturing process 1-3 and we also heard manufacturing process by producers incubation for 5 min. Next, the extract was passed through a 0.45-μm membrane filter (Advantec Toyo Kaisha, Ltd.). Micelles were prepared by addition of methyl linolate (0.2 vol%) to 5 ml of 0.1 mol/L cholic acid with vortexing for 2 min. After that, 0.02 ml of the tea extract was added to the micelles with mixing. Then, 5 mmol/L of a radical initiator, AAPH, was added with incubation at 37°C. A decrease in oxygen concentration caused by oxidation of methyl linolate was measured by means of an Oxygen monitor with Clark's oxygen electrode (model 5300, YSI/Nanotech Ltd., Kawasaki, Japan) as a function of time.
| Reproducibility
In order to confirm reproducibility, experiments were conducted at least two times.
| Concentrations of caffeine and polyphenols in tea extracts
The extraction conditions recommended by a producer are different for each fermented tea. In this study, however, each postfermented tea was prepared under the same conditions: 100 ml of boiling water was added to 1.5 g of tea leaves with incubation for 5 min. Concentrations of caffeine and catechins per 100 ml of each tea extract are shown in Table 1.
Compared with green tea as a control unfermented tea, caffeine content of postfermented tea was low. Catechin content of postfermented tea was also lower than that of green tea. Among the postfermented teas, the concentration of all catechins was the highest in Ishizuchikurocha. In particular, the concentration of EGC in Ishizuchi-kurocha was high. On the other hand, Batabata-cha hardly contained catechins.
The concentration of catechins was the lowest in Awa-bancha. As for Ishizuchi-kurocha and Awa-bancha, two kinds of tea leaves that were produced by different companies were analyzed. Differences in caffeine and catechins content between the leaves from the two producers were small. Concentrations of caffeine and catechins depend on the fermentation process. As for Goishi-cha and Batabata-cha, tea leaves produced by some producers were blended into one product.
| Evaluation of antioxidant activity of postfermented tea
Antioxidant activities of postfermented teas were measured by three methods. The reducing efficiency of tea extracts against Cu(II) was
Fermentation method
Caffeine EGC EGCg EC GCg Ecg
Total catechins
Green tea Nonfermentation 43.7 a 24.5 a 45.9 a 9.5 a 0.9 a 6.9 a 87.8 a measured first ( Table 2). The reducing efficiency was the strongest in green tea. In ascorbate equivalents, the antioxidant activity of green tea was 2.6 mmol/L. The antioxidant activity of Ishizuchi-kurocha 1 was 2.1 mmol/L. The antioxidant activities of Goishi-cha, Awabancha, and Ishizuchi-kurocha 2 were approximately 1.2-1.9 mmol/L.
In contrast, the antioxidant activity of Batabata-cha was 0.1 mmol/L.
Next, antioxidant activity was evaluated as radical-scavenging capacity. A fluorescent probe and a radical initiator were mixed with each tea extract and temporal decay of fluorescence as a result of oxidation of the probe was measured. Because reactivity of fluorescein with radicals is low, if antioxidants exist in the solution, oxidation of fluorescein by radicals is inhibited and fluorescence is retained. Therefore, antioxidant activity of the tea extracts was evaluated by means of time before decay of fluorescence (lag phase). The lag phase determined for each tea extract is shown in Figure 2. The slope of linear approximation is proportional to radical-scavenging capacity. Green tea showed the highest radical-scavenging capacity. Ishizuchi-kurocha, Goishicha, and Awa-bancha also showed radical-scavenging capacity. On the other hand, Batabata-cha hardly showed any radical-scavenging capacity in this assay. Overall, radical-scavenging capacity of the postfermented teas was ranked as follows: green tea > Ishizuchi-kurocha > Goishi-cha > Awa-bancha > Batabata-cha. This order is similar to the ranking of copper ion-reducing efficiency.
Additionally, radical-scavenging ability was measured using PGR, which is a high-reactivity probe for radicals. All postfermented tea extracts showed a certain radical-scavenging rate, but there were no significant differences. The effect was not concentration dependent.
Moreover, we examined antioxidant activity of the postfermented tea extracts by means of oxygen consumption. The radical initiator, AAPH, was added to a mixture of methyl linolate, cholic acid, and a tea extract, and then the oxygen consumption was measured. Although oxygen in these mixtures is consumed by AAPH, if an antioxidant compound is present, the consumption of oxygen is reduced. Results are shown in Figure 3. Green tea inhibited oxygen consumption as efficiently as it reduced copper ions and scavenged radicals. Overall, the ranking of inhibitory effects on oxygen consumption among the tea extracts was as follows: green tea > Ishizuchi-kurocha > Goishi-cha = Awa-bancha > Batabata-cha.
| DISCUSSION
In this study, we measured catechin content of Japanese traditional postfermented teas and evaluated their antioxidant activities. It is reported that catechins are involved in the antioxidant activity of green tea (Matsuzaki & Hara, 1985). The concentration of catechins in the postfermented teas was 29% of the concentration in green tea at a maximum (Ishizuchi-kurocha), and 0.5% at a minimum (Batabata-cha).
In Ishizuchi-kurocha, EGC content was high. Although catechin content of the postfermented teas was only ~30% relative to green tea August. For postfermented tea, grown leaves including branches and nuts are harvested. Therefore, there is a possibility that the concentration of caffeine and catechins during picking of tea leaves is already different between green tea and postfermented tea before the fermentation process. On the other hand, it is reported that contents of catechins were reduced in Awa-bancha and Ishizuchi-kurocha during fermentation (Kato et al., 1993(Kato et al., , 1995. In this study, we compared concentrations of caffeine and catechins among tea extracts as a "final product." Changes in caffeine and catechins content during a fermentation process were not analyzed. This is a task for a future study.
Of course, postfermented tea is a favorite beverage, and its unique taste and flavor are important. Nevertheless, sometimes consumers select foods and drinks based on physiological effects such as antioxidant properties. Antioxidant activity is one of the beneficial properties, which contributes to a favorable brand image of foods. Strong antioxidant activity and low caffeine content are hailed as good properties of postfermented teas. Although Batabata-cha hardly showed any antioxidant activity, it is not inferior to other postfermented teas.
From another perspective, caffeine content is lower than in Ishizuchikurocha and Goichi-cha. This is another good property for a favorable brand image. Thus, postfermented teas have a potential for favorable physiological effects. The results suggest that anaerobic fermentation process is beneficial at least for antioxidative activity.
F I G U R E 3 Evaluation of inhibition of oxygen consumption by fermented tea. Antioxidant activity of the tea extracts was assessed towards oxidation of methyl linolate in micelles of cholic acid (pH 7.4). The tea extract and 5 mM AAPH were added to the micelles with incubation at 37°C. A decrease in oxygen concentration caused by oxidation of methyl linolate was measured by means of an Oxygen monitor with Clark's oxygen electrode as a function of time | v3-fos-license |
2016-05-09T02:22:10.723Z | 2015-11-13T00:00:00.000 | 1954435 | {
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} | pes2o/s2orc | Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode
Background Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here. Methodology/Principal Findings FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAHT488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH. Conclusions/Significance The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.
Introduction
Fatty acid amide hydrolase (FAAH) is a hydrolytic enzyme belonging to the family of serine hydrolases. It has a wide substrate specificity, capable of hydrolysing N-acylethanolamines and N-acyltaurines. Structurally, the enzyme is a membrane-bound homodimer, whereby the substrate accesses its catalytic active site via a membrane channel [1]. The catalysis of the mostwell studied substrate, the endogenous cannabinoid ligand anandamide (arachidonoylethanolamide, AEA) is brought about by the formation of an acyl-enzyme intermediate in association with substrate binding to a triad of amino acids, Lys 142 , Ser 217 and Ser 241 [2,3]. Knowledge of the structure of rat FAAH, and subsequent studies using humanised rat FAAH, have resulted in a series of computational, mutagenesis and structural studies investigating the different classes of FAAH inhibitors with the enzyme (see e.g. [3][4][5][6][7][8][9][10][11][12][13]; for a review, see [14]).
In 1996, Paria et al. [15] reported that the non-steroidal anti-inflammatory drug (NSAID) indomethacin reduced the ability of uterine microsomes to hydrolyse AEA following treatment both ex vivo (100 μg/mouse s.c.) and in vitro (10 μM; [15]). The ability of indomethacin to inhibit FAAH is shared by a number of other NSAIDs including ibuprofen [16], flurbiprofen [17] and carprofen [18]. In an X-ray crystallography study of FAAH complexed with carprofen, Bertolacci et al. [11] reported that this NSAID was able to bind to an area at the entrance of the channel allowing the substrate access to the active site. In support of this model, the authors showed further that the potency of carprofen was lower in mutant FAAH T488A (a key residue in the interaction of carprofen with the enzyme) than in the wild-type FAAH [11].
Although the NSAIDs like ibuprofen, flurbiprofen and carprofen inhibit FAAH, their potencies are modest (in the middle to high micromolar concentrations) at physiological pH values. However, a number of structure-activity relationship studies have identified more potent compounds [13,[19][20][21][22]. Of these, the amide derivative of ibuprofen with 2-amino-3-methylpyridine (Ibu-AM5) is particularly interesting, since it retains the cyclooxygenaseinhibitory properties of the parent compound, and in vivo can alleviate visceral pain without producing overt ulcerogenic effects [19,21,23], as can the combination of an NSAID with an FAAH inhibitor [24]. The corresponding amide analogue of flurbiprofen (Flu-AM1) can also inhibit FAAH in submicromolar concentrations and retains the cyclooxygenase (COX)-inhibitory properties of the parent compound [22]. Most recently, a compound with elements of flurbiprofen and a carbamate-based FAAH inhibitor, that inhibits both FAAH and COX and which shows anti-inflammatory and gastroprotective properties, has been disclosed [25].
FAAH shows pronounced enantioselectivity towards inhibition by chiral irreversible phenyl alkylcarbamates, azetidine urea inhibitors and slowly reversible 1,3,4-oxadiazol-2-one inhibitors and by ibuprofen itself [8,9,13,17]. Both Ibu-AM5 and Flu-AM1 retain the chiral centre of the parent profens, and in a recent study published in this Journal [26], we reported that the two enantiomers of Flu-AM1 had similar potencies towards mouse brain FAAH. That paper was primarily focussed upon the COX-inhibitory properties of the Flu-AM1 enantiomers rather than upon FAAH, and the Ibu-AM5 enantiomers were not investigated. In the present study, we have investigated in detail the interaction between the enantiomers of Ibu-AM5, Flu-AM1 and rat FAAH using biochemical, molecular biological, and molecular modelling methodologies.
Ethics statement
Ethical permission for the animal experiments was obtained from the local animal research ethical committee (Umeå Ethical Committee for Animal Research, Umeå, Sweden). 1) were synthesised as described elsewhere [26]). AEA and URB597 (cyclohexylcarbamic acid 3 0 -carbamoylbiphenyl-3-yl ester) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Carprofen was obtained from Sigma-Aldrich Inc, St. Louis, (MO, U.S.A.). Substrates were dissolved in ethanol or DMSO as appropriate.
Synthesis of the enantiomers of Ibu-AM5 and ibufenac-AM1
Enantiomerically pure (R)(-)-Ibu-AM5 and (S)(+)-Ibu-AM5 (see Fig 1) were synthesized using a slight modification of the procedure previously described for the racemate [23]. Commercial (R)(-)-ibuprofen and (S)(+)-ibuprofen were coupled with 2-amino-3-methylpyridine in the presence of 1-(3-dimethylaminopropyl)-3-ethylcardodiimide hydrochloride (EDC) and hydroxybenzotriazole (HOBt) in acetonitrile solution. Melting points were determined on a Stuart Scientific Melting point SMP1 and are uncorrected. 1 H NMR spectra were recorded on a Varian Inova 500 spectrometer. The chemical shifts (δ) are reported in part per million downfield from tetramethylsilane (TMS), which was used as internal standard. Infrared spectra were recorded on a Bruker Vector 22 spectrometer in Nujol mull. The main bands are given in cm -1 . Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing Finnigan MAT 95 instrument with BE geometry. All products reported showed 1 H NMR spectra in agreement with the assigned structures. The purity of tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara, Italy, with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at least 95% as compared with the theoretical values. Optical rotations were assessed at 10 mg/mL in methanol with a Perkin Elmer 241 polarimeter in a 10 cm water-jacketed cell at 25°C. Reaction courses and product mixtures were routinely monitored by TLC on silica gel (precoated F 254 Merck plates), and compounds were visualized using an UV lamp. Reagents and solvents were purchased from the Sigma Chemical Co (St. Louis, MO, USA).
Preparation of rat and mouse brain homogenates
Brains (minus cerebella) from adult Wistar or Sprague-Dawley rats (killed by decapitation) and from male B6CBAF1/J mice (killed by cervical dislocation), stored at -80°C, were thawed, weighed and homogenized in cold buffer (20 mM HEPES, 1 mM MgCl 2 pH 7.0). Homogenates were centrifuged (35,000 g at 4°C for 20 min) before the pellet was resuspended in cold homogenization buffer. Centrifugation and resuspension was repeated twice. The suspension was incubated at 37°C for 15 min to degrade any endogenous substrate able to interfere with the FAAH assay. After centrifugation (35,000 g at 4°C for 20 min), the pellet was resuspended in cold buffer (50 mM Tris-HCl, 1mM EDTA, 3 mM MgCl 2 , pH 7.4). The protein concentration was determined according to [27] after which the samples were frozen in aliquots at -80°C.
Cloning and expression of FAAH wt and FAAH T488A in HeLa cells
The recombinant plasmid (pcDNA4) containing rat Flag-FAAH gene was kindly provided by Prof. Dale Deutsch (Department of Biochemistry and Cell Biology, Stony Brook University). The single mutant FAAH (T488A) was obtained using a Quick Change site directed mutagenesis kit (Agilent Technologies). The insertion of the corrected mutation was confirmed by DNA sequencing. The recombinant plasmids pcDNA-FAAH wt and -FAAH T488A were used to transfect HeLa cells by Lipofectamine Ltx (Life Technologies). After 24h of expression, HeLa cells were detached from the flask with trypsin, washed twice with phosphate buffered saline and resuspendend in 0.5 mL of the same buffer. Then, the cells were homogenized by means of a pellet pestle (Sigma). Particulate matter was removed by centrifuging at 3500 g for 20 min. Protein content was determined by [27] and wt or T488A FAAH expression was visualized by Western Blot analysis using an anti-FLAG antibody (Sigma Aldrich).
Assay of [ 3 H]AEA hydrolysis in the homogenates and lysates
The assays were performed according to [28] with minor modifications. Briefly, homogenates or cell lysates in assay buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.4) were mixed with substance dissolved in ethanol on ice. Hydrolysis reactions were initiated by addition of substrate ([ 3 H]AEA in 10 mM Tris-HCl, 1mM EDTA, pH 7.4 containing 10 mg ml -1 fatty acid-free bovine serum, assay concentration 0.5 μM unless otherwise stated) and incubation at 37°C. For the brain homogenates, hydrolysis at 37°C was allowed to proceed for 10 min (assay volume was 200 μL, with 1 and 0.3 μg/assay protein concentrations of the rat and mouse homogenates, respectively). For the transfected cells, assay conditions are given in the Figure legend. For the kinetic experiments, the AEA: fatty acid-free bovine serum albumin ratio was kept constant at 1:~4.5 (μM). Following incubation, reactions were stopped by addition of 400 μL activated charcoal (80 μL activated charcoal + 320 μL 0.5 M HCl) and placement of samples on ice before centrifugation at 2500 rpm for 10 min. Aliquots (200 μL) of the supernatant were analyzed for tritium content by liquid scintillation spectroscopy with quench correction. Sample sizes were chosen on the basis of previous data investigating FAAH inhibitory properties of compounds.
Computational studies
Direct interactions between compounds and FAAH were investigated combining molecular docking studies with molecular dynamics simulations. The simulations were performed with deprotonated Lys 142 , as proposed for the catalytic mechanism of FAAH [1]. A preliminary validation of the computational protocol was performed by reproducing the experimentally determined binding mode of the pyrrolopyridine inhibitor found in the crystal structure 3QK5, which showed the reliability of the procedure not only to reproduce the ligand binding, but also the position of key water molecules within the active site.
Molecular docking. Rigid docking calculations were performed using the software Autodock 4.2 [29]. The 3D structures of compounds were generated with pymol. Docking was performed on a single monomer of rat FAAH (PDB code: 3QK5 [30], monomer A), after crystallized ligand removal. Rigid docking was performed using a cubic docking box of 60 3 grid points, centred on the position of the original ligand (3- The rotatable bonds of the compounds were detected automatically by Autodock using default parameters. For each docking run, 100 iterations were performed using default parameters of Lamarckian genetic algorithm (GALS). The results were clustered on the basis of RMSD criterion ( 4 Å). The representative poses of the best two clusters of each system were used for further analysis.
Molecular Dynamics. MD simulations were performed with Amber12 [31] to refine best docking results. To this end, the representative poses of the best two clusters retrieved from docking calculations, representing the A-and B-mode for each ligand, were loaded in the two monomers of the dimeric form of rat FAAH (PDB code: 3QK5,) after removal of the pyrrolopyridine derivative and water molecules. Hence, a total of 8 systems were subjected to molecular dynamics analysis. A preliminary validation of the computational protocol was obtained by predicting the experimentally determined binding mode of the pyrrolopyridine derivative in the 3QK5 crystal structure. The non-covalent ligand of the X-ray structure 3QK5 was chosen as reference due to the higher structural similarity toward our compounds with respect to carprofen. It is worth noting that the computational procedure successfully reproduced the binding mode of the pyrrolopyridine inhibitor, as well as the distribution of water molecules around the ligand in the binding cavity (see S1 Fig).
Each complex was immersed in a pre-equilibrated octahedral box of TIP3P water molecules, and the system was neutralized. The final systems contained about 80000 atoms. All simulations were performed with the ff99SBildn force field [32] for the protein and the gaff force field [33] for the ligands. The charge distribution of the inhibitors was refined using RESP charges [34] fitted to the B3LYP/6-31G(d) electrostatic potential obtained with Gaussian09 [35]. For each complex, the geometry was minimized using convergence criterion for the energy gradient set to 0.01 kcal.(mol.Å) -1 in three steps, which involve: i) hydrogen atoms in the system (5000 steps of steepest descent and 10000 steps of conjugate gradient), ii) hydrogen atoms, water molecules and counterions (2000 steps of steepest descent and 18000 steps of conjugate gradient), iii) finally the whole system (2000 steps of steepest descent and 18000 steps of conjugate gradient). Thermalization of the system was performed in four steps of 60 ps, increasing the temperature from 50 to 298 K. Concomitantly, the atoms that define the protein backbone were restrained during thermalization using a variable restraining force. Thus, a force constant of 30 kcal.mol −1 .Å −2 was used in the first stage of the thermalization and was subsequently decreased by increments of 5 kcal. mol −1 .Å −2 in the next stages. Then, an additional step of 250 ps was performed in order to equilibrate the system density at constant pressure (1 bar) and temperature (298 K). Finally, an extended trajectory was run using a time step of 2 fs. SHAKE was used for those bonds containing hydrogen atoms in conjunction with periodic boundary conditions at constant pressure and temperature, particle mesh Ewald for the treatment of long range electrostatic interactions, and a cutoff of 10 Å for non-bonded interactions. The structural analysis was performed using in-house software and standard codes of Amber 12. All the systems were subjected to 50 ns of molecular dynamics. One run for (R)-Flu-AM1 and one for (S)-Ibu-AM5 were extended to 100 ns in order to confirm the structural integrity of the proposed binding mode for the most active enantiomers.
Homology building. The amino acid sequence of mouse FAAH was retrieved from the Universal Protein Resource database (http://www.uniprot.org accession ID O08914). The 3D structure of the target protein was modelled using SWISSMODEL [36]. The X-ray determined structure of rat FAAH 3QK5 was used as template (covered sequence 100%; sequence identity of 91.7%). . The model assuming competitive interactions (where α!1) was also used, and the best fit was chosen by Akaike's informative criteria. Robust linear regressions for the Dixon plots, which are less sensitive to outliers than the standard least-squares methodology [37] were undertaken using the same computer programme.
Inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5
The inhibition of [ 3 H]AEA hydrolysis in rat brain homogenates by the enantiomers of Flu-AM1 and Ibu-AM5 is shown in Fig 1 and summarised in Table 1. For Flu-AM1, the potencies of the two enantiomers were very similar, with IC 50 values of 0.74 and 0.99 μM being found for the (R)-and (S)-enantiomers, respectively. In contrast, for Ibu-AM5, the (S)-enantiomer (IC 50 value 0.59 μM) was an order of magnitude more potent than the (R)-enantiomer (IC 50 value 5.7 μM). Ibufenac-AM1, lacking the methyl group on the C-2 carbon atom and thus lacking the chiral centre of Ibu-AM5, was a weak inhibitor of rat brain FAAH, with a pI 50 value of 4.17±0.04 (IC 50 value of 68 μM) as compared with the pI 50 value of 5.92±0.09 (IC 50 of 1.2 μM) for racemic Ibu-AM5 (Fig 1F). Time-dependency and reversibility experiments were undertaken for (R)-Flu-AM1 (Fig 2). There was a small degree of time-dependency for (R)-Flu-AM1, but it was less pronounced than for the irreversible inhibitor URB597. Thus, for example, the % activity remaining in the presence of 0.7 μM (R)-Flu-AM1 was 50±1% and 39±4% after preincubation for 0 and 60 min at 37°C, respectively. The corresponding values for 30 nM URB597 were 52±1% and 19±3%, respectively. Further, dilution experiments conducted after 60 min of preincubation indicated that the inhibition produced by (R)-Flu-AM1 was completely reversible in nature (Fig 2). The ability of the enantiomers of Ibu-AM5 to inhibit FAAH was also investigated in mouse brain homogenates ( Table 1). The pattern of enantiomeric selectivity for Ibu-AM5 was also seen in the mouse brain homogenates. However, the potencies of the enantiomers of Ibu-AM5 were about an order of magnitude lower in the mouse brain homogenates compared with the rat brain homogenates, and the same is true for the enantiomers of Flu-AM1. To aid the reader, the pI 50 and hence IC 50 values obtained in [26] for inhibition of mouse brain FAAH by the Flu-AM1 enantiomers are given in Table 1. (Fig 3A, left panel). For (S)-Flu-AM1, the corresponding values were 0.79±0.28 μM and 5.6±3.6, respectively (Fig 3B, left panel). Dixon plots were also constructed for the data (Fig 3A and 3B right panels). For a mixed-type inhibitor, the straight lines obtained at each substrate concentration should intersect at a value which, when projected onto the x-axis, corresponds to -K i [38,39]. For eight substrate concentrations as here, there are 28 different intersections. In an analogous situation, the direct linear plot of Eisenthal and Cornish-Bowden [40], those authors used the median values to solve the issue of undue influence of outliers on intersections. Using this approach, K i values for (R)and (S)-Flu-AM1 of 0.28 and 0.86 μM, respectively, were found.
Mode of inhibition of rat brain FAAH by (R)-and (S)-enantiomers of Flu-AM1 and
The two enantiomers of Ibu-AM5 were also investigated, and again, both datasets were better fitted by the linear mixed inhibition model than by a pure competitive inhibition model. (S)-Ibu-AM5 inhibited rat brain [ 3 H]AEA hydrolysis with K i and α values of 0.80±0.21 μM and 3.2±1.5, respectively, and the Dixon plot gave a K i value of 0.89 μM (Fig 3C). The corresponding values for the (R)-enantiomer were 10±2.5 μM and 3.1±1.1, respectively; Dixon plot K i value 9.8 μM (Fig 3D).
Multiple inhibition experiments. Experiments where two inhibitors are investigated together are a useful way of determining whether the compounds act in a mutually exclusive manner or in a cooperative manner [39]. In essence, the activity of the enzyme in the presence of different concentrations of both compounds is assessed and the data plotted as 1/v vs. the concentration of one of the compounds, i.e. a Dixon plot. For two compounds acting in a mutually exclusive manner, the Dixon plots should show a series of parallel lines, whereas for two compounds interacting in a co-operative manner, the lines should form a "V"-shape towards the y axis [39]. We investigated the interaction between either (R)-Flu-AM1 or (S)-Ibu-AM5 and carprofen (Fig 4). When analysing the data for carprofen per se, we noted that the Hill slope, n H was greater than expected (see S1 Appendix). However, this can satisfactorily be explained in terms of its interaction with the fatty acid-free bovine serum albumin in the assay, whereby the free carprofen concentration available for interaction with FAAH is not directly proportional to the added concentration (for analysis see S1 Appendix). Given the uncertainty concerning the free carprofen concentrations, we have presented the data showing the -AM compound concentrations on the x-axes and with the different lines corresponding to each added carprofen concentration (Fig 4). In this way, parallel lines vs. a "V"-shape can be identified without making assumptions about the free carprofen concentrations. There was no obvious evidence of a "V"-shape in the Dixon plots, suggesting that the inhibitors act upon FAAH in a mutually exclusive manner.
Inhibition of wild-type FAAH and FAAH T488A by (R)-Flu-AM1 and carprofen The simplest explanation for the mutual exclusivity of the -AM compounds and carprofen described above is that they share a binding site on the FAAH molecule. Given the report by Bertolacci et al. [11] that the potency of carprofen was lower towards FAAH T488A than wildtype FAAH, consistent with their model of carprofen interaction with the enzyme, we investigated (R)-Flu-AM1 and carprofen in HeLa cells transfected with the two recombinant FAAH proteins.
Initial time courses indicated that the catalytic activity of the FAAH T488A lysates was very low. In consequence, a long incubation time (18 h at 37°C) was necessary. Under these conditions, (R)-Flu-AM1 indeed inhibited AEA hydrolysis by lysates expressing wild-type FAAH more potently than by lysates expressing FAAH T488A (Fig 5A). In order to check that this difference was not a reflection of the different assay protein contents (lower for the wild type FAAH lysates than for the FAAH T488A lysates in view of their different catalytic activities), the inhibition of a mixture of wild-type lysates and lysates containing the empty vector (which lacked overt AEA-hydrolytic activity) were combined to give the same protein concentration as the FAAH T488A lysates. Addition of the lysates containing the empty vector did not affect the observed potency of (R)-Flu-AM1 towards wild-type FAAH (Fig 5A). Carprofen was also less potent in the FAAH T488A lysates than the wild-type FAAH lysates (Fig 5B), but the steep inhibition curves for the samples will tend to minimise changes in potency, assuming that the high n H values reflect saturation with fatty acid-free bovine serum albumin as a limiting factor (S1 Appendix). As for (R)-Flu-AM1, addition of the lysates containing the empty vector did not affect the observed potency of carprofen towards wild-type FAAH (Fig 5B). It was noted that the potency of (R)-Flu-AM1 towards wild type FAAH was lower than seen in the rat brain homogenates, but it is hard to compare these values given that the experimental conditions are so different. Indeed, in preliminary experiments using another batch of wild-type FAAH, 1.5-2 μg/assay and incubation times of 20 min at 37°C, the potencies of (R)-and (S)-Flu-AM1 (1.6 and 2.4 μM, respectively) and (R)-and (S)-Ibu-AM5 (8.8 and 0.53 μM, respectively, data from two experiments) were reasonably similar to the rat brain FAAH values.
Molecular dynamics studies
The binding modes of the enantiomeric forms of Flu-AM1 and Ibu-AM5 to the rat FAAH enzyme were studied by combining docking studies and MD simulations using a protocol which was successful in predicting the binding mode of non-covalent ligands (see Materials and Methods section). The docking results were ranked considering the cluster population and the binding energy. The results showed that the ligands bind to a region formed by the acyl chain binding (ACB) channel and the membrane access channel (MAC), filling the cavity experimentally found for other non-covalent ligands [10,11]. Two binding modes that differ in the orientation of the amide moiety were found. Thus, the amide moiety points toward either the catalytic triad (A-mode) or the membrane interacting helices α18-α19 (B-mode). The Bmode was found to be the most populated cluster for the two enantiomers, but the scores of A-and B-modes were highly similar, thus preventing a firm distinction between the ligand orientations (S1 Table).
To check the structural integrity of two binding modes, the best poses of each docking cluster were submitted to 50 ns of all atom MD simulation. As a general trend, the simulations starting from the B-mode remained stable along the whole trajectory, whilst the ligand showed significant rearrangements when the simulation started from the A-mode. As an example, this is reflected in the positional root-mean square-deviation (RMSD) profiles obtained for (R)and (S)-Flu-AM1, which show much larger fluctuations in the A-mode binding (S2 Fig). Moreover, the B-mode binding generally led to a consistent pattern of interactions in the binding pocket, whereas the A-mode exhibited less consensus in the interactions between ligand and protein. As an additional test, an independent MD simulation was run for (R)-Flu-AM1 bound to FAAH dimer starting from the B-mode binding, and the results confirmed the structural integrity of the ligand arrangement (see below). Therefore, in the following the discussion is limited to the structural features of the B-mode simulations. Three out of the four ligand-bound systems run for (R)-Flu-AM1 converged to a common binding mode, which is characterized by a stable hydrogen bond between the hydroxyl group of Thr 488 and the carbonyl unit of (R)-Flu-AM1 (Fig 6A), as noted in the similar arrangement obtained upon superposition of representative snapshots (see S3 Fig). The ligand is closely packed in the binding site, forming interactions that are preserved along most of the trajectory (Fig 6A). The biphenyl moiety fills a hydrophobic cavity, with the distal ring pointing to the centre of the anionic hole and forming van der Waals contacts with hydrophobic residues lining the ACB channel (Ile 491 , Phe 381 , Leu 380 , Ile 238 and Phe 194 ). No specific interactions were observed for the methyl group on the chiral centre. On the other side, transient hydrogen bonds were observed between the pyridine nitrogen and either the backbone of Asp 403 or alternatively Gly 485 (through a water molecule), and between the amide NH unit and the backbone of Leu 401 (S3 Fig). Van der Waals contacts are also formed with Met 436 and Ile 407 . The structural integrity of this binding mode was maintained upon extension of the trajectory up to 100 ns (data not shown).
The MD trajectories run for (S)-Flu-AM1 led to similar ligand poses in the two monomers of FAAH. The results showed that the amide bond establishes stable hydrogen bonds between the amide NH unit and the backbone of Gly 485 , and between the amide carbonyl with the Thr 488 side chain ( Fig 6B). Moreover, the biphenyl system formed van der Waals contacts with hydrophobic residues in the ACB channel (Leu 192 , Ile 491 , Phe 381 , Leu 401 , Phe 432 ). The methyl group on the chiral carbon pointed toward Leu 404 and Leu 401 .
Comparison of (R)-and (S)-Flu-AM1 binding modes is shown in Fig 6, which highlights how the enantiomers were bound in slightly different arrangements in the same site located between ACB and the MAC channels. Nevertheless, the enantiomers showed a very similar pattern of interactions: i) the aromatic rings formed a number of van der Waals contacts mainly involving aliphatic side chains, ii) the fluorine atom weakly interacted with Phe 381 , and iii) the amidopyridine moiety established hydrogen-bonds with Gly 485 and Thr 488 .
MD simulations of (S)-Ibu-AM5 B-mode converged to similar binding modes in the two monomers of FAAH. Extension of the MD simulation to 100 ns confirmed the stability of the B-mode, which remained stable for the last 70 ns of the simulation. (S)-Ibu-AM5 binds at the bottom of the ACB channel and the MAC (Fig 7A) with the pyridine nitrogen fitting the position of the carprofen carboxylic group (PDB code 4DO3) (S4 Fig). The pyridine ring is firmly packed in a hydrophobic cavity in the proximity of the membrane, interacting with hydrophobic residues at the gorge of the MAC (Trp 531 , Leu 429 and Ile 407 ). The amide unit of the ligand formed hydrogen-bonds with the backbone carbonyl of Gly 485 and to the Thr 488 side chain ( Fig 7A). The profen moiety is placed in the apolar gorge of the ACB with the methyl group on the chiral carbon pointing toward Leu 404 and Leu 401 .
Simulations run for (R)-Ibu-AM5 also supported the B-mode binding, leading to similar ligand arrangements in the two monomers. Compared to the (S)-enantiomer, most of the differences arise from the pattern of interactions established by the amidopyridine moiety, since a hydrogen bond was formed between the carbonyl unit of the amide group and the hydroxyl group of Thr 488 and the pyridine nitrogen formed a water-mediated contact with Gly 485 (Fig 7B), in addition to hydrophobic contacts with Trp 531 , Ile 407 and Phe 381 .
Discussion
In the present study, the interaction of the enantiomers of Flu-AM1 and Ibu-AM5 with rat FAAH have been extensively studied in order to determine the importance of the presence of the chiral centre and its configuration. (R)-Flu-AM1 was marginally more potent than the corresponding (S)-enantiomer as an inhibitor of FAAH, a result also seen for flurbiprofen [17]. In contrast, the (R)-enantiomer of Ibu-AM5 was 10-fold less potent an inhibitor of FAAH than the (S)-enantiomer, in direct contrast to ibuprofen, where the (R)-enantiomer is more potent than the (S)-enantiomer [17]. Interestingly, the presence of the methyl at the C-2 carbon atom of Ibu-AM5 is more important than its configuration for FAAH inhibitory activity, since ibufenac-AM1 was less potent an inhibitor of FAAH than Ibu-AM5.
The computational studies suggested that Flu-AM1 and Ibu-AM5 bind a region located between the ACB channel and the entrance of the MAC, filling the binding site of other noncovalent inhibitors, such as carprofen [11] (PDB code 4DO3) and the pyrrolopyridine inhibitor in the 3QK5 crystal structure (S4 Fig). Among the two possible binding modes found in docking studies, MD simulations showed that only the B-mode forms a relevant interaction with Thr 488 . This conclusion was supported by the finding that (R)-Flu-AM1 is a less potent inhibitor of the FAAH T488A mutant enzyme than of the wild-type enzyme. The data with the wild type and FAAH T488A mutant with carprofen are consistent with those of [11], although they found a larger shift despite a lower assay albumin concentration, and lend support to their model that carprofen interacts with Thr 488 at the entrance to the substrate binding pocket for rat FAAH. On the other hand, the proposed binding mode differs from the one proposed for a series of 1,3,4-oxadiazol-2-ones derivatives of ibuprofen and flurbiprofen [13]. A reasonable explanation of differences in the binding mode can be found in the higher polarity of the 1,3,4-oxadiazol-2-one moiety compared to the pyridine unit, which would make the coordination with the oxyanion hole the main driving force upon binding.
The comparison of the best representative poses of (R)-and (S)-Flu-AM1 showed very different binding modes for the two enantiomers, with the (S)-enantiomer binding deeper in the MAC. The same behaviour was observed from the MD simulations of (R)-and (S)-Ibu-AM5 (S5 Fig) suggesting that chirality is a main determinant of the binding mode. Interestingly, this behaviour seems to be associated to the conformational change of Phe 432 . In the MD trajectories of (S)-enantiomers the Phe 432 χ 1 dihedral switched from 90°to 180°, allowing the packing of the methylpyridine ring in a hydrophobic cavity formed by Trp 531 , Leu 429 and Ile 407 (S2 Table). The same Phe 432 rotamer (χ 1 = 180°) was found in the crystal structure of the rat FAAH in complex with an anandamide analogue (PDB code 1MT5). In contrast, in the binding of the (R)-enantiomers, Phe 432 assumes the same conformation found in 3QK5 and 4DO3 crystal structures (χ 1 = 90°).
The enzymatic assays showed that these differences in the binding mode slightly affected the activity of Flu-AM1 enantiomers, while it has more remarkable effect on the Ibu-AM5 enantiomers. The binding modes of (R)-and (S)-Flu-AM1 were characterized by a similar pattern of hydrophobic interactions and two hydrogen bonds that is in full agreement with the similar activities showed by these enantiomers. On the contrary, the (S)-Ibu-AM5 binding mode was more stable in the light of the higher member of H-bonds.
The above discussion has only considered a single binding site for the compounds. However, the kinetic experiments suggested a mixed type of inhibition, with a value of α in single figures. A mixed-type inhibition has often been suggested to imply an allosteric mode of inhibition, but this is not necessarily so, and there are many examples of compounds that produce kinetics that are not competitive despite their binding to the active site [41]. One possible mechanism, seen with some NSAIDs such as flurbiprofen (but not ibuprofen) and their interactions with COX, is a two-step reaction whereby an initial competitive interaction is followed by a slower tight-binding or even irreversible inhibition. However, in these cases, the timedependency is very marked, occurring over minutes [42]. In the present case, there was some apparent time-dependency with (R)-Flu-AM1, but it was very slow, and we have previously not seen such time-dependency with racemic Flu-AM1, despite its mixed-type mode of inhibition [22]. We thus do not favour this mechanism as an explanation for our data. A simpler mechanism for a fully reversible linear-mixed type inhibitor is for the compound to bind to two mutually exclusive sites [39]. Such a model would be consistent with the data, since all that is required in the multiple inhibition experiments with carprofen is mutual exclusivity rather than a specific locus of action [39].
We noted a difference in potencies between inhibition of rat and mouse FAAH by the enantiomers of Flu-AM1 and Ibu-AM5. Pronounced species-dependency has also been reported for carbamate and urea FAAH inhibitors [7,43], although these studies did not compare rat with mouse FAAH. Interestingly, the inhibition of mouse brain FAAH by (R)-Flu-AM1 is competitive rather than mixed [26]. In theory, differences in the amino acid compositions in the binding site of rat and mouse FAAH may account for the potency differences of the enantiomers in the two species. The alignment of the mouse and rat FAAH structures revealed only two differences within the (R)-FluAM1 binding site, Phe 194 to Tyr and Ile 407 to Val. We built a 3D model of the mouse FAAH to evaluate the effects of these mutations on the binding site. These mutations did not change the binding site shape, as shown in S6 Fig, and therefore they should not affect to a dramatic extent the inhibitory potency of (R)-FluAM1. This may mean that there is a species difference in the structure of the enzyme at this additional site. Further experiments are required to be able to identify the site on the enzyme.
Conclusions
The present study has used a range of biochemical, pharmacological, molecular biological and computational methods to explore the interaction of the enantiomers of Flu-AM1 and Ibu-AM5 with FAAH. Given the potentially useful properties of compounds with combined FAAH-COX inhibitory properties [25,44], the characterisation of the binding of the compounds to the substrate access channel of FAAH may provide vital information for the optimisation of such compounds. | v3-fos-license |
2020-05-07T09:03:30.076Z | 2020-02-12T00:00:00.000 | 219132064 | {
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} | pes2o/s2orc | The effect of additive containing an organic form of iodine on the physiological-biochemical parameters of the body of cows
P. G. Demidov Yaroslavl State University, Sovetskaya st., 14, Yaroslavl, 150003, Russia. Tel.: 48-52-797-702. E-mail: [email protected] Bogdanova, A. A., Alekseev, A. A., Flerova, E. A., & Konovalov, A. V. (2020). The effect of additive containing an organic form of iodine on the physiological-biochemical parameters of the body of cows. Regulatory Mechanisms in Biosystems, 11(1), 54–59. doi:10.15421/022007
Introduction
Stable increase in the production of livestock products requires well-organized provision of a nutritious and balanced diet to agricultural animals, one of the main factors of which is satisfaction of the organism's requirement for mineral substances. Insufficient and excessive intake of macro-and microelements has an inhibiting effect on the organisms of animals, decreasing their growth rates, productive qualities and causing various diseases (Hunchak et al., 2018). One of the vital microelements for the organisms of animals and humans is iodine (Voogt et al., 2010;Rasmussen et al., 2011;Hu et al., 2012;Flachowsky et al., 2014;Weng et al., 2014). Many scientists have noted the deficiency of this component in the soil and water in different countries. Iodine deficiency is observed in Africa, Asia, and also most of the world's developed industrial centers such as the Russian Federation and Austria, New Zealand, USA and Europe, which is reflected in the iodine concentration in plants and animal fodders made from them (Hu et al., 2012;Leung et al., 2012;Fuge, 2013;Vavilina & Rybkin, 2013). Organic introduction of iodine with the main diet into the organism of animals can cause malfunctioning of the thyroid as a result of insufficient formation of thyroxine hormone, which in turn leads to the development of endemic diseases, reduction of productive qualities, and decrease in the reproductive ability of animals (Nudda et al., 2009;Pattanaik et al., 2011;Rasmussen et al., 2011;Zimmermann & Boelaert, 2015).
To solve this problem, currently, in the feeding of agricultural animals, most often the non-organic form of iodine is used as an iodinecontaining component of mineral additives. In such form iodine is unstable and can oxidize, leading to decrease in its content in the feeds. Moreover, during the use of non-organic iodine in mineral mixes, it can take part in reactions with biologically active substances and form compounds which the organism is unable to digest (Vereshchak et al., 2012;Liu et al., 2015;Petrov & Gnezdilova, 2015). This way of including iodine in the diet makes the control of its ingression into the organism of animals impossible. As an alternative source of iodine, it is possible to use organic compounds of this element in which iodine is bound to a protein or amino-acid. Digestion of iodine present within such complexes is controlled by the system of homeostasis. This contributes to the cleavage of this microelement depending on the organism's requirement for it, and the excesses of organic iodine are removed naturally (Vereshchak et al., 2012;Gorlov et al., 2014;Rey-Crespo et al., 2014).
The biochemical control of the satisfaction of animals' requirements for iodine requires study of physiologic-biochemical parameters. Cur-rently, there are data on the impact of fodder additives which contain non-organic and organic forms of iodine on the organisms of cattle. Nonetheless, their effect on the biochemical parameters of blood, reproductive functions and dairy productivity of animals is studied insufficiently.
The objective of our studies was the impact of iodine-containing preparations on the physiologic-biochemical parameters of the organism of lactating cows.
Materials and methods
For the research, carried out at the PLC Agrofirm Pahma of Yaroslavsky District, using the method of pair analogues, in the period of milking, control (I) and experimental (II) groups of Ayrshire cows were formed, each containing 15 individuals . They were kept in stanchiontied stables during the winter stall period.
The animals of the control and experimental groups received a daily diet, including (kg/individual): cereal-legume silage -12, maize silage -13, cereal hay -1, combined fodder -10, flattened grain -1; soybean grist -2, premix -0.1, barley hay -1, salt -0.12, monocalcium phosphate -0.05; fodder chalk -0.05. The nutrition value of the diet equaled 257.6 mJ. The diets corresponded to the norm for this age period (Kalashnikov, 2003). Together with the main diet and water, the cows received 10.43 mg of iodine per individual a day, equaling 0.45 mg of iodine per 1 kg of dry matter of the diet at the norm of 0.88-1.00 mg of iodine per 1 kg of dry matter of the diet (Kalashnikov, 2003, Romanenko et al., 2014. As an additional source of iodine, non-organic iodine is used, which contains iodine potassium. The daily norm of this preparation for one animal was two tablets of 0.2 mg each according to the recommendations for this preparation. Over 30 days of the preparation period, both groups of lactating cows received the basic diet with potassium iodine (Technical Conditions 9347-016-46811620-2008), and in the following 60 days the animals of the experimental group were given feed with organic form of iodine instead of potassium iodine, and the control group continued to receive the same diet. In the final period (19-120th days) all the animals of both groups were fed with the basic diet with potassium iodine and the effect of the consequences of adding organic iodine-containing feed was studied (Table 1). Feeding with the fodder in the form of powder and potassium iodine in the form of tablets was carried out manually once a day during the morning feeding together with the basic diet. The amount of feed with organic iodine was calculated following the recommendations of the USA "Nutrient Requirements of Dairy Cattle" (National Research Council et al. Nutrient requirements of dairy cattle, 2001) and the All-Russia Research Institute for Animal Husbandry (VIZh) (Kalashnikov et al., 2003). Because 1 g of feed contains no less than 7 mg of organic iodine, the daily dose of it was estimated at 1.5 g per animal. In the control and experimental groups, on the 1st, 30th, 60th, 90th and 120th days, before the morning feeding, blood from the jugular vein was taken for determining the biochemical parameters. Activity of alkaline phosphatase, amount of urea, magnesium, phosphorus, total protein, cholesterol, albumin, activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the level of glucose in blood serum were determined using a STAT FAX 3300 biochemical analyzer with Diakon (Russia) and Vektor-Best (Russia) reagents kits. Calcium in the blood serum was determined using the complexometric method with indicator of fluorescein according to Vichev, Karakashev, the reserve alkalinity in blood serum was determined using the diffuse method with two flasks interconnected by a tube, and carotene in blood serum was determined according to Karp and Preis as modified by Yudkin (Kondrahin, 2004), the protein index was calculated, and iodine in blood serum was determined using the method of mass-spectrometry with inductively bond argon plasma. De Ritis coefficient was calculated as ratio of AST to ALT.
Record and evaluation of dairy productivity of lactating cows of both groups were performed by estimation of daily milking according to the data of monthly control milking made during the experiment. Physical-chemical properties of milk: dry matter, contents of fat, protein, lactose and the number of somatic cells were determined using a Ben-tleyCombi 150 analyzer.
During the evaluation of reproductive ability of the cows we considered the following parameters: the number of days before the first insemination, percentage of the animals inseminated over the first 90 days, service-period, index of insemination (number of inseminations necessary for productive insemination). In the study we used the data of zootechnical control.
The results were statistically analyzed in Statistica program (Stat Soft Inc., USA). For the evaluation of the surveyed parameters we used statistical methods. We calculated the mean arithmetic value and the standard error. The differences between the values in the control and experimental groups were determined using ANOVA, where the differences were considered reliable at P < 0.05. The results were determined as mean ± standard error (x ± SE).
Results
The parameters of the protein metabolism of lactating cows of the experimental and control groups were within the physiological norm (Table 2). During the course of the experiment, on the 90th day we observed a reliable increase in the parameter of total protein in blood serum of the animals which received organic form of iodine in the content of feed with organic form of iodine, which remained also on the 120th day (P < 0.05). Increase in the content of total protein in lactating cows of the experimental group occurred as a result of rise of the level of albumin by 15.4% and 7.9% on the 90th and 120th days respectively (P < 0.05) compared with the parameters of animals of the control group. As a result, on the 60th day of the experiment the coefficient of ratio of albumin and globulin or protein index in the experimental group of animals exceeded this parameter of the cows in the I group by 1.7%, and by the 90th and 120th days it was higher than such in the control by 14.5 and 7.0% respectively (P < 0.05, Table 2).
Research on the mineral metabolism in the animals that took part in the experiment did not reveal deviations in the surveyed parameters from the physiological norm (Table 3). It should be noted that the content of iodine in the blood serum of animals of the experimental group exceeded the value of this parameter in the control on the 90th and 120th days of the experiment by 13.4 and 8.8% respectively (P < 0.05). On the 90th day of the experiment, in the blood of lactating cows, the level of phosphorus increased by 7.4%, equaling 5.9% on the 120th or 30th days after feeding the animals with the organic iodine compared with the data of the control group.
As a result of our studies, we found no deviations from the values of the physiological norm of the parameters of activity of the enzymes in the blood serum of animals from the experimental and control groups (Table 4). On the 90th day of the experiment, in the blood serum of animals which received organic iodine, we observed increase in the parameter of alanine aminotransferase (ALT) by 11.6% compared with such value in the cows of the control group. On the 120th day, the experimental group was observed to have a significant increase in the activity of ALT equaling 16.5% compared with the parameter of the control.
On the 30th day after feeding with organic iodine was stopped, the animals of the II group were determined to have a decrease in the values of the de Ritis ratio equaling 13.5% compared with the parameter of the control group. The value of the alkaline phosphatase in animals of the II group of lactating cows increased by 11.3% on the 90th day compared with the animals of the I group, on the 120th day this tendency was maintained, the difference from the same parameter of the control group equaled 11.1% (P < 0.05). The level of glucose in the animals of the II group exceeded the same parameter in the animals of the control group by 3.1% and 2.3% on the 60th and 90th days of the experiment respectively (Fig. 1). According to the data of our research, the content of cholesterol in blood serum of cows of the experimental group was lower by 10.9% on the 60th and on the 90th day reliably reduced by 16.9% compared with the control (P < 0.05), and on the 120th day the decrease in this parameter accounted for 6.1% compared with the control (Fig. 2).
As a result of feeding with organic iodine, the lactating cows of the II group were observed to have an increase in the content of carotene by 9.1% on the 60th day and 10.3% on the 90th day of the study, and on the 120th the value of this parameter was 10.6% compared with the data of the control group (P < 0.05; Fig. 3).
According to the results of our studies, by the 60th day of the experiment, mass of the share of fat in both groups of cows increased (Table 5). The number of the somatic cells (NSC) in the milk of animals in the experiment did not exceed the allowable normative values. We should note that in the milk of animals which received organic iodine this parameter decreased by 19.1% and 28.4% on the 60th and 90th days respectively. This tendency was observed also on the 30th day after termination of providing feed containing the organic iodine.
Percentage of the inseminated animals after the first insemination comprised 60% in the experimental group of animals and 46.7% in the control. Analysis of the duration of the service-period revealed that in animals which received organic iodine this parameter accounted for 88 days on average and in the animals of the control -95.5 days, and a positive tendency towards duration of service-period was seen in the experimental group. It was determined that the index of insemination in the lactating cows of the II group was reliably lower than such in animals of the control group and accounted for 1.6 (Р < 0.05).
Discussion
This study demonstrated that the use of feed additive with organic iodine in the amount of 1.5 g per individual daily in feeding of the lactating cows during 60 days contributes to the activation of the hormoneforming functions of the thyroid, which is a stimulating factor for the intensification of the metabolic processes in the organism of animals.
The animals which received organic iodine were observed to have increase in the serum proteins, indicating absence of impaired metabolism. This is related to the fact that iodine introduced into the organism of animals positively affects the functioning of their thyroid, resulting in synthesis of hormones, most of which are bonded with blood proteins (Mellen & Hardy, 1957;Bartalena & Robbins, 1993;Power et al., 2000).
Different sections of the thyroid produce hormones directly involved in the phosphorus-calcium and mineral metabolism, and in particular are responsible for homeostasis and metabolism of calcium, phosphorus and magnesium (Mosekilde et al., 1990;Çaksen et al., 2003;Cardoso et al., 2014). Our study revealed increase in the parameters of mineral metabolism in the blood serum of cows which received organic iodine. Note: * P < 0.05 at comparison with the parameters of the control group. The study demonstrates the increase in AST and ALT activities in the blood serum of lactating cows the diet of which included organic iodinecontaining feed. This to some extent allows us to diagnose the absence of disorders and dysfunctions of the liver for these enzymes are the main liver enzymes. The activity of this organ is related to the thyroid hormones which regulate the main metabolism of hepatocytes, the cells of the parenchyma of the liver, which in turn regulates endocrine functions of the thyroid hormones (Malik & Hodgson, 2002;Chalasani et al., 2004;Moustafa et al., 2009;Mariani & Berns, 2012). During our experiment, the animals which received organic iodine were observed to have a decrease in the de Ritis Coefficient. This parameter in lactating cows of the experimental and control groups was more than one, which could indicate intensified activity of the heart muscle, but is considered the norm for highly-productive animals (Lapteva & Suvorov, 2018).
As a result of feeding the cows with an organic iodine-containing additive we determined increase in alkaline phosphatase in the blood of animals of the experimental group. This parameter is an indicator according to which one can judge the cell penetrability, regulation of fat and protein metabolisms, and also the digestion of mineral substances from the blood by the tissues (Wei et al., 2004). Increase in the content of glucose in the blood of the experimental group of cows indicates an increase in the process of gluconeogenesis, which mainly occurs in the liver, the functioning of which, as described above, is closely related to the correct functioning of the thyroid (Sinha et al., 2014;Jayanthi & Srinivasan, 2019).
Decrease in the cholesterol level in the blood of lactating cows which received organic iodine represents the activity of hormones of the thyroid towards the increase of lypolysis, implicating decrease in the concentration of cholesterol and triglycerides in the blood (Shin & Osborne, 2003;Gagnon et al., 2010). Furthermore, this indicates insufficient production of thyroxine hormone which activates the enzyme which, in turn, regulates the intensity of the cholesterol synthesis (Angelin et al., 2015;Balabaev & Derho, 2017).
Increase in the content of carotene was determined in the blood serum of animals which received the additive with organic iodine, representing increase in the vitamin metabolism in lactating cows. This may be an indicator of normal hormone-producing function of the thyroid (Zimmermann et al., 2007;Al-Rubae'I & Al-Musawi, 2011;Gunchak & Grimak, 2014).
The obtained results of the study indicate the correspondence of the action of the proposed iodine additive to the actions of the existing analogues (Krasnoslobodceva, 2010;Moschini et al., 2010;Gorlov et al., 2014;Bykova et al., 2017). Moreover, according to data of control milkings, we determined an increase in the dairy productivity and quality parameters of milk from cows of the experimental group, where the animals received organic iodine, compared with the control animals. Use of the feed with organic iodine led to the 3.9% increase in the daily yield of milk with 4.0% fat, 2.9% increase in the milk protein and reduction of NSC measuring 19.1% on the 60th day, and the tendency towards improvement of some parameters of dairy productivity remained also on the 90th day (milk yield of milk with 4.0% was 3.5% higher, NSC decreased by 28.4%). As a result of the effect of the additive, on the 120th day of the experiment the amount of the milk yield from the lactating cows of the experimental group exceeded the control values by 6.8%, and the content of milk fat was 5.1% higher at decrease in NSC by 21.2%. This indicates the fact that sufficient production of thyroid hormones positively correlates with the parameters of the dairy productivity of cows in the biosynthesis of the constituents of milk (Blum et al., 1983;Krasnoslobodceva, 2010).
The hormone regulation of the thyroid is known to directly affect the reproductive function of animals. The hormones of the thyroid have a stimulating effect on the differentiation of the ovarian follicles and intensify the production of luteinizing hormone which contributes to ovulation, formation of the corpus luteum and the development of the embryo (Pop-pe et al., 2007;Balabaev & Derho, 2016). According to the results of our studies, we found an improvement of the reproductive function of cows which received organic iodine compared with the animals of the control group: the parameters of the service-period and index of insemination of the animals which received the organic iodine-containing additive reduced by 8.5% and 15.8% respectively. This characterizes the surveyed additive as an efficient preparation for improvement of physiological-biochemical parameters of the organism and productive qualities of the animals.
It should be noted that in the 30 days after finishing the feeding with the fodder with organic iodine, the animals of the experimental group were observed to experience the continuing effect of the additive, i.e. higher values of the biochemical parameters of blood, increase in dairy productivity and improvement of the reproductive qualities compared with such of the control.
Conclusion
Thus, as a result of the research conducted, we determined that use of feed with organic iodine in the amount of 1.5 g per individual in the feeding of lactating cows over 60 days contributed to uniform ingression of iodine into the organism of animals and increased in its parameter in the blood serum. Furthermore, according to the results showing an increase in the parameters of all types of metabolism in the organism of animals which received the feed with organic iodine, we can presume that the additive has an effect on the hormone-producing function of the thyroid. It was found that all these factors together had a positive effect not only on the increase in the parameters of dairy productivity of animals which received organic iodine in the additive but also on their reproductive function. Nonetheless, to recommend this feed as a preparation which helps eliminate iodine-deficiency in feeding cows and for accurate analysis of its impact on the hormone-producing function of the thyroid of animals, further studies on the level of thyroid hormones in blood are needed. | v3-fos-license |
2021-05-11T00:06:52.507Z | 2021-03-18T00:00:00.000 | 234327158 | {
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} | pes2o/s2orc | Upconversion-based nanosystems for fluorescence sensing of pH and H2O2
Hydrogen peroxide (H2O2), a key reactive oxygen species, plays an important role in living organisms, industrial and environmental fields. Here, a non-contact upconversion nanosystem based on the excitation energy attenuation (EEA) effect and a conventional upconversion nanosystem based on the joint effect of EEA and fluorescence resonance energy transfer (FRET) are designed for the fluorescence sensing of H2O2. We show that the upconversion luminescence (UCL) is quenched by MoO3−x nanosheets (NSs) in both systems due to the strong absorbance of MoO3−x NSs in the visible and near-infrared regions. The recovery in UCL emissions upon addition of H2O2 enables quantitative monitoring of H2O2. Benefiting from the non-contact method, hydrophobic OA-NaYF4:Yb,Er can be used as the luminophore directly and ultrahigh quenching efficiency (99.8%) is obtained. Moreover, the non-contact method exhibits high sensitivity toward H2O2 with a detection limit of 0.63 μM, which is lower than that determined by simple spectrophotometry (0.75 μM) and conventional upconversion-based nanocomposites (9.61 μM). As an added benefit, the same strategy can be applied to the sensing of pH, showing a broad pH-responsive property over a range of 2.6 to 8.2. The successful preparation of different upconversion-based nanosystems for H2O2 sensing using the same material as the quencher provides a new design strategy for fluorescence sensing of other analytes.
Introduction
Hydrogen peroxide (H 2 O 2 ), an important bioactive molecule in living systems, plays an essential role in the physiological process including signal transduction, cell proliferation, differentiation, and maintenance. 1,2 Abnormal production or accumulation of H 2 O 2 will lead to severe damage to DNA and proteins, causing a series of serious diseases, [3][4][5][6][7] such as diabetes, Alzheimer's and Parkinson's disease, cardiovascular disorders, and even cancer. Additionally, H 2 O 2 is widely used as a bleaching agent and sterilant in industrial and environmental elds, 8,9 such as food processing, drinking water treatment, packaging, and organic pollutant degradation. However, exposure to high concentrations of H 2 O 2 is a great threat to organisms. 10,11 Therefore, quantitative detection of H 2 O 2 is of great importance for monitoring its potential risk.
Optical methods via uorescence changes have attracted considerable attention, as the uorometric approach is a nondestructive method that can be simply and rapidly performed with high sensitivity and selectivity. 12 In contrast to conventional uorescence probes (such as organic dyes, carbon nanomaterials, and semiconductor quantum dots), upconversion nanoparticles featuring large anti-Stokes shis, excellent chemical-and photo-stability, sharp multicolor emissions, and low toxicity have been regarded as a promising class of luminophores. 13 Up to now, a variety of functional materials including organic dyes, [14][15][16] noble metals, [17][18][19] quantum dots, [20][21][22] carbon nanomaterials, [23][24][25] and two-dimensional materials [26][27][28] has been employed to couple with upconversion nanoparticles to construct uorescence probes, realizing quantitative detection of inorganic ions, [29][30][31] pH, 32-34 small molecules, [35][36][37][38] and nucleic acids. [39][40][41][42] Most of the upconversion-based probes rely on the uorescence resonance energy transfer (FRET) process, in which a very short distance between the upconversion nanoparticles and absorbers is required. Moreover, in order to obtain high-sensitivity detection, high-quality upconversion nanoparticles with strong emission and high upconversion efficiency are employed, which are commonly prepared by applying oleic acid (OA) as the ligand. The oleate-capped upconversion nanoparticles are hydrophobic and prone to disperse in nonpolar solvents, whereas hydrophilic upconversion nanoparticles are required for typical sensing applications of interest. Therefore, the hydrophobic-to-hydrophilic transition of upconversion nanoparticles is essential. 43 Herein, we propose different upconversion nanosystems for H 2 O 2 sensing using MoO 3Àx nanosheets (NSs) as the energy acceptor based on either the excitation energy attenuation (EEA) effect or the joint effect of the EEA and FRET, owing to the strong absorbance of MoO 3Àx NSs in both visible and nearinfrared (NIR) regions. By coupling of MoO 3Àx NSs solution and oleate-capped NaYF 4 :Yb,Er upconversion nanoparticles (abbreviated as OA-UCNPs) solution, a EEA-based upconversion nanosystem for sensing of H 2 O 2 in the non-contact mode is designed, where MoO 3Àx NSs act as the energy acceptor of the incident light for the activation of UCNPs. Additionally, this system can be used for pH sensing as well. Beneting from the non-contact method, hydrophobic OA-UCNPs can be used directly for the sensing and ultrahigh quenching efficiency (99.8%) can be reached. Meanwhile, by the integration of hydrophilic UCNPs and MoO 3Àx NSs, we are able to prepare conventional upconversion-based nanocomposites for H 2 O 2 sensing via the joint effect of the EEA and FRET, where MoO 3Àx NSs act as the energy acceptor of not only the 980 nm exciting light for UCNPs but also uorescence emissions of UCNPs. To the best of our knowledge, this is the rst upconversion-based nanoprobe for the sensing of one analyte by two different systems while using the same material as an energy acceptor.
Characterization
Fourier transform infrared (FT-IR) spectra were recorded in transmission mode on a Thermo Scientic Nicolet iS5 FT-IR spectrometer with the KBr method. X-ray photoelectron spectroscopy (XPS) was measured with a Thermo Fisher Scientic ESCALAB 250Xi instrument. Transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS) were performed on the FEI Tecnai G2 20 S-TWIN with a LaB 6 cathode operated at 200 kV. UV-vis absorption spectra were acquired on a CARY 50 spectrophotometer. Powder X-ray diffraction (XRD) measurements were performed on a Philips X'Pert MPD Pro Xray diffractometer at a scanning rate of 4 min À1 in the 2q range from 10 to 80 (Cu Ka radiation, l ¼ 0.15406 nm). z-Potential measurements were carried out on an Anton Paar Litesizer™ 500 instrument. Upconversion luminescence (UCL) emission spectra were obtained on a ber-coupled spectrometer (Ocean HDX, Ocean Optics) with an external 980 nm continuous-wave (CW) laser (0-5 W, Roithner Lasertechnik GmbH) at room temperature (RT). Quartz cuvettes (0.7 mL, 10 mm  2 mm light path) were used for UV-vis absorption and UCL measurements.
Synthesis of MoO 3Àx NSs
MoO 3Àx NSs were prepared according to the previous publication with minor modications. 44,45 In a typical process, 1.5 g bulk MoO 3 powder was ground with 0.3 mL of acetonitrile for 30 min and then added to a water/ethanol solution (25 mL, v/v ¼ 1/1). The dispersion was then probe-sonicated for 2 h at 100 W (Branson Digital Sonier W-250D) at a 5 s ON and 2 s OFF pulse. To avoid overheating of the solvent, the beaker lled with MoO 3 dispersion was immersed in an ice bath during sonication. The light blue supernatant containing a high concentration of MoO 3 NSs (denoted as S-MoO 3 NSs) was collected via centrifugation at 7000g for 30 min. For the preparation of MoO 3Àx NSs, the supernatant dispersion was lled into a quartz glass vial and irradiated with a UV lamp (254 & 365 nm, 15 W) for 5 h, dark blue MoO 3Àx NSs solution was nally obtained, and the MoO 3Àx NSs solution was then diluted to 2 mg mL À1 by water and ethanol (v/v ¼ 1/1) solution, and stored at 4 C for further use.
Synthesis of OA-UCNPs
As previously reported, the synthesis of oleate-capped NaYF 4 : 20 mol% Yb, 2 mol% Er was carried out by employing OA as ligand via a high-temperature coprecipitation method. 46 Briey, in a 100 mL round ask, 3.12 mL of Y(CH 3 COO) 3 (0.2 M), 0.8 mL of Yb(CH 3 COO) 3 (0.2 M) and 0.8 of mL Er(CH 3 COO) 3 (0.02 M) were mixed with 6 mL of OA and 14 mL of ODE at RT. The mixture solution was rst heated to 110 C for 30 min to evaporate the water and then heated to 160 C for 40 min to form lanthanide-oleate complexes, followed by cooling down to 50 C. A methanolic solution (10 mL) containing 3.2 mmol of NH 4 F and 2.0 mmol of NaOH was slowly added and then stirred at 50 C for 30 min. Aer evaporating the methanol, the solution was heated to 310 C at a rate of 10 C min À1 and maintained for 30 min under nitrogen atmosphere. Aer cooling down to RT, OA-UCNPs were precipitated out with the addition of excess ethanol, collected aer washing three times with the ethanol, and nally dissolved in cyclohexane for further use.
Preparation of ligand-free UCNPs
Ligand-free UCNPs were prepared using our previously reported method. 47 5 mmol of formic acid was directly added to 2 mL of cyclohexane solution containing 20 mg of OA-UCNPs, ligandfree UCNPs were precipitated out aer shaking for 10 s at 3000 rpm on a vortex mixer. Bare UCNPs were obtained aer centrifugation and washing once with ethanol and three times with water and nally dissolved in water.
Synthesis of UCNPs/MoO 3Àx nanocomposites
To synthesize UCNPs/MoO 3Àx nanocomposites, PEI-capped UCNPs (abbreviated as PEI-UCNPs) was rst prepared. Typically, 4 mL ligand-free UCNPs solution (5 mg mL À1 ) were added to a vial containing 4 mL PEI solution (10 mg mL À1 ), followed by overnight stirring. PEI-UCNPs were collected aer centrifugation at 16 000g for 30 min and washing three times with water, and nally dispersed in water with a concentration of 1 mg mL À1 . UCNPs/MoO 3Àx nanocomposites were prepared by mixing 0.5 mL PEI-UCNPs solution with an appropriate amount of MoO 3Àx NSs solution, the mixture was rst shaken for 3 min (3000 rpm) on a vortex mixer and then ultrasonicated for 5 min. UCNPs/MoO 3Àx nanocomposites were then collected by centrifugation at 7000g for 30 min, washed three times with water, and redispersed in water.
Non-contact uorescence sensing of pH
To detect pH in the non-contact mode, OA-UCNPs dispersed in cyclohexane with a concentration of 1 mg mL À1 were sealed in a quartz cuvette, the cuvette was then aligned with the other cuvette containing 1 mg mL À1 MoO 3Àx NSs solution with different pH. The pH was adjusted by either 50 mM NaOH or 50 mM HCl ethanol/H 2 O (v/v ¼ 1/1) solution. The cuvette containing MoO 3Àx NSs was put in front of the other one containing OA-UCNPs solution, and the UCL spectra were collected under the excitation of a 4 W 980 nm CW laser.
Non-contact uorescence sensing of H 2 O 2
The non-contact sensing procedure for the H 2 O 2 was similar to that of the non-contact pH sensing, except that MoO 3Àx NSs were dissolved in acetate buffer (50 mM, pH 4.
Fluorescence sensing of H 2 O 2 by UCNPs/MoO 3Àx nanoassemblies
To detect H 2 O 2 , 0.5 mg mL À1 of UCNPs/MoO 3Àx aqueous solution (0.35 mg mL À1 MoO 3Àx NSs) and different concentrations of H 2 O 2 (0.4 mL) were added to 0.1 mL acetate buffer (50 mM, pH 4.5, DMF/H 2 O, v/v ¼ 1/1), The mixture was then incubated at RT for 2 h, and the UCL spectra were measured under the excitation of a 4 W 980 nm CW laser.
Design principle of upconversion-based nanosystems for H 2 O 2 and pH
The design strategy of UCNPs/MoO 3Àx nanocomposites for uorescence sensing of H 2 O 2 is based on the modulation of MoO 3Àx NSs-induced reduction in UCL emissions by H 2 O 2 through the joint effect of EEA and FRET. In contrast, the pH and H 2 O 2 dual-responsive upconversion-based nanosystem is realized by the direct adjustment of the excitation energy for UCNPs in the non-contact mode (Fig. 1a).
Without modications, UCNPs give rise to green and red luminescence emissions under 980 nm excitation. Aer the reduction of MoO 3 by UV light, the oxygen-decient MoO 3Àx NSs exhibit strong absorption in both visible and NIR regions, overlapping well with the UCL emissions of UCNPs and the excitation wavelength for UCNPs of 980 nm (Fig. 1b). Owing to the strong NIR absorption of MoO 3Àx NSs attached on UCNPs, the EEA will rst take place in the UCNPs/MoO 3Àx system when activated by the 980 nm light, resulting in a lowered intensity of excitation light arriving at the UCNPs, thus weakening the resulting luminescence emissions. Moreover, the efficient FRET process occurs through the spectral overlap between the absorption of MoO 3Àx NSs and the UCL of UCNPs in the visible region, leading to a further decrease in the intensity of luminescence emissions. Thus, the quenching in UCL of UCNPs is efficiently achieved by the joint effect of the EEA and FRET. However, upon the addition of H 2 O 2 , the oxygen-decient MoO 3Àx NSs can be oxidized back to MoO 3 (denoted as H-MoO 3 ), leading to the decrease of absorption in the visible and NIR regions (Fig. 1b), resulting in the recovery of UCL emissions via the reduction in EEA and FRET. Additionally, XPS was performed to evaluate the valence state of Mo in these nanosheets. As shown in Fig. 1c, the doublet peaks (235.9 eV and 232.8 eV) in the pristine MoO 3 sample are assigned to the binding energies of the 3d 3/2 and 3d 5/2 orbital electrons of Mo 6+ . Aer treatment by tip-sonication, two new peaks at lower binding energies (234.7 eV and 231.6 eV) appear in the obtained S-MoO 3 NSs, which can be assigned to the Mo 5+ oxidation state, and the integral area ratio of Mo 5+ /Mo 6+ is calculated to be 17.1% from the XPS spectrum. This phenomenon indicates that the MoO 3 is slightly reduced during the exfoliation process, showing weak absorption ability of S-MoO 3 NSs in visible and NIR regions (Fig. 1b) MoO 3Àx enables the ability of UCNPs/MoO 3Àx nanoprobes for H 2 O 2 sensing with high sensitivity. Additionally, the adjustment of pH or addition of H 2 O 2 in the acidic environment will lead to the variation of MoO 3Àx NSs in NIR absorption, and thus uorescence sensing of pH and H 2 O 2 can be achieved through the direct modulation of MoO 3Àx absorption-induced EEA in the non-contact mode.
Characterization of UCNPs, MoO 3Àx NSs, and UCNPs/MoO 3Àx nanocomposites Hydrophobic OA-UCNPs are synthesized by employing OA as the ligand via the high-temperature coprecipitation method. 46 OA-UCNPs present uniform hexagonal shape with a mean diameter of about 28 nm, which is revealed by the TEM measurement (Fig. 2a). The XRD pattern of the obtained OA-UCNPs with well-dened diffraction peaks agrees well with the standard data of hexagonal-phase NaYF 4 (JCPDS no. , demonstrating their high crystallinity (Fig. S1 †). Ligandfree UCNPs are prepared by direct addition of formic acid to the cyclohexane solution containing OA-UCNPs through the vortexing method and sequential modication with PEI to obtain PEI-UCNPs. 47 TEM images demonstrate unchanged morphology and size aer ligand removal and polymer functionalization (Fig. S2 †). The transition of OA-UCNPs to ligand-free UNCPs and further to PEI-UCNPs are conrmed by FT-IR. As shown in Fig. S3, † the transmission bands at 2926 and 2852 cm À1 can be assigned to asymmetric and symmetric methylene (-CH 2 -) stretching, and those at 1561 and 1460 cm À1 can be attributed to the vibrations of the carboxylate groups, indicating the presence of oleate ligand on the surface of OA-UCNPs. However, the disappearance of these characteristic peaks conrms the removal of surface ligand aer treatment by formic acid. When further modied by PEI, new peaks appear at 3396 cm À1 (N-H stretching), 2930 and 2854 cm À1 (asymmetric and symmetric -CH 2 À stretching), and 1545 cm À1 (N-H bending). Accordingly, the FT-IR results verify the success in ligand removal of OA-UCNPs and further attachment of PEI on bare UCNPs. Aer ligand exfoliation and polymer modication, ligand-free UCNPs and PEI-UCNPs are easily dispersed in water, and the z-potentials are measured to be +35.7 mV and +32.8 mV, respectively (Fig. S4 †), indicating the formation of stable colloidal solutions.
To prepare UCNPs/MoO 3Àx nanoassemblies, MoO 3 NSs are rstly prepared by tip sonication of bulk MoO 3 , and oxygen-decient MoO 3Àx NSs are easily obtained by UV irritation. 45 As shown in Fig. 2b, the nanostructure of the MoO 3Àx sample is comprised of NSs with lateral diameters in the range of 20-300 nm. UCNPs/MoO 3Àx nanoassemblies are then constructed by assembling the positive charged PEI-UCNPs and negatively charged MoO 3Àx NSs (Fig. S4 †) via electrostatic interactions, as characterized by TEM (Fig. 2c). Furthermore, the EDS spectrum of UCNPs/MoO 3Àx nanocomposites implies the presence of Na, F, Y, Yb, Er, Mo, and O. These results prove the successful assembling of UCNPs and MoO 3Àx NSs (Fig. 2d).
Next The optical properties of MoO 3Àx NSs solutions (1 mg mL À1 ) at different pH are rst investigated by UV-vis spectroscopy. As represented in Fig. 3a, the absorption intensity in the visible and NIR regions becomes weakened with increasing pH, and the maximum of the absorption peak gradually redshis from 744 to 866 nm. However, no absorption peak is found in the visible and NIR region above pH 7. Moreover, the absorption at 980 nm shows the same trend as well (Fig. S5a †). This phenomenon arises from the reduction of Mo in the reduced state (returning to the Mo VI state) by the addition of OH À to the MoO 3Àx NSs solution, leading to the reduction of free carrier concentration, and thus reducing the absorption in visible and NIR regions. 48,49 Next, the luminescence properties are investigated by placing MoO 3Àx NSs solutions (1 mg mL À1 ) with different pH in front of the OA-UCNPs solution (1 mg mL À1 ) and illuminate it then with the light of 980 nm wavelength at RT, where the 980 nm light rst passes through the MoO 3Àx NSs solution and then reaches OA-UCNPs (Fig. 1a). The luminescence intensity rises generally with increasing pH and remains constant above pH 8.2, as is presented in Fig. 3b. The luminescence intensity at 658 nm grows slowly when pH < 4.4, then increases remarkably in the range of 5.0 to 8.2, and the UCL shows no signicant change aerward. However, the UCL intensity at 658 nm shows a nonlinear relationship with the pH, which is different from typical upconversion sensors based on the FRET process. [32][33][34] Notably, we nd that the logarithm of luminescence intensity at 658 nm exhibits three-separate linear regions with pH, and the linear correlation coefficient of each calibration curve is calculated to be 0.992 (pH 2.6-4.4), 0.988 (pH 5-6), and 0.998 (6.3-8.2), respectively (Fig. 3c). Thus, this upconversion-based sensor shows broad pH responsiveness in the range of 2.6 to 8.2. To investigate the reversibility of this pH sensor, the pH value of MoO 3Àx NSs was adjusted from 8.2 to 2.6 and back to 8.2 by NaOH and HCl solutions for 5 cycles. As shown in Fig. S6, † the uorescence intensity shows good reversibility of the two-way switching processes aer the second cycle of pH adjustment. A slight increase in the uorescence intensity at pH 2.6 was noticed aer the rst pH adjustment from 8.2, which may result from a lower reduction degree of Mo(VI) in the acidic environment than under exposure to the UV light.
Non-contact uorescence sensing of H 2 O 2
The sensing ability of the upconversion-based nanosystem for H 2 O 2 in the non-contact mode is evaluated by the UV-vis absorption and UCL spectroscopy. As can be seen in the absorption spectrum (Fig. 4a) (Fig. 4b). The linear correlation coefficients of these two calibration curves are larger than 0.99, and the limit of detection (LOD) is calculated to be 0.75 mM. The luminescence properties are then studied using similar procedures as the above-mentioned pH sensing, except that MoO 3Àx solutions (1 mg mL À1 in acetate buffer, pH 4.5) with different added H 2 O 2 concentrations are placed in front of the OA-UCNPs solution. The quenching efficiency (denoted as (F 0 À F)/F 0 , where F and F 0 represent the luminescence intensity at a specic wavelength in the presence and absence of MoO 3Àx NSs, respectively) at 658 nm reaches 99.8% when 1 mg mL À1 MoO 3Àx NSs solution is aligned in front of 1 mg mL À1 OA-UCNPs solution. When H 2 O 2 is added in the range from 0 to 0.8 mM, the absorption intensity of MoO 3Àx NSs solution at 980 nm shows a continuous decrease (Fig. S5b †). As a result, the UCL intensity of OA-UCNPs experiences a gradual uptrend in both red and green regions upon 980 nm excitation with the increasing addition of H 2 O 2 (Fig. 4c). This can be ascribed to the oxidation of MoO 3Àx to MoO 3 by H 2 O 2 , leading to the reduction in excitation energy depletion by MoO 3Àx NSs at 980 nm, and resulting in more excitation energy reached by OA-UCNPs. Similarly, like the above-discussed pH sensing in non-contact mode, the uorescent intensity exhibits a nonlinear relationship with the H 2 O 2 concentration as well. In addition, the logarithm of luminescence intensity at 658 nm is linearly correlated with the H 2 O 2 concentration in the range of 0-200 mM (R 1 2 ¼ 0.993) and 250-500 mM (R 2 2 ¼ 0.997), respectively ( Fig. 4d). According to the 3s rule, the detection of H 2 O 2 can be down to 0.63 mM, providing a lower detection limit than those reported by other upconversion-based nanoprobes (Table 1).
To further estimate the selectivity for H 2 O 2 in the noncontact mode, the uorescence responses of the nanosystem toward various interfering species including cations, anions, and amino acids were investigated. As shown in Fig. 4e, only the addition of H 2 O 2 results in the recovery of the UCL emission, whereas no obvious change in luminescence intensity is observed aer the addition of large excesses of the other interfering species, such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , F À , Cl À , CO 3 2À , NO 3 À , SO 4 2À , cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Furthermore, competition experiments exhibit the recovery in UCL intensities at 658 nm, performed by adding H 2 O 2 to MoO 3Àx NSs solutions containing other interfering species (Fig. 4f). The results indicate that the sensing of H 2 O 2 is barely affected by these coexistent species. Therefore, this system can serve as an upconversion uorescence nanoprobe for H 2 O 2 with high selectivity in the non-contact mode.
Application in real sample analysis
For a practical application of the non-contact upconversionbased sensor, we studied the detection of H 2 O 2 residue in contact lens solution, as H 2 O 2 is usually applied in the contact lens disinfection processes and is harmful to human eyes. The results are summarized in To quantitatively analyze the quenching ability of MoO 3Àx NSs on PEI-UCNPs, a series of MoO 3Àx NSs modied PEI-UCNPs nanocomposites (the concentration of PEI-UCNPs is xed at 0.5 mg mL À1 ) is prepared by changing the MoO 3Àx NSs content (from 0 to 0.4 mg mL À1 ). The overlap integral (J(l)) between the normalized emission spectrum of the donor (UCNPs) and the absorption spectrum of the acceptor (MoO 3Àx NSs) is dened by the equation as follows: where l is the wavelength in nm, F D is the 980 nm laseractivated UCL spectrum of PEI-UCNPs normalized to an area of 1, 3 A is the extinction coefficient spectrum of MoO 3Àx NSs in units of M À1 cm À1 . The J(l) value for the donor acceptor pair is calculated to be 2.79 Â 10 13 M À1 cm À1 nm 4 . The effect of different MoO 3Àx NSs loading on PEI-UCNPs is evaluated by UCL spectra. As shown in Fig. 5a, the red emission intensity of UCNPs/MoO 3Àx nanoassemblies experiences a signicant decrease with the increasing addition of MoO 3Àx NSs. Additionally, the green emission intensity of upconversion-based nanoassemblies with different loading of MoO 3Àx NSs shows a similar tendency, but with a slower downward trend. As shown in Fig. 5b To elucidate the effect of EEA-induced reduction in the UCL of UCNPs by MoO 3Àx NSs, a non-contact mode is designed. The MoO 3Àx NSs solution (0.35 mg mL À1 ) is sealed in the quartz cuvette, aligning in front of another quartz cuvette containing 0.5 mg mL À1 PEI-UCNPs solution, and the UCL spectra are depicted in Fig. S7. † Upon the excitation by a 980 nm CW laser, the incident light rst passes through the MoO 3Àx NSs solution, and the energy-reduced light then reaches the UCNPs, resulting in the loss of intensity in UCL emissions. Ideally, the UCL intensity at 658 nm reduces by 72.3% compared with the control experiment.
Notably, the EEA effect will affect the intensity in all emissions, and the red-to-green emission ratio (R/G, where the red emission is integrated from 600-700 nm and the green emission is integrated from 500-600 nm) keeps its stability, which is conrmed by the activation of PEI-UCNPs (0.5 mg mL À1 ) with different power of the 980 nm laser. As presented in Fig. S8, † the red and green emission intensities increase with increasing laser power, and the R/G remains stable. However, a gradual decrease in the R/G values is observed for the UCNPs/MoO 3Àx nanocomposites with the increasing loading content of MoO 3Àx NSs (inset of Fig. 5a). This phenomenon can be attributed to the FRET-induced uorescence quenching by MoO 3Àx NSs, where the quenching ability by MoO 3Àx NSs in red emission is more pronounced than in the green region. As discussed above, the uorescence quenching of UCNPs by MoO 3Àx NSs is achieved by the joint effect of EEA and FRET, owing to the strong absorbance ability of MoO 3Àx NSs in both visible and NIR regions.
The sensing performance of UCNPs/MoO 3Àx nanoassemblies toward H 2 O 2 is investigated by UCL emission spectroscopy. As shown in Fig. 5c, the UCL emission intensity in red and green regions increases with the increasing addition of H 2 O 2 solution. As discussed above, the addition of H 2 O 2 leads to the oxidation of MoO 3Àx , resulting in the reduction in the absorption in both visible and NIR regions, and thus inhibiting the EEA effect at 980 nm and FRET process from the UCL of UCNPs to absorption of MoO 3Àx in the visible region, corresponding to the enhancement of UCL emission intensity. However, the uorescence intensity shows no obvious changes if more than 3.0 mM H 2 O 2 are added. The uorescence intensity at 658 nm exhibits a linear correlation to the H 2 O 2 concentration in the range of 0-0.8 mM (R 1 2 ¼ 0.990) and 1.0-2.5 mM (R 2 2 ¼ 0.996), respectively (Fig. 5d)
Conclusions
In summary, we have designed two different methods (i.e., a non-contact method and a conventional method) for upconversion uorescence sensing of H 2 O 2 . The non-contact method relies on the MoO 3Àx NSs absorption-induced EEA effect and operates by placing the MoO 3Àx NSs solution in front of UCNPs solution, whereas the conventional upconversion-based uorescence nanoprobe, based on the joint effect of EEA and FRET, was constructed by the integration of UCNPs and MoO 3Àx NSs via electrostatic interactions. An advantage of the noncontact method is that the valuable sensor particles do not become consumed or contaminated during the measurement and can be reused for a long time. The MoO 3Àx NSs act as the quencher in both nanosystems, owing to the strong absorptive capacity of MoO 3Àx in both visible and NIR regions. However, the addition of H 2 O 2 leads to the oxidation of MoO 3Àx , resulting in the recovery of UCL emissions, and thus enabling the quantitative detection of H 2 O 2 by both methods. Beneting from the non-contact method, hydrophobic OA-UCNPs can be applied as the luminophore directly and ultrahigh uorescence quenching (99.8%) is obtained. Moreover, the non-contact method exhibits high sensitivity toward H 2 O 2 down to 0.63 mM, which is lower than that determined by the spectrophotometry of MoO 3Àx (0.75 mM) and conventional UCNPs/MoO 3Àx nanocomposites (9.61 mM). Additionally, pH sensing can be achieved by employing the non-contact mode as well, which has shown a broad pH-responsive range from 2.6 to 8.2. We believe that these results could provide new insights into the design of upconversion-based nanosystems for uorescence sensing of other analytes.
Conflicts of interest
There are no conicts to declare. | v3-fos-license |
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} | pes2o/s2orc | EFFICIENCY OF IMPREGNATION WITH SALT SOLUTIONS IN THE RESISTANCE OF Corymbia torelliana AND Eucalyptus cloeziana WOODS TO FUNGUS Postia placenta
The research aimed to evaluate the efficiency of salt solutions (sodium chloride, lithium chloride, sodium carbonate, magnesium sulfate, zinc sulfate, and copper II sulfate) impregnated in Corymbia torelliana and Eucalyptus cloeziana woods on biological resistance to brown rot (Postia placenta) fungus under laboratory conditions. Wood test samples of 2.0 x 2.0 x 3.0 cm (tangential x radial x longitudinal) from each species were taken of the planks obtained in the heartwood region on base-top direction. The samples were impregnated with 5% concentration of solutions and submitted for 16 weeks to the P. placenta fungus under laboratory conditions. The wood of E. cloeziana was more resistant to decay than C. torelliana, both impregnated with salt solutions as not impregnated. The treatments with saline solutions were satisfactory as regards resistance of wood degradation, and may be used as a parameter for the evaluation of the wood, the xylophagous tested fungus.
INTRODUCTION
Wood is a building material from renewable forest resources and, being a material of biological origin, its service request is subject to the effects of the weather and changes in environmental conditions and even those of recognized natural durability are prone to attack of xylophagous organisms. According to Araújo; , wood resistance to deterioration is the inherent ability of the species to withstand the action of deteriorating agents, including biological, physical and chemical agents.
Knowledge of the natural resistance is important to recommend the most appropriate use, saving unnecessary expenses with the replacement of parts and reducing the impacts to the environment (PAES et al., 2008). To evaluate the natural biological resistance of wood, accelerated laboratory tests are an alternative in which wood samples are exposed to xylophagous fungi that cause white or brown rot.
The xylophagous fungi are the organisms responsible for the greater losses caused to the wood intended for use in solid products. Among the fungi responsible for the rotting of wood, the basidiomycetes class stands out, in which are found the fungi responsible for white and brown rot, having the Postia placenta among them. They decompose the polysaccharides of the cell wall, and the attacked wood has a residual brownish coloration (RINGMAN et al., 2015).
Due to the increasing demand for use of Eucalyptus woods, which demonstrably have high dimensional instability (PAES et al., 2015), some new techniques for improving the characteristics of wood as your hygroscopicity, and which have characteristics fungicides (PAES et al., 2013). However, due to the environmental pressures, the development of products of wood preservatives that are less harmful to the environment, when compared to traditional and effective products, which are made up of base metals such as copper, chromium, arsenic, boron and amines, which are not corrosive to metals , in order to meet, more broadly, the timber market (MARCHESAN, et al. 2015).
The purpose of this research was to evaluate the efficiency of saline solutions (sodium chloride, lithium chloride, sodium carbonate, magnesium sulfate, zinc sulfate and copper sulfate -II) impregnated in the Corymbia torelliana and Eucalyptus cloeziana woods on biological resistance to brown rot fungus (Postia placenta) under laboratory conditions.
Obtaining wood, production and impregnation of samples
The woods used in the research were Corymbia torelliana and Eucalyptus cloeziana that were in the form of planks, coming from forest plantations with 15 years of age, located in the region of Vale do Rio Doce in the state of Minas Gerais. The wood of Pinus sp., obtained in local businesses, Jerônimo Monteiro, state of Espírito Santo, Brazil, was also used as control.
From each eucalypts species and pinus wood, samples of 2.0 x 2.0 x 3.0 cm (tangential x radial x longitudinal) were taken, over the wooden planks obtained in the heartwood region on base-top direction. A total of 112 test samples free of defects such as nodes and gum resins, more the control, were used.
The samples were dried in an air circulated kiln, maintained at 103 ± 2ºC, until constant mass, weighed in scale with an accuracy of 0.01g. The dry samples were impregnated with solutions at 5% concentration (mass/volume) of the grade of salt purity (sodium chloride, lithium chloride, sodium carbonate, magnesium sulfate, zinc sulfate and copper (II) sulfate) in a desiccator under intermittent vacuum, during 96 hours. The saline solution absorption (%) by woods was determinate and dry salt retention (kg m -3 of wood) was calculated according to American Society for Testing and Materials -ASTM D-1413 (2008).
The wood of the species studied had basic density of 0,55 g cm -3 (C. torelliana) and 0.63g cm -3 (E. cloeziana), as reported by Paes et al. (2015) in studies to examine the effect of saline solutions tested on wood dimensional stability.
Determination of the biological resistance of wood
The determination of the biological resistance of Corymbia torelliana and Eucalyptus cloeziana woods was carried out by means of a laboratory accelerated rotting test, as described by the American Society for Testing and Materials -ASTM D-1413 (2008).
The assay took place in 600 mL bottles, filled with 300g of soil with 6.87pH and water retention capacity of 138.76%, as designated by ASTM D-1413ASTM D- (2008. The soil of each bottle was moistened, according to water retention capacity and two seeder strips of Pinus sp. were added in each bottle. The bottles were sterilized at 121 °C, 1.1 kPa, for 30 minutes. After cooling of the bottles, fragments were obtained from pure cultures of the fungus Postia placenta (Fr.) M.J. Larsen & Lombard (Mad 698) from the Forest Products Laboratory, United State Department of Agriculture, and put on the seeder strips. After the development of the fungus in the Pinus sp. feeders and colonization in the soil, the test samples were added at the rate of two samples per bottle and eight replicates for each forest species and saline solution, including the control (Pinus sp. wood samples).
The assay was kept in an acclimatized room (28 ± 2 °C and 75 ± 5% relative humidity) for 16 weeks. After this period, the samples were dried under the same conditions prior to the assay and the mass loss was calculated as described in ASTM D-2017ASTM D- (2008. The exposure time (16 weeks) was enough that samples of Pinus sp. wood, of same dimension, had mass loss ≥ 50%. This shows that the culture of the fungus was vigorous, and the exposure time sufficient to server deterioration of wood samples.
Statistical analysis of results
The saline solution absorption (%) by woods and dry salt retention (kg m -3 of wood) were evaluated by descriptive statistic and the arithmetical average and coefficient of variation were assessed. For analysis of the wood biologic resistance, a completely randomized design was used in a factorial arrangement, containing the To enable statistical analysis, data on percentage of mass loss were transformed into arcsen [square root (mass loss / 100)], as suggested by Stell;Torrie (1980). Such transformation was necessary to allow the homoscedasticity of the variances. For the factors and interactions detected as significant by the F test (p < 0.05), the Tukey test was used (p < 0.05). The normality of the data was verified by the Lilliefors test and the homogeneity of the variances by the Cochran test.
RESULTS
The time of 96 hours under vacuum was not enough for the wood test samples stay completely saturated, as postulated by Siau (1995). But, reached moisture contents were more than fiber saturation point (FSP), on the basis of the saline solution absorption ( Table 1). The dry salt retentions (kg m -3 of wood) varied little among the saline solutions used, with maximum values of 3.04 kg m -3 (Corymbia torelliana) and 2.94 (Eucalyptus cloeziana), Table 1. The differences in retention and absorption values of the saline solutions are related to wood density (0.55 g cm -3 -C. torelliana and 0.63g cm -3 -E. cloeziana). Thus, highest densities promoting more absorptions by fibers and, consequently, greater salt retention. The analysis of variance of the mass loss data (%) caused by the degradation by xylophagous fungus tested, for the different saline solutions impregnated in the woods of Corymbia torelliana and Eucalyptus cloeziana showed significant results by the F test, for the treatments, forest species and for the interaction among these factors. The effect of the interaction was outspread and averages compared (Table 2). A better effect of the saline solutions in the improvement of the resistance of the wood of C. torelliana in relation to that of E. cloeziana can be observed. This result was pronounced, since E. cloeziana wood was naturally resistant to the fungus tested. This is probably associated with the quantity and type of extractive in this wood, as reported by Paes et al. (2016).
DISCUSSION
The variation of retention among saline solutions can be considered homogeneous (± 3 kg m -3 ), Table 1, and so little influence on the consumption of treated wood by the fungus tested. The result is associated with the efficiency of the salt and natural resistance of the tested wood.
With the base on the values contained in Table 2, the all salt solutions improve the C. torelliana wood resistance to deterioration caused by Postia placenta fungus. This was pronounced, primarily by biological resistance of the E. cloeziana to the Postia placenta that was higher than 21.87% when compared to the C. torelliana. The average value of biological resistance of wood C. toreliana to Postia placenta brown rot fungus was within the range observed for Lopez et al (2018) for the round wood with diameter of 10 to 12; and 12 to 14 cm, that ranged from 22.56 a 26.51%, respectively, the authors classified the wood as moderate resistance, on the basis of the ASTM D-2017 (2008).
For Corymbia torelliana wood, the non-impregnated samples showed a deterioration of 84.85% caused by the fungus in relation to the impregnated ones, with magnesium sulfate proportionally providing the lowest mass loss for the wood of this species. Thus, all the saline solutions tested had potential to be indicated in the impregnation of the wood minimizing the deterioration caused by the attack of the brown rot fungus tested, being one of the fungi responsible for large losses of wood exposed in direct contact with the soil or in humid conditions that propitiate its attack. However, it is still needed assays to evaluate the lixiviation, effectiveness in field conditions and environmental pollution problem, caused by possible leaching or metal corrosion caused by salts.
It is observed, in general, that the Eucalyptus cloeziana wood presented the smallest mass loss caused by the degradation of the Postia placenta fungus, both for the non-treated samples and for the other salts used. Although not detected statistical differences between the means for the treated and untreated samples, for some salts the deterioration caused in the wood was more than double that observed for the untreated one. This may be related to chemical reactions with wood that promoted the removal of some constituents responsible for the inhibition of the growth of tested fungus. Even so, the efficiency of some salts, such as magnesium and zinc sulfates, was observed in improving wood resistance, reaching values of 63.43% (zinc sulfate) in relation to the untreated wood.
In the literature, the use of fungi causing brown rot in the deterioration of the wood, Postia placenta being one of them, there are divergences between the results obtained by several authors for the different woods analyzed (BRITO et al., 2008;ANDRADE et al. 2012;LOPES et al., 2018). The divergences in the results may be related to the force of fungus strain used, regions of the trunk, age and growth sites for the evaluated woods. This is in accordance with the observation of Hill (2006), that in natural system of interaction between biological materials (fungus and wood); there will always be variability in results and even contradictory, unexpected and inexplicable results. Paes (2002) demonstrated such a relationship when evaluating the natural resistance of wood of the genus Corymbia to xylophagous fungi, obtaining mass loss values from 5.12 to 20.62%, depending on the region evaluated in pith-bark direction into tree trunk.
Another explanation would be the type of wood exposed to the degradation, as well as the extractive content present in the wood, since the wood has high variability of its constituents both in the base-top and in the pith-bark directions. The fungi initially consume the sapwood and later, after the loss of some extracts of the heartwood caused by evaporation, leaching and reactions caused by the environment, they initiate the heartwood attack. According to Rayner;Boddy, (1995) the attacks of xylophagous organisms are associated with the class, quantity and location of the extractives in the wood.
For the same woods used in the present work, Paes et al. (2016) obtained values of extractives soluble in ethanol: toluene (2: 1 v / v) of 4.65 and 5.40% for C. torelliana and E. cloeziana, respectively. The lower amount of extractives may have favored deterioration caused by the tested fungus on C. torelliana wood.
The fungi usually have a greater capacity for deterioration of sapwood, since there are phenolic substances with fungicidal properties and insecticides in the heartwood. However, the extractives do not distribute homogeneously in the stem, presenting their highest concentration and, consequently, the greater natural resistance in the wood of the external parts of the heartwood and near the base of the tree. In the case of sapwood, the natural resistance is lower (MELO et al., 2010;SILVA et al., 2014, COSTA et al., 2017.
The use of saline solutions provided a beneficial effect on the resistance of C. torelliana wood to the deterioration caused by the Postia placenta fungus. This improvement may be related to the fungicidal and antibacterial effects of sodium compounds, copper, zinc and magnesium sulfates and lithium chloride. In some of them, such as copper and zinc compounds, they are part of formulations used for the protection of wood against the action of decay fungi and xylophagous insects. It is interesting for new research with these substances in order to provide improvements in the resistance of the wood of other forest species, especially those coming from the Eucalyptus and Pinus genus to expand their uses in the country's forest-based industries.
CONCLUSIONS
• The untreated Eucalyptus cloeziana wood obtained less mass loss than that of the untreated Corymbia torreliana, when submitted to fungus attack. • The impregnation with the salts promoted improvements in the biological resistance of the wood of Corymbia torreliana, making it more resistant to the tested fungus. • The Eucalyptus cloeziana wood impregnated with sodium chloride, lithium chloride and sodium carbonate had deterioration caused by fungus Postia placenta more than double that observed for the untreated wood. | v3-fos-license |
2018-04-03T01:01:50.039Z | 2014-08-04T00:00:00.000 | 205574463 | {
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} | pes2o/s2orc | Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II
The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.
Introduction
DNA topoisomerases are essential enzymes that solve the topological problems of double-stranded DNA during various DNA-related events, such as replication, transcription, and chromosome segregation, by directing the cleavage and religation of DNA strands (Ghilarov & Shkundina, 2012;Schoeffler & Berger, 2008;Vos, Tretter, Schmidt, & Berger, 2011;Wang, 2002). Topoisomerases are the targets of some of the most successful anticancer and antibacterial drugs (Bailly, 2012;Bradbury & Pucci, 2008;Pommier, Leo, Zhang, & Marchand, 2010). Some of these drugs stabilize the topoisomerase-DNA covalent complexes and/or inhibit the religation step, which induces the accumulation of the complexes, toxic intermediates (Pommier et al., 2010). Therefore, considerable attention has been paid to the mechanism underlying the cleavage/ religation reaction. There are several DNA topoisomerase families. Type IA and II topoisomerases (topo IA and topo II) possess similar catalytic domains for the cleavage/religation reaction, (Aravind, Leipe, & Koonin, 1998;Berger, Fass, Wang, & Harrison, 1998) and their catalytic mechanisms are expected to be similar (Liu & Wang, 1999). It is believed that this reaction proceeds via a general acid/ base catalysis (Supplementary Figure 1) . During the DNA cleavage step, a general base deprotonates the catalytic tyrosine residue to activate the oxygen for nucleophilic attack on the DNA backbone phosphate, and the subsequent substitution of deoxyribose produces its covalent intermediate. A general acid enhances the scission of the P-O3′ bond by protonation of the O3′ atom. The religation reaction is believed to be the reverse of the DNA cleavage reaction (Schmidt, Burgin, Deweese, Osheroff, & Berger, 2010). Despite numerous experimental studies in recent years, it is still not defined for the general acid/base in topo IA and II.
In topo II, possible candidates for the general base involved in DNA cleavage are conserved histidine residues near the catalytic center and the metal-associated water Noble & Maxwell, 2002), whereas candidates for the general acid are water and an undefined amino acid in the catalytic site . In topo IA, a charge relay mechanism was proposed (Perry & Mondragon, 2002;Zhang, Cheng, & Tse-Dinh, 2011). In this mechanism, a conserved glutamic acid in the TOPRIM domain acts as the general acid and base in the cleavage and relegation reactions, respectively, and the reactions are promoted by hydrogen bonds between the conserved aspartic acid in the TOPRIM domain and the conserved histidine. However, mutation of the glutamic acid to glutamine did not abolish the cleavage/religation activity (Chen & Wang, 1998).
By contrast, a substrate-assisted mechanism has been examined theoretically in DNA polymerase (Wang & Schlick, 2008;Wang, Yu, Hu, Broyde, & Zhang, 2007), which catalyzes a phosphoryl transfer reaction similar to that of religation reaction in topo IA and II. In this mechanism, a proton migrates from the O3′ to a leaving group via the α-phosphate of deoxynucleoside triphosphate. This mechanism can explain why mutational studies failed to identify the general acid and base involved in the cleavage/religation reaction, because external general acids and bases are not required in this mechanism. Thus, in this study, we examined the possible substrateassisted mechanism for the DNA religation reaction of topo II using quantum mechanics/molecular mechanics (QM/MM) calculations.
Modelling of the reactant state
The initial coordinates were taken from the crystal structure of yeast topo II-covalent intermediate (PDBID: 3L4K), which is the only one PDB structure containing two divalent ions in its active site (Schmidt et al., 2010). The structure is considered to be the best for the reactant state of the religation reaction, although artificial atoms were introduced into the crystal structure to determine the covalent intermediate state, i.e. Zn 2+ and 3′ sulfur atoms. We replaced the Zn 2+ and terminal S3′ atoms with Mg 2+ and O atoms, respectively. The topo II model was immersed in a TIP3P water box. A 500 ps molecular dynamics (MD) simulation was performed with T = 300 K and P = 1 bar to construct the hydrogen bond network in the active center with (i) positional restraints for all the heavy atoms except for water molecules and (ii) a distance restraint between the O2P oxygen of the reactive phosphate and the O3′ H atom. All MD simulations were performed using GROMACS 4.5.4 (Hess, Kutzner, van der Spoel, & Lindahl, 2008) with the ff99SBildn force field (Lindorff-Larsen et al., 2010).
QM/MM calculation
A water droplet model was used for the QM/MM calculations. The droplet model contained approximately 50,000 solvent water molecules, 15 Na + ions, 16 Mg 2+ ions, and approximately 25,000 atoms of the DNA and topo II. During the QM/MM calculations, important atoms in the active site were included in the QM region, whereas the remaining atoms were handled by MM. The QM region included the DNA backbone, two Mg ions, eight active site water molecules, and the side chains of Glu449, Asp526, Asp528, Arg781, and Tyr782 (a total of 134 QM atoms, see Figure 1(a)). The QM/MM calcu-lations were carried out using the NWChem program package (Kendall et al., 2000). The QM level of theory employed was hybrid DFT (B3LYP) with the 6-31 + G ⁄ and 4-31G basis sets for Mg ions and all other atoms, respectively, whereas the MM level was handled by the ff99 force field (Wang, Cieplak, & Kollman, 2000). We have used hydrogen cap atoms in the QM/MM boundary region. More details of computational methods and set-ups are provided in the Supporting Information.
Modelling of the reactant state
The hydrophilicity of the active site of the cleavage/ religation reaction should allow binding of several water molecules in the active site. Unfortunately, they were not resolved in the crystal structure because of the limited resolution (2.98 Å). Furthermore, the terminal 3′ oxygen of the substrate DNA was substituted by sulfur to trap the covalent intermediate (Schmidt et al., 2010). In order to hydrate the active site and generate an appropriate hydrogen bond between the terminal 3′ oxygen and the O2P of the reactive phosphate, we performed a sufficiently long MD simulation of 500 ps with restraints, as described in the Methods section. Then, a QM/MM geometry optimization was performed without any restraints. The QM/MM optimized structure showed that one and four water molecules were coordinated to metals A and B, forming 5-and 6-coordinated structures (see Figure 1(b). Stereoview figures are provided in the Supporting Information). Interestingly, the distance between the terminal O3′ and the O2P of the reactive phosphate was reduced from 3.85 to 2.78 Å, and the distance between Mg A (2+) and O3′ was increased from 2.05 to 2.40 Å. These distance changes are attributed to the reconstruction of the active site after the replacement of artificial atoms.
QM/MM simulation for the DNA religation reaction
Starting from the QM/MM optimized structure for the reactant, possible reaction pathways were searched using the QM/MM geometry optimization with harmonic potential restraints. The most favorable pathway had two characteristic activation barriers in the first and last reaction steps. The overall energy profile is shown in Figure 2.
We found three intermediate states (I1-I3) and four transition states (T1-T4) between the reactant (R) and product (P) states. Schematic illustrations and the threedimensional structures of these key states are shown in Figure 3(a) and Supplementary Figure 2. The first step (R-I1) was initiated by the nucleophilic attack of the terminal O3′ on the reactive phosphate, and a proton transfer occurs from the terminal 3′ OH to the O2P oxygen of the reactive phosphate. The calculated activation energy of ΔE # = 18.9 kcal/mol is the highest energy barrier in the overall reaction. The calculated activation energy is comparable to the experimental value of ΔG # = 20 kcal/mol, which is estimated by the experimental reaction rate constants (Mueller-Planitz & Herschlag, 2008). Figure 3(b-c) show the three-dimensional structures of the high energy transition states, TS1 and TS3. In TS1, the proton transfer was already completed and the hydrogen bond distance between O3′ and the H of O2P was 1.50 Å. Another characteristic feature of this step was a significant decrease in the Mg A-O3′ distance from 2.40 to 2.06 Å, which was associated with a stabilization of the free O3′ via its direct coordination to Mg A. TS1 was the highest energy state; hence, Mg A promoted the overall religation reaction and the nucleophilic attack of the terminal O3′ against the reactive phosphate. This was consistent with the experimental observations that metal ions were required in the religation reaction of topo II and topo IA (Goto, Laipis, & Wang, 1984;Tse-Dinh, 1986).
The first intermediate state (I1) of the religation reaction was stabilized by another hydrogen bond between the O5′ of the (À1) phosphate and the H atom of the reactive phosphate that migrated from O3′. The relative QM energy of I1 was calculated as E I1 = 7.7 kcal/mol (Figure 2, Supplementary Figure 2).
In the second step, the H atom of the reactive phosphate moved from the O5′ of the (À1) phosphate to the O atom of water 1. Water 1 was stabilized close to the reactive phosphate via hydrogen bonds with the (À1) phosphate and Arg781 (Supplementary Figure 2). This step had a low activation barrier of 3.6 kcal/mol, and the resulting intermediate I2 was slightly better stabilized than I1 (E I2 = 5.5 kcal/mol).
The third step was a proton transfer from the O2P of reactive phosphate to the water 1, and this reaction also had a small activation barrier. In I2, the distance between the O2P of reactive phosphate and the oxygen of water 1 was 2.46 Å (Supplementary Figure 2). In this case, the close contact between the O atoms facilitated a low activation barrier proton transfer, known as a low-barrier hydrogen bond (LBHB). A remarkable structural feature of I2 was that the water 1 was sandwiched by two phosphate groups, reactive phosphate and (À1) phosphate. The stable hydrogen bonds around water 1 supported the LBHB interaction. In I3, a hydronium ion of water 1 is formed, which was stabilized by the tetra-and pentacoordinated phosphates with formal charges of À1 and À2, respectively. The highly charged pentacoordinated phosphate was stabilized by three cation groups, the hydronium ion, Mg A on the opposite side of the hydronium ion, and Arg781. Arg781 closed to the protondonor O2P of the reactive phosphate where R O, H = 2.45 Å to R O··H = 2.11 Å (Supplementary Figure 2).
The H atom of Arg781 was involved in this salt bridge and it transferred to Tyr782 during the next step. The last step (I3-TS4-P) involved a concerted double proton transfer and the dissociation of Tyr782. The double proton transfer occurred between: (1) the hydronium ion and Arg781 and (2) Arg781 and Tyr782. The calculated energy barrier of this step was 8.5 kcal/mol, which was the second highest step in the overall DNA religation reaction. The evolution of this step was monitored based on the interatomic distances and the relative QM energy along the reaction path (Figure 4). It was shown that the hydrogen bonds required for the double proton transfer were formed in TS4. In TS4, one N atom of Arg781 side chain was changed its hybridization from a planar (sp2) form to a tetrahedral (sp3) form, as shown in Figure 3(c). Furthermore, the salt bridge was weakened between the hydronium ion and the O2P of the highly charged pentacovalent phosphate, i.e. from R H··O = 1.31 Å (R O··O = 2.39 Å) and h O··H-O = 162 to R H··O = 1.71 Å (R O··O = 2.52 Å) and h O··H-O = 135 for I3 and TS4 (Supplementary Figure 2 and Figure 3(c)), respectively. Therefore, the energy barrier of TS4 was attributed to two factors: the formation of tetrahedral NH 3 in Arg781 and the weakening of the hydronium ion-pentacovalent phosphate salt bridge. These structural changes led to the formation of hydrogen bonds that allowed the concerted double proton transfer. After the formation of these hydrogen bonds in TS4, the two proton transfer reactions and the dissociation of Tyr782 occurred in a completely concerted manner (Figure 4). The optimal reaction pathway described above used an indirect proton transfer path via water 1 and Arg781 from the pentacovalent phosphate to Tyr782. The energy barrier of the direct proton transfer path was calculated as 23.16 kcal/mol, which was too high; hence, this direct pathway could not be the route of the proton migration.
Discussion
A two-metal mechanism has been used in polymerases and phosphodiesterases (Yang, Lee, & Nowotny, 2006). In this mechanism, metal A assists the deprotonation of the nucleophile and metal B enhances the protonation of the leaving group, and both ions contribute to stabilize the excess charge of the pentacovalent transition state. Several experimental studies of topo II have suggested the active role of two metal ions during the cleavage/religation reaction (Deweese, Guengerich, Burgin, & Osheroff, 2009;Noble & Maxwell, 2002;West et al., 2000).
Schmidt et al. proposed a unified two-metal mechanism for the cleavage/religation reaction of topo IA and topo II (Schmidt et al., 2010) based on the crystal structure of a covalent intermediate of topo II, where the metal B was located far from the reactive phosphate. In the unified catalytic mechanism, the two metal ions were expected to have a key role in the general acid/base catalysis where one metal ion, Mg A, stabilizes a pentacovalent transition state directly and promotes the leaving and deprotonation of the 3′ hydroxyl during the cleavage and religation reactions, respectively, whereas another metal in the active site, Mg B (2+), anchors the substrate DNA.
Our results show that Mg A promotes both deprotonation of the nucleophile and protonation of the leaving group, i.e. Mg A stabilized the deprotonated O3′ in TS1 with the close coordination value of R MgA··O3′ = 2.06 Å and the highly charged pentacoordinated phosphate in TS4. The coordination geometries of Mg A in each transition states are consistent with two-metal mechanism proposed by Schmidt et al. It is notable that phosphoryl transfer reactions have been classified into associative and dissociative mechanisms (Lassila, Zalatan, & Herschlag, 2011) depending on the dissociation time of the leaving group. As shown in Figure 3, the religation reaction of topo II is a typical case of the associative mechanism.
The arginine residue involved in substrate-mediated proton transfer is strictly conserved between topo IA and topo II (Narula et al., 2011;Okada et al., 2000), although the role of the arginine residue is not well understood. In yeast topo II, the substitution of Arg781 with alanine reduced the catalytic activity greatly (Liu & Wang, 1998), whereas an experimental study of the human enzyme showed that a lysine substitution rescued the catalytic activity (Okada et al., 2000). Topo IA behaved similarly in the corresponding arginine (Arg321) mutant (Chen & Wang, 1998;Narula et al., 2011). Furthermore, a mutation of arginine to a bulky hydrophobic residue resulted in a complete loss of religation activity of topo IA and an accumulation of covalent intermediates (Narula et al., 2011). Based on our results, these experiments can be explained as follow. The participation of surrounding water molecules and the lysine side chain mediate the proton transfer pathway in the less active alanine and the active lysine mutants, respectively, whereas the bulky hydrophobic side chain completely disrupts the pathway. Thus, the religation reaction of topo IA and topo II can be explained by the substrate-mediated proton relay (SMPR) mechanism elucidated in the present study.
Interestingly, the DNA cleavage reaction remains active in topo IA, although its religation activity is abolished completely by the bulky hydrophobic substitutions (Narula et al., 2011). This implicates that another proton transfer pathway such as the charge relay mechanism is present in topo IA.
The catalytic activity of topo IA has a bell-shaped pH-dependency but this trend is not observed in a His365 mutant (Perry & Mondragon, 2002). This indicates that His365 participates in general acid-base catalysis, although His365 does not make direct contact with the reactive phosphate in the crystal structure of topo IA. These results implicate the charge relay mechanism (Perry & Mondragon, 2002).
In the charge relay mechanism, O3′ is protonated and deprotonated during the cleavage and religation by the glutamic acid in the TOPRIM domain, whereas the pKa of the glutamic acid is controlled by a hydrogen bond network formed by the active site of aspartic acid and histidine (Perry & Mondragon, 2002). However, the charge relay mechanism is inconsistent with the fact that the mutation of Glu to Gln still maintains the DNA relaxation activity in topo IA (Chen & Wang, 1998;Perry & Mondragon, 2002).
Based on these experimental results and our computational results, the charge relay mechanism and the SMPR mechanism alone cannot explain the DNA cleavage reaction of topo IA fully. In conclusion, we suggest that both proton pathways must coexist in the topo IA DNA cleavage reaction.
No clinically used drugs are targeted at topo IA, but it was recently identified as a target for antibiotics that are expected to lead to the accumulation of toxic covalent intermediates (Tse-Dinh, 2009). Thus, the disruption of the proton transfer pathway in the religation reaction of topo IA may be an attractive strategy for the rational design of novel antibiotics.
Conclusion
We found a SMPR mechanism of the religation reaction in topo II, and the SMPR mechanism can be applied to the cleavage and religation reaction in both topo IA and topo II. In the SMPR mechanism, Mg A promotes both deprotonation of the nucleophile and protonation of the leaving group, i.e. Mg A stabilizes the transient negative charges during the nucleophilic attack and promotes the dissociation of the leaving group. The proton relay pathway in the SMPR mechanism is formed by the terminal O3′, reactive phosphate, water, Arg781, and Tyr782. This pathway is consistent with a series of Arg781 mutations in topo IA and topo II.
For the cleavage reaction of topo IA, the experimental results and our calculations suggested the coexistence of the charge relay mechanism and the SMPR mechanism. These mechanisms imply their irreversibility, which might be related to their highly sophisticated biological functions. We also expect that this irreversible proton transfer pathway will provide new insights for drug design. | v3-fos-license |
2017-05-04T22:18:03.136Z | 2016-08-31T00:00:00.000 | 13426794 | {
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} | pes2o/s2orc | Flavonoid C-glucosides Derived from Flax Straw Extracts Reduce Human Breast Cancer Cell Growth In vitro and Induce Apoptosis
Flax straw of flax varieties that are grown for oil production is a by product which represents a considerable biomass source. Therefore, its potential application for human use is of high interest. Our research has revealed that flax straw is rich in flavonoid C-glucosides, including vitexin, orientin, and isoorientin. The objective of this study was to evaluate the cytotoxicity and possible proapoptotic effect of flax straw derived C-glucosides of flavonoids in the human breast adenocarcinoma cell line (MCF-7). The effects of flax straw derived flavonoid C-glucosides on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B assays. The expression of apoptosis-related genes was assessed by real-time PCR. Our data revealed that flax C-glucosides as well as pure compounds are cytotoxic toward MCF-7 cells and inhibit their proliferation. Moreover, the induction of apoptosis was correlated with the changes in the mRNA level of pro-apoptotic genes. Increased expression of bax and caspase-7, -8, and -9 and decreased mRNA expression of bcl-2 was observed, whereas the mRNA levels of p53 and mdm2 were not altered. These results clearly demonstrated that flax straw metabolites effectively induced growth inhibition and apoptosis in human breast adenocarcinoma cells.
INTRODUCTION
Flavonoids are among the most abundant polyphenols in plants. Based on their structures, they can be grouped into several subclasses such as flavonols, flavones, isoflavones, flavanones, and flavan-3-ols. Normally they are accumulated in the vacuoles of plant tissues as O-linked glycosidic conjugates, but also as C-glycosides (Harborne, 1993). The flavonoid glycosides mainly occur as 3 or 7 O-glycosides, but the C-5, 6, 8 and 4 positions are glycosylated as well. The transformation of flavonoid aglycones to glycosides plays a crucial role in flavonoid biosynthesis, and their glycosylation increases their solubility and stability relative to flavonoid aglycones (Plaza et al., 2014). The sugars of C-glycosides are attached directly to the flavonoid skeleton by a C-C bond that is resistant to acid hydrolysis. Most flavonoid C-glycosides are flavones that have been found in bryophytes, ferns, gymnosperms, and angiosperms (Harborne, 1993). In cereals such as rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays), C-glycosylflavones are the main class of accumulated flavonoids (Brazier-Hicks et al., 2009). It was found that in buckwheat the direct precursors of flavone-C-glycosides are 2-hydroxyflavanones, which underwent catalyzed C-glucosylation (Kerscher and Franz, 1988). Thus, the flavanones, which are core intermediates of the flavonoid pathway, are the most likely precursors of C-glucosides. It is known that cereal crops synthesize C-glucosylated flavones through the activity of C-glycosyltransferases and dehydratase acting on activated 2-hydroxyflavanones (Brazier-Hicks et al., 2009).
In plants, flavone-C-glycosides function as antioxidants (Kitta et al., 1992), insect feeding attractants (Kim et al., 1985), antimicrobial agents (Dinda et al., 2006), and promoters of mycorrhizal symbioses (Akiyama et al., 2002). It was observed that accumulation of C-glycosylflavones was enhanced by UV-B light in a UV-tolerant rice cultivar but absent in a susceptible cultivar, which might suggest their possible UV light protective roles (Markham et al., 1998). Interestingly, they also possess some favorable activities for humans, as they can protect cells from oxidative stress and counteract inflammation (Manthey et al., 2001;Kim et al., 2005). For example, isovitexin extracted from rice bran was shown to prevent reactive oxygen species damage in an in vitro system (Lin et al., 2002) and was able to reduce the production of hydrogen peroxide induced by LPS in mouse macrophages (Lin et al., 2005). The flavone C-glycosides from blueberry are able to reduce IL-8 production, which indicates their potential as anti-inflammatory agents (Flores et al., 2012). There are some reports on the important role of flavonoid C-glycosides in cancer development. Dietary flavone was shown to alter expression of genes that regulate cancer cell proliferation, cycle, and apoptosis (Ullmannova and Popescu, 2007). Tricin has in vitro antiproliferative activities toward breast and colon cancer cell lines at submicromolar concentrations (Hudson et al., 2000). Vitexin is suggested to serve as a therapeutic agent for the treatment of human leukemia, as it promoted the expression of cleaved caspase-3 and cleaved caspase-9 in leukemia cells and simultaneously reduced Bcl-2 expression and therefore induced apoptosis (Lee et al., 2012).
Various flavonoid C-glucosides and di-C-glucosides were found in flax (Dubois and Mabry, 1971), including orientin and isoorientin and their derivatives (Wagner et al., 1972;Mierziak et al., 2014). Flax straw was found to be a rich source of valuable metabolites, including C-glycosylated flavonoids. Flax straw is one of the by-products of flax oil production and increases the biomass reservoir; hence, its putative application for human use is of high interest.
In this study, we identified and determined the level of C-glycosylated flavonoids in flax straw extracts. For the purpose of our research we used different genetically modified flax types in which the secondary metabolite accumulation in the plant organs was increased. Three genetically modified flax types (named W94, GT4, and L9) were generated and described previously (Lorenc-Kukuła et al., 2005Boba et al., 2011). W92 flax overexpresses three genes of the phenylpropanoid pathway encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). This modification resulted in an increase in phenylpropanoid content: flavonoids (kaempferol and quercetin), anthocyanins, phenolic acids (coumaric, ferulic, and synaptic acids) and lignan (Żuk et al., 2011). GT4 flax overexpresses the glucosyltransferase gene and is characterized by overproduction of phenylpropanoids such as kaempferol and quercetin glycosides, anthocyanins, proanthocyanidins, phenolic acids and their glucoside derivatives, and SDG (Lorenc-Kukuła et al., 2009;Czemplik et al., 2012). L9 flax, also used in this study, was obtained by suppression of the endogenous flax gene coding for lycopene β-cyclase, which resulted in a decrease in carotene content, but also in an increase in accumulation of other terpenoids and tocopherols (Boba et al., 2011).
Combinations of natural molecules or extracts that effectively inhibit tumor development and progression are nowadays actively researched (Pan et al., 2010). In this study, we investigated the in vitro effect of straw extracts from three transgenic flax types (W92, GT, and L) and one non-transgenic one (Linola) on the growth, proliferation and apoptosis of cells from the human breast cancer cell line MCF-7. We also tested the effects of the pure compounds of the main constituents of flax straw on human breast cancer cells.
Plant Material
Flax seeds (cv. Linola 947) were obtained from the Flax and Hemp Collection of the Institute of Natural Fibers in Poland. The GT plants overexpress the glucosyl transferase gene SsGT1 from Solanum sogarandinum under a seed-specific napin promoter. W92 plants overexpress three genes of the phenylpropanoid pathway: CHS, CHI, and DFR. L plants were transformed with lycopene b-cyclase (lcb) from Arabidopsis thaliana. GT transgenic plants line #4 (GT4) and line #5 (GT5) and W92 transgenic plants line #40 (W92.40) and line #72 (W92.72), L transgenic plants line #9 (L9) and Linola, the non-transgenic control type, were grown in a field and were harvested 4.5 months after sowing. After the separation of seed capsules, the straw was used for further experiments.
Phenolic Component Extraction and UPLC Analysis
A 5 g amount of flax straw was ground using a Retsch mill and extracted three times with methanol. Fractions were pooled and evaporated at 40 • C under a vacuum and then resuspended in 1 mL of water. The extract was analyzed on a Waters Acquity UPLC system with a 2996 PDA detector, using an Acquity UPLC column BEH C18, 2.1100 mm, 1.7 µm. The mobile phase was acetonitrile (solution A) and 0.1% formic acid (solution B) as the eluent. The separation was done at 4 ml/min flow ratio with gradient flow: 1 min-95% A and 5% B, 2-12 min-gradient to 70% A and 30% B, 12-15 min-gradient to 0% A and 100% B, and 15-17 min-gradient to 95% A and 5% B. The compounds were identified based on the retention times and absorbance spectra, and their quantities were calculated in comparison to the standards (Sigma-Aldrich, USA). The detection was done at k = 280 nm.
Cell Culture
The human breast adenocarcinoma cell line (MCF-7) was obtained from the Laboratory of Nuclear Proteins of the Faculty of Biotechnology, University of Wrocław, Poland and was grown as a monolayer in Eagle Minimum Essential Medium (Lonza, Switzerland) supplemented with 10% fetal bovine serum (Lonza, Switzerland), 1% L-glutamine (Invitrogen, USA) and a 1% antibiotic mixture (Invitrogen, USA) at 37 • C in a 5% CO 2 atmosphere.
Cell Proliferation Assay
Cells were seeded in a 96-well plate at a concentration of 5 × 10 3 cells/mL. After 24 h, GT4, GT5, W92.40, W92.72, L and Linola flax straw extracts were added to the plate at varying volumes: 5, 10, and 20 µL. Each flax type straw extract was prepared from an equal amount (5 g) of dry weight of straw and the flavonoid content in each sample was presented in Table 2. Additionally, the pure compound standards of vitexin (0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 µg), isoorientin (20, 40, 60, 80, 100, 120, 140, 160 µg), and orientin (0.1, 0.25, 0.5, 1, 1.5 µg) were added at different concentrations corresponding to the amount in the flax straw extracts. To assess the proliferation potential, MCF-7 cells were incubated and assayed after 24 and 48 h. After this period of treatment, 10 µL of MTT stock solution (4 mg/mL) was added to each well to give a total reaction volume of 550 µL. After 4 h of incubation, the medium with MTT solution was removed from the plate. The formazan crystals in each well were dissolved in 50 µL of DMSO and incubated for 30 min with gentle shaking. The absorbance at 540 nm was read on a Varioskan Flash Microplate Reader (Thermo Scientific, USA). The MTT assay was performed in four repetitions. The results were presented as a % in reference to the control (100%).
Cell Cytotoxicity Assay
In vitro cytotoxicity against human cancer cell line MCF-7 was determined using the sulforhodamine B assay (SRB) as described previously (Skehan et al., 1990). Cells were seeded in a 96-well plate at concentration of 5 × 10 3 cells/mL. After 24 h, GT4, GT5, W92.40, W92.72, L and Linola flax straw extracts were added to the plate at varying volumes: 5, 10, and 20 µL. Each flax type straw extract was prepared from an equal amount (5 g) of dry weight of straw and the flavonoid content in each sample was presented in Table 2. Additionally, the pure compound standards of vitexin (0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 µg), isoorientin (20, 40, 60, 80, 100, 120, 140, 160 µg), and orientin (0.1, 0.25, 0.5, 1, 1.5 µg) were added at different concentrations corresponding to the amount in the flax straw extracts. To assess the cytotoxicity, the MCF-7 cells were incubated and assayed after 24 and 48 h. After incubation, the cells were fixed by gently layering trichloroacetic acid (50 µL/well, 50% w/v) on top of the medium in all the wells and incubated at 4 • C for 1 h. The plates were washed five times with distilled water and air-dried. The staining was performed with sulforhodamine B dye (0.4% w/v in 1% acetic acid, 50 µL/well). The unbound dye was washed five times with 1% acetic acid and the plates were air-dried. The adsorbed dye was dissolved in Tris buffer (150 µL/well, 10 mM) and the plates were gently shaken for 10 min on a mechanical shaker. The absorbance at 530 nm was read on a Varioskan Flash Microplate Reader (Thermo Scientific, USA). The cytotoxicity effect was calculated by subtracting the mean OD values of the respective blank from the mean OD value of the experimental set. The percentage growth in the presence of the test extract was calculated considering the growth in the absence of any test extracts as 100%.
Detection of Apoptosis by Annexin V Labeling
The detection of surface phosphatidylserine by FITC-annexin V binding was performed with use of Annexin V-FITC Fluorescence Microscopy Kit (BD Pharmingen TM ). MCF-7 cells were seeded on 24-well plate at concentration of 20 × 10 5 cells/mL. After 24 h, Linola flax straw extracts (Lin.1,Lin.2,and Lin.3) were added to the plate. The positive control consisted cells treated with 1 µM of staurosporine (known to cause an apoptotic effect on a variety of human tumor cell lines) and the negative control consisted non-treated cells. After 24 h of treatment, the -treated, positive-and negative-control cells were washed twice with cold phosphate-buffered saline (PBS) and once with annexin binding (AB) buffer. Cells were then incubated for 15 min with phosphatidylserine labeling solution (Annexin V-FITC diluted 1:10 in AB buffer). Additionally, the nuclear morphology was analyzed by staining the cells with 5 µg/ml Hoechst (Invitrogen, Matrigel TM ) in PBS for 15 min. Cells were then washed with AB buffer and observed immediately by fluorescence microscopy. An Olympus FV500 confocal laser scanning microscope was used for fluorescence observation.
Western Blotting Analysis
MCF-7 cells were seeded on 24-well plate at concentration of 20 × 10 5 cells/mL. Linola flax straw extract (Lin.3) was added to the plate. After 24 h of incubation the cells were washed twice with PBS and then scraped at 4 • C in lysis buffer: 100 mM Tris-HCl, pH 7.4, 1% Triton X-100 and protease inhibitor cocktail (Bioshop Canada, Inc.). Samples were sonicated three times for 30 s and stored at −20 • C for further experiments. The protein concentration of the samples was determined by the standard Bradford (Bradford, 1976) procedure. Identical amounts of protein (50 µg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted electrophoretically onto nitrocellulose membranes (Schleicher and Schuell). After the transfer, the membrane was sequentially incubated with a blocking buffer (5% dry milk) and then with monoclonal mouse anti-caspase 7 antibody (1: 1000 dilution) and polyclonal rabbit anti-caspase 8 antibody (1: 1000 dilution) overnight at 4 • C. An antibody for GAPDH (Sigma-Aldrich, USA) was used as an internal control. Alkaline phosphatase-conjugated goat anti mouse IgG and anti-rabbit IgG served as a second antibody, and was used at a dilution of 1: 25000. Immunoreactivity was visualized by Alkaline Phosphatase Substrate Kit (Bio-Rad) according to the protocol supplied with the kit.
Statistical Analysis
Each test procedure (MTT and SRB test, RT-PCR) was performed three times, whereas in each single test, four repetitions were performed. The results were averaged and a standard deviation was calculated so that each bar seen in the diagrams represents the mean value of four separate experiments ± standard deviation. Analysis of variance (ANOVA) was performed using the Tukey test. Statistica 9 (Statsoft) software was used for the statistical analysis.
Identification and Quantification of Flavonoid C-glucosides in Flax Straw
The UPLC analysis of the methanol extracts of flax straw revealed the presence of three flavonoid C-glucosides: vitexin, orientin, and isoorientin. The amounts of those components were determined in straw from transgenic flax type GT4, GT5, W92.40, W92.72, L9 and a control, non-transgenic flax line (Linola). The results are presented in Table 1. Generally, the level of the three identified flavonoid C-glucosides in all flax types did not differ dramatically, but some differences were observed. In all flax types the most abundant compound was isoorientin, which reached 9 mg/gFW in Linola type. In all transgenic types, the level of isoorientin was slightly lower, with the lowest amount in the W92.72 type (4.53 mg/gFW). Vitexin was the most elevated in the GT4 flax straw (0.083 mg/gFW), and was lower in the W92.72 type (0.037 mg/gFW) in comparison to the control. W92.72 flax straw was characterized by the highest level of orientin (0.062 mg/gFW). For the cell in vitro studies, we used three different volumes (20, 10, and 5 µl) of straw extracts derived from the same amount of tissue (5 g) of each flax type, and their biochemical composition is presented in Table 2.
Effects of Flax Straw Extracts on Proliferation of Human Breast Carcinoma Cells and Cytotoxic Effect
The proliferation of human breast carcinoma cells treated with flax straw extracts was assessed using the MTT test (Figure 1). The proliferation level of MCF-7 cells was diminished to 80% in comparison to the control, after 24 h of treatment, for most of the flax types tested. Generally, no significant differences among different flax types were observed ( Figure 1A). After 48 h of treatment, the inhibitory effect was stronger, and 10 and 20 µl volumes of all preparations tested lowered the proliferation level by around 30%. The most effective were 20 µl of W92.72 and L9 extracts ( Figure 1B). However, the lower volumes of flax straw extracts exhibited a pro-proliferative effect on MCF-7 cells after 48 h of treatment. A 5 µl of W92.72, W92.40, GT4, GT5, L9, 10 and 5 µl of Linola straw extract induced proliferation potential with the highest increase of 18% for 5 µl of W92.40 straw extract ( Figure 1B).
Cell cytotoxicity was assessed using the SRB assay. After 24 h of treatment, addition of 20 µl of flax straw extracts (apart from the L9 type) resulted in a cytotoxic effect of up to 46% for Linola extract, in comparison to the control (Figure 2A). For smaller extract volumes such an effect was not observed, but on the contrary, the anti-cytotoxic effect was noticed for the following samples: 10 and 5 µl of W92.72, W92.40, GT4, GT5, Linola and all samples of L9 flax straw extracts. After 48 h of treatment, the 5 µl of W92.72, 10 and 5 µl of W92.40 and 5 µl of Linola straw extract exhibited no cytotoxic effect on human breast cancer cells ( Figure 2B). The strongest cytotoxic effect was observed after 48 h of treatment for all other preparations. Treatment of MCF-7 cells with the highest amount of straw extracts resulted in a 70% decrease in protein content in comparison to the nontreated cells. The Linola preparation was again the most effective ( Figure 2B). The results showed that each of the tested flax straw extracts exhibited anti-cancer effects on MCF-7 cells.
Influence of Main Constituents of Flax Straw Extracts on Human Breast Cancer Cells
To investigate which of the flax straw extract components might be lower proliferation of MCF-7 cells, we verified the activity of the three main C-flavonoids: orientin (O), isoorientin (I), and vitexin (V). The amounts of the tested compounds correspond to the amounts in the previously tested preparations. The proliferation of MCF-7 cells in the MTT test was significantly decreased in the higher amounts of tested compounds, and this effect was stronger after 48 h of incubation. The vitexin, isoorientin, and orientin reduced MCF-7 proliferation by up to 40% in comparison to the control after 24 h of treatment (Figures 3A-C). The inhibition was stronger after 48 h of treatment and showed that vitexin reduced proliferation by up to 40% of the control value, while isoorientin inhibited proliferation by up to 50% (Figures 3D,E), and orientin by up to 55% (Figure 3F). However, the lower amounts of the tested compounds exhibited a stimulatory effect on the proliferation potential of MCF-7 cells. Vitexin (0.2, 0.4, 0.6 µg) caused the slight increase in the proliferation potential after 24 h of treatment, as well as isoorientin (20, 40 µg) and orientin (0.1 µg; Figures 3A-C). Such an effect was not observed after 48 h of treatment (Figures 3D-F).
All tested compounds exhibited a cytotoxic effect on MCF-7 cells. Interestingly, lower amounts of vitexin (0.2, 0.4, 0.6, 1 µg after 24 h, and 0.2, 0.4 µg after 48 h) and isoorientin (20, 40 µg after 24 and 48 h) caused an anti-cytotoxic effect (Figures 4A,B,D,E). For 0.8 and 1.2 µg of vitexin in the 24 h of treatment no significant changes were observed (Figure 4A). Similarly, for treatment with 0.1, 0.25, and 1 µg of orientin no significant alteration occured. After 24 h of treatment, addition of 1.4 µg of vitexin, 120, 140, and 160 µg of isoorientin and 0.5 and 1.5 µg of orientin resulted in a cytotoxic effect up to 27, 79, 83, and 66%, respectively, in comparison to the control (Figures 4A-C). The strongest effect was observed after 48 h of treatment (Figures 4D-F). After 48 h of treatment, addition of 0.6-1.4 µg of vitexin, 60-160 µg of isoorientin, and 0.1, 0.5, 1.5 µg of orientin resulted in a cytotoxic effect. The strongest effect was noted for 140 µg of isoorientin and was up to 86%, in comparison to the control (Figures 4E). These results indicate that all of the tested C-glucosyl derivatives of flavonoids exhibited a significant inhibitory effect toward MCF-7 cells.
Flax Straw Extract Promoted Expression of Pro-apoptotic Genes of Human Breast Cancer Cells
To explore whether flax straw extracts induced expression of pro-apoptotic genes, the quantitative analysis of expression of apoptosis-related genes in MCF-7 cells was performed. Realtime PCR analyses were used to measure the mRNA expression of Bcl-2, Bax, caspase-7, -8, -9, p53, and mdm2, as well as the expression of the GAPDH (an internal standard), after the treatment of human breast cancer cells with the flax straw extract. As the previous experiments did not show significant differences among individual flax extracts regarding their influence on MCF-7 cells, for the purpose of this study, the Linola flax straw extract was chosen. Its effect on apoptosis of human carcinoma breast cancer cells was studied after 6, 12, and 24 h of treatment. For the experiment three different volumes of Linola flax straw extracts were used (Lin.1,Lin.2,Lin.3), which corresponded to the amounts in the previous experiments of this study.
In all samples tested, the treatment with flax straw extracts resulted in significant changes in the expression of the bcl-2 and bax genes. A reduction in the transcript level of the bcl-2 gene was observed in all samples, with the lowest level at 24 h for Lin.2 and Lin.3 samples (Figure 5C). In contrast, the bax gene was overexpressed in all samples tested up to twofold. To determine the caspases involved in apoptosis enhancement, the analysis of expression level was performed for caspase-7, -8, and -9. At 6 h, the mRNA level of all caspases was elevated in all samples (Figure 5A). At 12 h, the caspase-7 and -8 were characterized by an increase of mRNA level, while caspase-9 exhibited no significant changes. At 24 h, the expression level of caspase-8 was elevated, whereas caspase-7 and -9 expression levels were not altered or slightly lowered ( Figure 5C). Some differences were also observed in the expressions of p53 and mdm2 genes of flax straw treated MCF-7 cells. A slight increase was observed in mRNA level for the p53 gene, while the mdm2 gene was increased in all samples at 24 h ( Figure 5C). The level of annexin V mRNA was not significantly altered upon flax straw extracts treatment. However, the increase in the expression level of this gene was observed for Lin1 extract after 12 h of treatment ( Figure 5B).
Induction of Apoptosis by Flax Straw Extract
To determine if the flax straw extract has an apoptotic effect, the annexin V assay was used to detect phosphatidylserine in the outer leaflet of the plasma membrane. Phosphatidylserine is localized in the inner leaflet of the plasma membrane, but while the cells undergo apoptosis, the asymmetry of the cell membrane is lost and phosphatidylserine is then also found on the outer surface of the cell membrane. Annexin V is a protein that binds to phosphatidylserine in the presence of calcium ions. Annexin V might be labeled with a fluorochrome to enable visualization of cells at the early stage of apoptosis. After the MCF-7 cells were treated with Linola straw extracts, phosphatidylserine was localized on the outer leaflet of plasma membrane (Figure 6). The morphology of nuclei (stained with Hoechst) of the cells with phosphatidylserine present in the outer leaflet of the membrane was altered and chromatin condensation was observed (Figure 6).
Apoptosis is a complex activity that mobilizes a number of molecules and is classified into caspase-dependent or caspase-independent mechanisms. To examine the molecular mechanism underlying apoptosis process, activation of caspase-7 and caspase-8 in response to flax straw extract was also analyzed by western blotting assay. The application of mouse monoclonal antibody targeted to active caspase-7 and rabbit polyclonal antibody targeted to active caspase-8 revealed positively stained MCF-7 cells after 6 h of exposure to Linola flax straw extract (Figure 7). An increase of caspase-7 and caspase-8 activity was noted in human breast carcinoma cells when treated with Linola flax straw extract. Our results were parallelly compared with those observed in the presence of 1 µM staurosporine, a known apoptosis induction agent.
DISCUSSION
Flax is a crop that is used in many branches of industry and is mainly grown for oil production and fiber processing. Flax metabolites are an object of investigation of many studies, but straw remains less investigated. Straw of flax varieties which are grown for oil production are leftovers that contribute to the waste material. Interestingly, they are a source of many health-oriented metabolites of the phenylpropanoids pathway.
We examined straw of three transgenic flax types (W92, GT and L) and a non-transgenic one (Linola), and our analysis revealed the presence of C-glucoside derivatives of flavonoids, including vitexin, orientin, and isoorientin. Isoorientin was the most abundant metabolite in the straw extracts. Some differences were observed in the level of identified flavonoid C-glucosides in all flax types in comparison to the control, but GT, W92, and L flax modifications did not significantly influence the content of flavonoid C-glucosides in straw. Further biochemical analysis of flax straw of different flax types revealed inter alia the presence of catechins, ferulic acid, gallic acid, caffeic acid, chlorogenic acids, p-coumaric acid, vanillic acid, vanillin, 4-hydroxybenzaldehyde, syringaldehyde, and apigenin FIGURE 5 | Expression levels of genes involved in the apoptosis of MCF-7 cells. The mRNA level of bcl-2 (B-cell CLL/lymphoma 2), bax (BCL2-associated X protein), p53, mdm-2 (MDM2 proto-oncogene), cas 7 (caspase 7), cas 8 (caspase 8), cas 9 (caspase 9) and annexin V(anex) in MCF-7 cells treated with Linola flax straw extracts (Lin.1,Lin.2,Lin.3) at 6 h (A), 12 h (B), and 24 h (C) was obtained from the real-time RT-PCR analysis. GAPDH was used as a reference gene and the transcript levels were normalized to those of the control non-treated cells (C = 1; not presented in the figure). Data represent the mean ± standard deviations from three independent experiments. derivatives (data not published). Additionally, the levels of the identified compounds were altered depending on the different stages of stem development. Mierziak et al. (2014) studied the biochemical composition of flax stems of W92, GT, and W92 × GT crossbreed plants, and observed an altered level of flavonoid C-glucosides in comparison to the control. Similarly, isoorientin was the most abundant compound in flax stems, but our analysis revealed higher amounts of this flavonoid in flax straw (9.01 mg/gFW in comparison to 1 mg/gFW for Linola flax). Interestingly, the level of vitexin was around 10 times lower in our studies. These analyses show the different biochemical composition of flax stems and straw of the same flax types.
For the purpose of these studies, we focused on the identification and determination of the level of flavonoid C-glucosides, as it was already reported that they possess inhibitory activity toward human cancer cells in in vitro studies. Vitexin has been shown to exhibit apoptotic actions on human U937 human leukemia cells (Lee et al., 2012). Another study showed that orientin and vitexin from Trollius chinensis possessed antitumor effects that may be associated with the regulation of the apoptosis-related gene expression of p53 and bcl-2 in esophageal cancer (An et al., 2015). The leaf extract of Celtis australis L. and Celtis occidentalis L. exhibited cytotoxic activity against human hepatocellular carcinoma, colon adenocarcinoma and gastric carcinoma cell lines (El-Alfy et al., 2011).
The use of flax straw, rich in flavonoid C-glucosides, for inhibiting the growth of human breast cancer cells is therefore reasonable. Our analysis indicated that flax straw extracts of different flax types are able to inhibit the proliferation of MCF-7 cells (up to 30%) and are cytotoxic toward this cell line (up to 70% decrease in protein content). The inhibition rate was proportionate to the dose and was more significant at 48 h than at 24 h of treatment. To detect synergism, we used only pure compounds in order to control the working concentrations and avoid effects due to unknown components, as it happens when whole extracts are used. Similarly, treatment of MCF-7 cells with flavonoid C-glucoside pure compounds reduced the proliferation and was cytotoxic toward human breast carcinoma cells. The inhibition rates of all compounds tested, vitexin, orientin and isovitexin, were comparable in the MTT proliferation test (up to 60% reduction). Isovitexin was the most effective in the cytotoxic assay (up to 89% reduction). We observed moderate cytotoxicity in MCF-7 cells after treatment with flax straw extract in comparison with the individual flavonoid C-glucoside treatment, supporting the hypothesis that there is no synergistic effect of action. Nevertheless, we assume that C-glucosides of flavonoids mainly contribute to the inhibitory activity toward MCF-7 cells, as the activity of these metabolites translates into the activity of the extract. Interestingly, we observed that the smaller volumes of some straw extract samples caused a proproliferative effect. A similar phenomenon was observed after treatment of breast cancer cell line BT-20 and bone cancer cell line MG63 with relatively low concentrations of root bark extract of Persea americana and leaves extract of Hunteria umbellata, traditional Nigerian medicinal plants, which caused an increase in proliferation up to 8 and 27%, respectively (Engel et al., 2011).
To elucidate the effect of flax straw extracts on the induction of apoptosis in MCF-7, we performed expression level analysis of crucial genes involved in this process. It is well-known that mitochondria are implicated as they are one of the apoptotic targets, and the balance between the expression levels of Bcl-2 and Bax proteins determines whether the cell responds to an apoptotic signal. Bax, a pro-apoptotic factor, integrates into the outer mitochondrial membrane and leads to the release of cytochrome-c from mitochondria, which further activates caspases-9 (Thomas et al., 2000). In contrast, Bcl-2 prevents this process by preserving mitochondrial integrity and promotes cell survival (Jeong and Seol, 2008). To reveal the effect of flax straw extracts on expression of Bcl-2 and Bax, RT-PCR was performed. It was observed that flax straw extracts were involved in the upregulation of Bax and downregulation of Bcl-2. There are two core signaling pathways that are involved in apoptosis, the intrinsic and the extrinsic pathway. The intrinsic pathway is a signal transduction pathway involving the mitochondria and the Bcl-2 family, which was observed in the MCF-7 cells treated with flax straw extracts, and therefore we suggest that flax straw extract might trigger the intrinsic pathway of apoptosis.
Caspase activation is considered to be a key hallmark of apoptosis. Caspases, a conserved family of cysteine proteases, are the central components of the apoptotic response that irreversibly commit a cell to die. Breast carcinoma cells of the MCF-7 line have lost expression of caspase 3 as a result of a 47 base pair deletion within exon 3 of the casp-3 gene (Liang et al., 2001). Despite the absence of detectable caspase 3, MCF-7 cells have been shown to undergo apoptosis. Moreover, it is known that caspase 7 is highly related to caspase 3 and shows the same substrate specificity in vitro (Talanian et al., 1997). We assessed the effect of flax straw extracts on the cascade of caspases, and the mRNA expression levels of caspase 7, 8, and 9 were determined in our experiment. Results from the present study demonstrated that mRNA expression levels of caspase 7, 8, and 9 were increased. In addition, the present study suggests that the extrinsic pathway may in part have contributed to the flax straw extract-induced apoptosis, as demonstrated by an increased expression level of caspase-8 in MCF-7 cells. It is suggested that caspase-8 might activate Bid protein (BH3 interacting domain), which might be a molecular bridge to intrinsic pathway of apoptosis, as it binds to the activated Bax protein on mitochondria surface, and thus contribute to the pore generation in this organelle (Kantari and Walczak, 2011). Interestingly, increased expression of p53 was not detected in the MCF-7 cells used in the present experiments, and the mdm2 expression was elevated in the late apoptosis. p53 is one of the most powerful tumor suppressor genes in human cancers, and both extrinsic and intrinsic apoptotic pathways are activated by p53, while mdm2 is an important negative regulator of p53 and is the primary cellular inhibitor of p53 (Maximov and Maximov, 2008). Our findings suggest that flax straw extracts induce p53-independent apoptosis through downregulation of Bcl-2. Similarly, it was shown that curcumin caused apoptosis in melanoma cell lines and in a human colon cancer cell line in a p53-independent pathway (Bush et al., 2001;Watson et al., 2010). We did not observed the significant changes in the transcript level of annexin V gene.
For further confirmation of apoptosis, we performed the method that uses the binding of FITC-labeled annexin V to phosphatidylserine which is exposed on the surface of apoptotic cells but not on viable cells. Our experiments indicated that flax straw extract induces apoptotic processes in MF-7 cells, as these cells were annexin V-positive. Moreover, the nuclei morphology visualized with Hoechst staining also indicates an apoptotic process upon flax straw treatment. Part of the flax straw treated cells exhibited a homogeneous intense staining of the nucleus or nuclear fragments, that is characteristic for apoptotic cells with nuclear condensation. All cells with nuclear condensation were also stained with annexin V at the cell membrane, whereas, none of the cells with a euchromatic nucleus were annexin V-positive. Therefore, we suggest that annexin V-positive cells are undergoing apoptosis.
Additionally, the apoptotic process occurrence upon flax straw extract treatment was confirmed by the assessment of the protein level of caspase-7 and caspase-8. The flax straw extract treatment resulted in an increase of activated form of the proteins of both examined caspases. We suggest that this treatment results in the apoptosis process activation, as the staurosporine, known a known apoptosis induction agent, treated MCF-7 cells exhibited the same level of caspase-7 and -8 protein as the flax straw extracttreated cells (Figure 7). Flax straw extract induce apoptosis via caspase-dependent mechanism.
Taken together, the results of the present study suggest that flax straw extracts inhibit the proliferation of MCF-7 cancer cells by increasing apoptosis and might provide a useful alternative to cancer chemotherapy. Due to the adverse effects and resistance of many anticancer agents that have been developed, there is a great interest in the use of plant-based compounds to develop safe and efficient antitumor factors.
AUTHOR CONTRIBUTIONS
AK, JS made substantial contributions to conception and design of the study and to data interpretation; AK performed UPLC analysis, MC carried out plant extracts preparation, cell culture proliferation, cytotoxicity experiments, gene expression levels analysis, Annexin V staining, western blot analysis and made substantial contributions to acquisition and data analysis and wrote the manuscript. JM performed the real-time PCR analysis, western blot analysis and preformed statistical analysis. All authors reviewed and approved the manuscript. | v3-fos-license |
2019-04-08T13:07:28.562Z | 2016-01-01T00:00:00.000 | 99596063 | {
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} | pes2o/s2orc | Green Synthesis of Barium Sulfate Particles Using Plant Extracts
The biological molecules in the extracts of four fruits or vegetables: kiwifruit, oranges, tomato and carrot, were used as templates to synthesize barium sulfate (BaSO4) particles. The products were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray power diffractometry. The results showed that, leaf-shaped barite BaSO4 crystals with toothed edge were obtained with kiwifruit extracts; thorn spherical barium sulfate crystals with diameter of 2-4 micrometers were produced with tomato extracts; rod-like or quasi-spherical BaSO4 crystals with size of several hundred nanometers to several micrometers were gained with orange extracts; while quasi-spherical BaSO4 nano-crystals were obtained with carrot extracts. The formation mechanism of BaSO4 is also discussed, showing that the proteins, carbohydrates, vitamins and organic acids in above four kinds of fruits or vegetables may provide nucleation sites, controlling the growth of BaSO4 crystals with different morphologies.
Introduction
Barium sulphate (BaSO 4 ) is generally white or colorless, chemically inert, insoluble in water, with high density, and the main source of barium [1]. It is widely used as a shading material for the x-ray photography, a gammaray absorber, a white pigment and etc because it is non-harmful to humans. BaSO 4 is also one of the most important fillers used in the plastics, rubber and paint industries, and is also used in pharmaceutical formulations [2]. It can be used for X-ray attenuation instead of shielding made from lead when incorporated into polymeric materials to form composite [3].
In recent years, more attention has been paid on synthesis of BaSO 4 nanoparticles for their multiple applications in the oil industry, electronics, TV screen, glass, car filters, paint industry, ceramics and medicine, etc. Many approaches have been chosen to control the size and morphology of BaSO 4 particles. These methods could be classified as direct precipitation, microemulsion, membrane separation and organic modification [1]. M.
Ismaiel et al have synthesized BaSO 4 nanoparticles by precipitation method using polycarboxylate as a modifier [4]. D. Adityawarman have obtained BaSO 4 nanoparticles in a non-ionic microemulsion [5]. G. Wu et al have precipited BaSO 4 nanoparticles through impinging streams [6]. A. Gupta have prepared BaSO 4 nanoparticles using sodium hexa metaphosphate as a stabilizer [7]. Very recently, the synthesis of BaSO 4 nanoparticles by a chemical precipitation route without polymer stabilizers has been reported [1].
In this paper, we have synthesized BaSO 4 particles by a very simple and green method.
The BaSO 4 particles especially nanoparticles obtained may be used as filler in plastics. The possible formation mechanism of the BaSO 4 particles directed by biomolecules of the fruits or vegetables including kiwifruit, oranges, tomato and carrot is discussed. This report may be very significant for biomineralization research and green synthesis of functional inorganic materials.
Materials and Instruments
Barium chloride dihydrate (BaCl 2· 2H 2 O), anhydrous sodium sulfate (Na 2 SO 4 ), and ethanol were purchased from reagent plant of Shanghai (Shanghai, China), and were analytically pure. They were used without further purification. Four kinds of fruits or vegetables: kiwifruit, oranges, tomato and carrot were purchased from a local farmer.
Methods
First, 80g of each fruit or vegetable were washed carefully, and then crushed to extract juice, and the juice was mixed proper amount of double-distilled water, obtaining 90 mL of the fruit or vegetable extracts aqueous solution. After that, 20 mL of each solution was mixed with 40 mL of 0.05 mol/L BaCl 2 , completely, obtaining 60 mL of mixed solution containing fruit or vegetable extracts and 0.0333 mol/L BaCl 2 . Then each of the mixed solution was blended with 40 mL of 0.05 mol/L Na 2 SO 4 very slowly. The white precipitates produced in the mixed systems. The control experiment was also performed without adding of fruit or vegetable extracts. The above five reaction systems were placed at room temperature for 24 hours.
The white precipitates produced in the reaction solutions were separated by centrifugation (centrifugating rate, 4000 rpm), washed three times with double-distilled water and ethanol, respectively, and then vacuum dried for further determination.
The sizes and morphologies of the precipitates were examined by scanning electron microscopy (SEM), while their crystal phases were determined by Fourier transform infrared spectroscopy (FT-IR) or X-ray powder diffractometry (XRD). Fig.1a and b, it can be seen that the particles obtained without any exracts are leaf-shaped with toothed edge. The size of each "leaf" is about 4-8 ȝm. Fig. 1c and d shows that the particles obtained with kiwifruit extracts are also leaf-shaped, but the sizes of them become slightly smaller. When in the presence of tomato extracts, many thorn spheres with diameters of 2-4 ȝm are produced ( Fig. 1e and f). While with orange extracts, quasi-spherical or rod-like BaSO 4 particles with sizes of several hundred nanometers are gained ( Fig. 1g and h). At last, it can be seen from Fig. 1i If seen clearly, the intensity of crystal face (020) in Fig. 3a is stronger than that of it in The extracts of fruits and vagetables including kiwifruit, tomato, orange and carrot contain mang kinds of organic macromolecules such as proteins, carbohydrates, vitamins and some organic acids. In previous reports, it is accepted that the soluble biomacromolecules such as acidic proteins, glycoproteins and polysaccharides in biological systems act as nucleators, growth modifiers, and anchoring units in the mineral formation [8]. Here, we speculate that Ba 2+
Summary
Green synthesis of BaSO 4 particles using the extracts of four fruits or vegetables including kiwifruit, oranges, tomato and carrot was performed. leaf-shaped barite BaSO 4 crystals with toothed edge were obtained in kiwifruit juice; thorn spherical barium sulfate crystals with diameter of 2-4 micrometers were produced in tomato juice; rod-like or quasi-spherical BaSO 4 crystals with size of several hundred nanometers to several micrometers were gained in orange juice; while quasi-spherical BaSO 4 nano-crystals were obtained in carrot juice. The formation mechanism of BaSO 4 was also discussed, showing that proteins, carbohydrates, vitamins and organic acids in above four kinds of fruits or vegetables can provide nucleation sites, controlling the formation of BaSO 4 particles with different morphologies. | v3-fos-license |
2017-06-30T15:17:29.682Z | 2014-04-01T00:00:00.000 | 18169644 | {
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} | pes2o/s2orc | Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles
Background Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. Results Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. Conclusions Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death.
Background
Cytotoxic drugs (such as, cis-and carboplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, out of a list of over 50) are still commonly used in chemotherapy to stop cancer cells from multiplying and dispersing. These drugs affect all growing cells, but since most normal cells do not divide as often as cancer cells they are proportionately less affected. Unfortunately, the lack of tumor specificity of these cytotoxins produces unwanted and often severe and dangerous side effects. One approach to overcome this is the development of nano-sized drug delivery systems (DDS), which have been shown to increase the drug accumulation in tumors via passive targeting, i.e., through the enhanced permeation and retention (EPR) effect [1][2][3]. Two types of nano-sized DDS, liposomes and albumin nanoparticles (NPs), i.e. Doxil® and Abraxane®, respectively, have caused notable improvements in the therapeutic efficacy of anticancer agents [4]. However, also these therapeutics still display significant side effects and only prolong life marginally. The lack of success in improving overall survival in many solid tumors and the still significant side effects of even the second generation drugs make it imperative to further develop and refine such DDS.
All currently US FDA approved chemotherapeutic DDS and most that are in current clinical trials or in vivo studies still employ traditional cytotoxic drugs as their therapeutic agents. Many of these chemotherapeutic agents (e.g., alkylating agents and antimetabolites) produce DNA damage which leads to p53-dependent apoptosis [5]. Indeed, the loss of the p53 tumor suppressor pathway contributes to the development of most human cancers [6]. Thus, inactivation of the apoptotic response provides an attractive explanation for the poor responsiveness of p53 mutant tumors to many traditional anticancer agents. Such limitations have spurred efforts to identify new and more effective chemotherapeutic agents that act independently of the p53 pathway. The high selectivity and low toxicity of many proteins make them attractive substitutes of cytotoxic drug. For example, Cytochrome c (Cyt c) is an important mediator of apoptosis when it is released from the mitochondria to the cytoplasm [7,8]. This process normally takes place in response to DNA damage, but in many cancer cells it is inhibited (most likely due to inactivation of the upstream components of the signaling pathway(s) that activates the Cyt c release, such as the p53 pathway). The targeted delivery of Cyt c directly to the cytoplasm could selectively initiate apoptosis in most cancer cell by circumventing inactivation of apoptosis due to damage to upstream events.
However, protein drugs also do have significant drawbacks, primarily related to their limited physical and chemical stability during storage and after administration [9][10][11]. Also, most proteins including Cyt c cannot cross lipid bilayer membranes [12]. This makes it necessary to develop methods allowing for the intracellular delivery of sufficient amounts of Cyt c to induce apoptosis in the target cells. The development of nanosized carriers for protein therapeutics allows maintaining protein stability and controlling release properties but having a sufficiently high protein loading remains challenging [11,13]. Recently our research group has been developing strategies for the intracellular delivery of Cyt c using nanosized DDS [14,15]. We demonstrated that Cyt c delivered from a smart nanosized system could potentially be an effective chemotherapeutic agent to treat cancer, but also recognized that the delivery system used needed improvement to achieve a better efficiency. Specifically, we demonstrated that mesoporous silica nanoparticles (MSN) could be utilized as efficient carrier for the delivery of apoptosisinducing Cyt c [15]. However, Cyt c-MSN conjugates were not able to induce cell death in HeLa cells during the first 48 h. They showed 45% cell viability after 72 h at a Cyt c concentration of 37.5 μg/ml. It was not possible to increment the Cyt c concentration since that resulted in a toxic concentration of MSN (>100 μg/ml) and the particles were already loaded to the maximum with the protein. This demonstrates fundamental limits of such drug-loaded systems in practical applications. Ideally, to avoid such limitations, the drug should itself form the DDS. Consequently, we evolved a delivery system in which the core consisted of Cyt c NPs and thus the drug itself.
Production of Cyt c NPs by a solvent displacement method is a straight-forward process (i.e., by solventinduced nanoprecipitation) [16]. However, such NPs would simply dissolve when injected into the blood stream and thus need to be stabilized for application, e.g., by applying high temperature and/or chemical cross-linkers [17]. NPs, which were stabilized via a chemical cross-linker agent with thiol-cleavability, have been successfully used for triggered drug release mediated by the reducing environment inside the cell [18]. In this work we tested a new method for stabilizing Cyt c NPs by covalently coating them with the hydrophobic polymer poly (lactic-co-glycolic) acid (PLGA). PLGA, a biocompatible, biodegradable, and non-toxic polymer, has been intensely studied in the field of DDS and has received FDA approval for various applications including drug delivery [19,20]. Our idea was to coat the Cyt c NPs with PLGA using a hetero-bifunctional linker, such as succinimidyl 3-(2-pyridyldithio) propionate (SPDP), that includes a disulfide bond to be able to shed the polymer shell in the reducing environment of the cell ( Figure 1A). Afterwards the Cyt c nanoparticle should dissolve in the cell thus inducing apoptosis ( Figure 1B). In this work we demonstrate the potential of the designed anti-tumor Cyt c nanoparticle in cancer treatment.
Results and discussion
Nanoprecipitation of Cyt c Cyt c, an apoptosis-initiating protein, could potentially be used to target and specifically destroy cancer cells if delivered to their cytoplasm. Protein-based nanomaterials have been studied as carriers of anti-cancer drugs because of their biodegradability, low toxicity, and multiple modification capacity [21]. It would be ideal to use the protein as both, nanosized delivery device and drug, simultaneously. To test this concept, in this study we generated and tested Cyt c-based NPs to kill cancer cells by inducing the apoptosis.
Cyt c nanoparticles were obtained by a solvent displacement method shown to induce a reversible nanoprecipitation of proteins without compromising their frequently fragile structure and function [14]. In a previous study we found that acetonitrile was a useful organic solvent for obtaining Cyt c nanoprecipitates when it was added in at least 4-fold excess to the aqueous protein solution [14]. In this work we optimized this procedure for obtaining Cyt c nanoparticles and tested the effect of protein concentration on Cyt c precipitation and stability. While no buffer-insoluble aggregates were formed regardless of the protein concentration (data not shown), the precipitation yield decreased at higher protein concentrations ( Figure 2). In agreement with our previous work for other proteins [14], we also found that at increasing protein concentration under otherwise constant conditions the particle size increased ( Figure 2). We selected a Cyt c concentration of 5 mg/ml for further experiments since it had the highest precipitation efficiency (100%) and smallest particle diameter (around 150 nm). Note that a particle diameter of less than 400 nm should result in particles with good passive delivery properties [2,22,23].
The capability of Cyt c to still interact with Apoptotic Protease Activating Factor 1 (Apaf-1) and induce apoptosis after nanoprecipitation was verified next. Since Cyt c is a cell membrane impermeable protein the experiment was conducted in a cell-free system. The addition of Cyt c to fresh cytosol produces caspase activation [15]. Thus, the integrity of the soluble protein after the nanoprecipitation procedure was compared with native Cyt c and was found to be around 90%. We tested the use of the excipient methyl-β-cyclodextrin (mβCD) to further increase the Cyt c integrity during the nanoprecipitation procedure ( Figure 3). The excipient mβCD has been used before to improve enzyme activity in organic solvents [24]. The excipient was co-dissolved with the protein previous to the solvent precipitation step with acetonitrile. Because mβCD is soluble in acetonitrile, the excipient was removed by repeated centrifugation/washing cycles. Our results show that the excipient further improved bioactivity to 96% and decreased the particle diameter to 90 nm.
The impact of the nanoprecipitation procedure on Cyt c tertiary structure was investigated by circular dichroism (CD) measurements after dissolving the NPs in buffer ( Figure 3B). The near-UV CD spectra show that the nanoprecipitation procedure did not cause irreversible changes in the protein conformation. The two minima at 286 and 293 nm, characteristic of native Cyt c [25], were present in all NP formulations. However, these two minima were somewhat reduced when mβCD was used at the very high 1:20 mass ratio during nanoprecipitation. Since these two minima correspond to Trp-59 [15] and cyclodextrins have the ability to sequester hydrophobic moieties on protein surfaces [26] it is possible that Cyt c was somewhat destabilized by the additive under these conditions. Such destabilization has been reported for some proteins under aqueous and non-aqueous conditions [24,27].
Scanning electron microscopy (SEM) was performed to investigate the shape of the NPs ( Figure 3C). The SEM images of lyophilized Cyt c NP formulations show that the powder particles obtained had a spherical shape and confirm that the particle size was in the nanometer range.
Above findings demonstrate that the nanoprecipitation method is useful in obtaining nano-scale Cyt c without introducing irreversible functional loss. We also demonstrated that the protein was still able to interact with Apaf-1 to induce apoptosis upon rehydration.
Surface modification of Cyt c NPs
To make the nanoparticles useful for delivery purposes we proceeded to modify their surface using a two-step procedure ( Figure 4). First, the NPs' surface was modified with the linker SPDP to allow for their coating with PLGA-SH. The level of SPDP modification was determined by measuring the release of pyridine-2-thione at 343 nm after addition of dithiothreitol (DTT) to the reaction products. An increasing amount of linker was attached to the NPs at increasing reagent concentration during the reaction ( Figure 5A). Since some of the target amino groups of Cyt c overlap with the Cyt c-Apaf 1 interaction site, increased levels of SPDP linked to Cyt c NPs reduced the ability of Cyt c to interact with Apaf-1 by about 20% ( Figure 5A). However, since a large amount of Cyt c should be delivered to each single target cell in the end (a complete NP with roughly over 100,000 molecules), this was acceptable for our application. Circular dichroism (CD) spectra showed that no major changes in Cyt c tertiary structure occurred upon modification with SPDP ( Figure 5B). We selected the 5 fold-SPDP modification level for PLGA attachment to have sufficient polymer attachment points.
Next, the PLGA polymer was attached to the linkermodified (activated) NPs in order to prevent them to dissolve in aqueous media (e.g., upon reconstitution in buffer). The level of modification accomplished with PLGA-SH was determined by spectrophotometric analysis of the 2-pyridyldithio group released upon synthesis ( Figure 4) at 343 nm. The level of modification of the NPs with PLGA was determined to be 2.06 ± 0.04 moles per mol of Cyt c under the conditions employed (see Methods Section for details). This construct is referred to from here on as PLGA-S-S-Cyt c NPs.
Finally, we verified whether the constructs obtained would behave in the manner we anticipatedstable in aqueous medium under oxidizing conditions and dissolving after reduction-induced polymer shedding under intracellular conditions. PLGA-S-S-Cyt c NPs were placed in release buffer using glutathione concentrations of 0, 1 μM, and 10 mM and incubated at 37°C simulating extra-and intracellular conditions [28]. At pre-determined times the supernatant was removed and the concentration of dissolved Cyt c determined by measuring the heme absorbance at 530 nm. The supernatant was replaced to maintain sink conditions. The concentration of the released protein was used to construct cumulative release profiles ( Figure 6).
The PLGA coated Cyt c NPs released ca. 10% of Cyt c during the experiment, most of it during the first few hours as a "burst release". The amount of burst compares well with most conventional PLGA-based sustained release devices. Release of >80% of Cyt c was accomplished under reducing conditions demonstrating that the system design idea proved correct.
PLGA-S-S-Cyt c NPs cytotoxic effects to HeLa cells
To investigate whether the synthesized NPs would display the desired effect on cancer cells, HeLa cells were incubated with PLGA-S-S-Cyt c NPs at 25, 50, and 100 μg/ml Cyt c concentration for 6 h. Cyt c NPs coated with PLGA induced a significant reduction in cell viability after 6 h of incubation, in particular at the 100 μg/ml Cyt c concentration (Figure 7). Several control experiments were performed. PLGA was conjugated to the SPDP linker and added to HeLa cells at the same concentration as in the corresponding experiments with the highest PLGA-S-S-Cyt c NPs concentration. No significant cytotoxicity was observed after 6 h ( Figure 7) and even 24 h of incubation (data not shown) with PLGA-SPDP. To further confirm that the cell death after treatment with PLGA-S-S-Cyt c NPs was due to Cyt c, we constructed the same PLGA coated NPs delivery using the non-apoptotic protein αlactalbumin (LA). The LA-based NPs had no effect on cell viability excluding cytotoxic effects of the delivery system per se (Figure 7). Similar to us, Zhao and coworkers designed a smart nanoparticulate DDS for an apoptotic protein (i.e., caspase-3). The protein was released mediated by intracellular reducing conditions. The study showed evidence of cell death after 48 h of incubation [18] in contrast to 6 h for our DDS.
The DDS designed by us herein also proved much more efficient in killing HeLa cells than our previous smart DDS based on silica NPs [15]. Making the drug Cyt c itself the nanoparticulate delivery system eliminated the main problem encountered with the other system, namely, increasing silica toxicity when trying to deliver sufficient Cyt c to the target cells. Similarly, NPs using other materials [29] could present similar issues as our silica-based delivery system.
Another potential advantage of the newly designed DDS is its negative charge. Although positively charged NPs have shown good cell internalization properties [18,30,31], they potentially bind to vascular endothelial cells reducing their tumor accumulation by means of the EPR effect (i.e., the vascular endothelial luminal surface is known to be negatively charged) [2]. In contrast, PLGA has a negative charge at physiological and alkaline pH and it has been demonstrated that it can be internalized by the cell and is able to escape from the endosomes [32]. Therefore, our protein-based NPs could be an alternative to the intracellular delivery also of other apoptotic proteins.
Investigation of apoptosis induction in HeLa cells by the intracellular release of Cyt c
To confirm that the Cyt c-induced cell death observed in the cell viability experiments was indeed due to apoptosis, we assessed the occurrence of nuclear segmentation and chromatin condensation. After HeLa cells were incubated with PLGA-S-S-Cyt c NPs or the PLGA-S-S-LA NPs control for 6 h, the cells were stained with PI and DAPI. Co-localization of DAPI and PI occurred when cells were incubated with Cyt c-based NPs (Figure 8), which points toward nuclear fragmentation and chromatin condensation in the cells indicative of ongoing apoptosis. In contrast, cells without treatment or treated with the PLGA-S-S-LA control showed no indication for dye co-localization and thus apoptosis. These results are in agreement with confocal results by Santra et al [7] who demonstrated that the delivery of Cyt c to the cytoplasm of the cancer cells results in the induction of apoptosis of such cells [7].
Conclusions
Therapeutic proteins have enormous potential in the treatment and prevention of human diseases but are of limited use because they frequently display low physicochemical stability. Specifically, when injected proteins usually quickly degrade and have short blood half-lives. Thus, the development of DDS able to protect the protein payload from degradation and in addition enabling targeted and controlled delivery is of considerable interest. Herein, we presented a new method for the delivery of Cyt c, an apoptotic protein, to model cancer cells. The main advantage of the system is that the DDS core consisted of Cyt c NPs and thus the drug itself thus overcoming payload limitations of drug-loaded systems. In order to stabilize the protein NPs we coated them with hydrophobic PLGA using a redox-sensitive linker. Only little Cyt c was released under oxidizing conditions, but 80% was released within hours under intracellular conditions. We furthermore demonstrated that PLGA-S-S-Cyt c NPs were able to induce apoptosis in a human cancer cell line while PLGA-S-S-LA NPs as control did not. Redox-responsive polymer-coated protein-based NPs are a simple and effective method for intracellular delivery of Cyt c for induction of apoptosis. We foresee that this system could potentially be used for the delivery of other proteins. However, we like to point out that our system was not tested in a non-cancerous cell line at this point in time because it does not include targeting ligands. Thus, it would probably show comparable toxicity in cancer and non-cancer cell lines since the EPR effect afforded by the nanoparticle formulation would not be relevant in vitro studies. It is also important to acknowledge that particle size is by far not the sole determinant of tumor specificity in in vivo studies. For example, immune cells can consume the drug delivery system and some tumors maybe very dense in the core disabling EPR-mediated targeting. To address such questions, experiments ongoing in our laboratory focus on in vivo experiments using animal models. Tumor selectivity and internalization kinetics and mechanism of PLGA-S-S-Cyt c NPs are being scrutinized as well as the decoration of the system with tumor specific ligands. Combining passive targeting with additional ligand-mediating targeting should not only amplify the specificity of the NPs but also facilitate their efficient cellular uptake.
Experimental procedures
Cytochrome c from equine heart, reduced glutathione ethyl ester, and a protease inhibitor cocktail were from Sigma-Aldrich (St. Louis, MO). Acetonitrile (HPLC grade) was from Fisher (Waltham, MA). Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) was from Proteochem (Denver, CO). Poly (lactide-co-glycolide)-SH with a thiol end cap (PLGA-SH, 30,000 Da, copolymer ratio 1:1) was from Akina, Inc (West Lafayette, IN). 4', 6-Diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were purchased from Invitrogen (Grand Island, NY). All the reagents were used without further purification. All other chemicals were from various commercial suppliers and were at least of analytical grade. HeLa cells were purchased from the American Type Culture Collection (Manassas, VA) and grown according to the vendor's instruction.
Protein nanoprecipitation
Protein nanoparticles were obtained using a similar method as described previously by us [14]. Briefly, Cyt c was solvent-precipitated from nanopure water in presence of the excipient methyl-β-cyclodextrin by adding acetonitrile at a 1:4 volume ratio. Different protein concentrations (5, 10, 20, 40 mg/ml) as well as different mass ratios of protein-to-excipient (1:4, 1:8, 1:20 and 1:40, w/w) were studied. The protein suspension obtained was centrifuged at 6,000 rpm for 15 min, the supernatant discarded, and the pellet vacuum dried for 30 min.
Determination of the nanoprecipitation yield
Protein concentration and amount of protein aggregates in Cyt c NPs were determined as described by us in detail [33]. In brief, the Cyt c NPs were suspended in 2 ml of potassium phosphate (PBS) buffer for 2 h to dissolve the buffer-soluble fraction. The samples were then subjected to centrifugation at 6,000 rpm for 15 min and the supernatant used to determine the protein concentration. Next, 1 ml of 6 M urea was added to the pellet to dissolve the buffer-insoluble protein fraction. The protein concentration was determined by quantitative ultraviolet (UV) spectrophotometric analysis at 280 nm and also from the heme absorbance at 408 nm. The precipitation yield was calculated from the actual and theoretical quantity of protein recovered after nanoprecipitation and rehydration (%w/w). Cyt c as obtained from the supplier in PBS or 6 M urea was used to construct a calibration curve. The experiments were performed in triplicate, the results averaged, and the standard deviations calculated.
Synthesis of SPDP-Cyt c NPs
Following the Cyt c nanoprecipitation, the SPDP linker was added directly to the resulting suspension (i.e., in 80% acetonitrile) to accomplish the NPs surface modification with the linker. Different Cyt c-to-SPDP molar ratios (1:2.5, 1:5, 1:10) were tested. After letting the mixture react for 30 min, the suspension was used to determinate the level of modification with SPDP. To stop the reaction and to remove unreacted SPDP and N-hydroxysuccinimide by-products, repeated centrifugation (6,000 rpm, 15 min)/washing cycles were performed. The pyridyldithiol-activated Cyt c (SPDP-Cyt c) NPs pellet was saved for further reaction and analysis. The supernatant was used to determine the concentration of unreacted SPDP by measuring the release of pyridine-2-thione at 343 nm after addition of 10 μL of 15 mg/ml DTT (the absorbance of the supernatant before reaction with DTT was used as blank). The amount of covalent modification of the nanoparticles with the linker was determined from the difference between the initial SPDP concentration and the unreacted SPDP. The experiments were performed in triplicate, the results averaged, and the standard deviations calculated.
Synthesis of PLGA-Cyt c NPs
HS-PLGA (50 mg) dissolved into 5 ml of Acetonitrile was added to SPDP-Cyt c NPs (5 mg) and reacted at room temperature for 18 h. After reaction, unreacted HS-PLGA and pyridine 2-thione by-products were removed by centrifugation at 6,000 rpm for 10 min. The supernatant was used to determine the level of modification by measuring the concentration of pyridine-2-thione at 343 nm.
In vitro release of Cyt c
The release of Cyt c from PLGA-S-S-Cyt c NPs was measured similarly as described by us [28]. Briefly, 0.25 mg of PLGA-S-S-Cyt c NPs powder were suspended by sonication in 1 ml of 50 mM PBS with 1 mM EDTA at pH 7.4 and glutathione (GHS) concentrations of 0, 0.001, and 10 mM. Incubation was performed for various times at 37°C, and the NPs were pelleted by centrifugation at 14,000 rpm for 10 min. The supernatant was removed and used to determine the concentration of released Cyt c. The pellet was resuspended in GHS-PBS buffer. The amount of protein released was used to construct cumulative release profiles. The experiments were performed in triplicate, the results averaged, and the standard deviations calculated.
Dynamic light scattering
Particle sizes of the different formulations of Cyt c NPs were determined by Dynamic light scattering using a DynaPro Titan. The samples were dispersed in DMF and subjected to ultrasonication at 240 W for 30 sec prior to the measurements.
Scanning electron microscope (SEM)
SEM of the different formulations of Cyt c NPs was performed using a JEOL 5800LV scanning electron microscope at 20 kV. The samples were coated with gold for 10 sec to a thinkness of 10 nm using a Denton Vacuum DV-502A.
Circular dichroism (CD) spectroscopy CD spectra were recorded using a JASCO J-1500 High Performance CD spectrometer at room temperature. The protein (Cyt c, Cyt c NPs, or SPDP-Cyt c NPs) was dissolved in nanopure water. CD spectra were acquired from 260 to 350 nm (tertiary structure) at a concentration of 1 mg/ml using a 10 mm quartz cuvette. Each spectrum was obtained by averaging two scans. Spectra of nanopure water blanks were measured prior to the samples and subtracted from the sample spectra.
Cell-free caspase-3 assay
The cell lysate was obtained as described by us [15]. Briefly, the cell-free reaction was initiated by adding Cyt c or the different Cyt c NP formulations (e.g. 100 μg/mL of Cyt c NPs or SPDP-Cyt c NPs) to freshly purified cytosol (3 mg/ml) in a total reaction volume of 100 μL. The reaction was incubated at 37°C for 150 min. Afterwards the caspase-3 assay was performed following the manufacturer's protocol (CaspACE™ assay; Promega, Madison, WI). The plate was incubated overnight at room temperature and the absorbance at 405 nm was measured in each well using a Thermo Scientific Multiskan FC. All measurements were performed in triplicate.
Cell culture
HeLa cells were maintained in accordance with the ATCC protocol. Briefly, the cells were cultured in minimum essential medium (MEM) containing 1% L-glutamine, 10% fetal bovine serum (FBS), and 1% penicillin in a humidified incubator with 5% CO 2 and 95% air at 37°C . All experiments were conducted before cells reached 25 passages. For the cell viability and confocal microscopy experiments, HeLa cells were seeded in 96-well plates or chambered cover-slides (4 wells), respectively, for 24 h in MEM containing 1% L-glutamine, 10% FBS, and 1% penicillin. Subsequently, cell growth was arrested by decreasing the FBS concentration in the medium to 1% for 18 h. Then, cells were exposed and incubated with the different bioconjugates for 6 h.
Investigation of apoptosis induction in HeLa cells by the intracellular release of Cyt c
HeLa cells (25,000 cells) were seeded in chambered coverglass (4-wells) as previously described by us [15]. The cells were incubated with PLGA-S-S-Cyt c NPs at a 25 μg/mL Cyt c concentration at 37°C for 6 h. For detection of apoptosisdependent nuclear fragmentation, the cells were washed with PBS (1X) and incubated initially with DAPI (300 nM) and thereafter with PI (75 μM) for 5 min each. HeLa cells were then fixed using 3.7% formaldehyde. The coverslips were examined under a Zeiss laser-scanning microscope 510 using a 67× objective. Co-localization of DAPI and PI upon internalization into HeLa cells was determined, which is representative of highly condensed and fragmented chromatin in apoptotic cells [7,34,35]. DAPI was excited at 405 nm and its emission was detected at 420-480 nm. PI was excited at 561 nm and was detected above 600-674 nm. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2015-05-14T00:00:00.000 | 18855161 | {
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} | pes2o/s2orc | Hippocampal memory enhancing activity of pine needle extract against scopolamine-induced amnesia in a mouse model
We evaluated the neuropharmacological effects of 30% ethanolic pine needle extract (PNE) on memory impairment caused by scopolamine injection in mice hippocampus. Mice were orally pretreated with PNE (25, 50, and 100 mg/kg) or tacrine (10 mg/kg) for 7 days, and scopolamine (2 mg/kg) was injected intraperitoneally, 30 min before the Morris water maze task on first day. To evaluate memory function, the Morris water maze task was performed for 5 days consecutively. Scopolamine increased the escape latency and cumulative path-length but decreases the time spent in target quadrant, which were ameliorated by pretreatment with PNE. Oxidant-antioxidant balance, acetylcholinesterase activity, neurogenesis and their connecting pathway were abnormally altered by scopolamine in hippocampus and/or sera, while those alterations were recovered by pretreatment with PNE. As lipid peroxidation, 4HNE-positive stained cells were ameliorated in hippocampus pretreated with PNE. Pretreatment with PNE increased the proliferating cells and immature neurons against hippocampal neurogenesis suppressed by scopolamine, which was confirmed by ki67- and DCX-positive stained cells. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (pCREB) in both protein and gene were facilitated by PNE pretreatment. These findings suggest that PNE could be a potent neuropharmacological drug against amnesia, and its possible mechanism might be modulating cholinergic activity via CREB-BDNF pathway.
result in damage to proteins, lipids, and nucleic acids in patients with neurodegenerative disorders 11,12 . Brain tissue is extremely sensitive to oxidative stress due to its high oxygen consumption, iron content, polyunsaturated fatty acids, and low antioxidant capacity 13,14 . Moreover, the hippocampus and amygdala are more sensitive to oxidative injury 15 , and excessive oxidative stress can lead to memory deficits by impairing hippocampal synaptic plasticity 16 .
Among numerous herbal plants, the pine needle (Pinus densiflora Sieb & Zucc.) is commonly used as an herbal medicine and health supplement in East-Asian countries, such as Korea and China 17 . The pine needle is utilized as an infusion of tea and supplementary health food, and is reported to be helpful in the treatment of patients with cancer, coronary heart diseases and neurodegenerative disease [17][18][19] . Despite the many uses of pine needle, there have been no studies of its neuropharmacological activities. Thus, in the present study we investigated the anti-amnesic effects of pine needle extract (PNE) on memory deficits in a mouse model of cognitive impairment caused by scopolamine.
Results
Compounds present in the 30% ethanolic pine needle extract. Four different kinds of flavonoids including catechin, quercetin dehydrates, astragalin, and kaempferol, when PNE sample were subjected to the HPLC. Among them, only catechin and astragalin were detected at 19.01 min and 31.34 of retention time (see Supplementary Fig. S1A and B online), and the concentration of catechin and astragalin were 0.35 6 0.01 and 2.68 6 0.04 respectively (see Supplementary Fig. S1C online). GC-MS data confirmed that terpenoids of a-pinene or b-pinene were not present in PNE (data not shown).
Effect on anti-amnesia in Morris water maze task. Scopolamine injection significantly prolonged the escape latency time and cumulative path length compared with the naïve group on fourth day of the acquisition period (approximately 3-and 4-fold respectively, P , 0.001). The time spent in the target quadrant was significantly shortened in the control group compared with naïve group on fifth day (P , 0.001). Pretreatment with PNE significantly ameliorated the escape latency (P , 0.001 for all groups) and the path-length (P , 0.001 for all groups; Fig. 1A and B) prolonged by scopolamine. On day five, PNE pretreatment groups exhibited significantly increased time spent in the target quadrant compared with the control group (P , 0.001 for both 25 and 50 mg/ kg, P , 0.01 for 100 mg/kg; Fig. 1C). Tacrine had effects similar to those of PNE pretreatment.
Effects on ROS levels in serum and hippocampus. Scopolamine injection significantly increased the ROS levels in both serum and hippocampal tissue compared with the naive group (approximately 1.6-fold, P , 0.001 in both samples). Pretreatment with PNE significantly decreased ROS levels in serum and hippocampal tissue compared with the control group (P , 0.001 for all groups; Fig. 2A and B); similar effects were observed in the tacrine group. Effects on NO and MDA level in hippocampus. NO level in hippocampal tissue was increased significantly by scopolamine injection compared with the naive group (approximately 1.5-fold, P , 0.001), while pretreatment with PNE significantly attenuated the increase of NO level (P , 0.001 for all groups; Fig. 2C).
MDA level was increased significantly by scopolamine injection in hippocampal tissue compared with the naive group (approximately 3.9-fold, P , 0.001), while pretreatment with PNE significantly attenuated the elevation of MDA level compared with the control group (P , 0.01 for 25 mg/kg, P , 0.001 for both 50 and 100 mg/kg; Fig. 2D). Tacrine had effects similar to those of PNE pretreatment.
Effects on antioxidant biomarker profiles in the hippocampus. Scopolamine injection significantly depleted the antioxidant activities in hippocampal tissue, including TAC capacity (P , 0.001), GSH content (P , 0.001), activity of GSH-Rd (P , 0.001), GST (P , 0.01), SOD (P , 0.05), and catalase activity (P , 0.001), compared with the naïve group. These changes were reversed by PNE pretreatment and for all biomarkers, 50 and 100 mg/kg PNE pretreatment were more effective than 25 mg/kg PNE treatment. Interestingly, SOD activity in response to PNE pretreatment was further enhanced compared with the naïve group. In contrast, tacrine had different effects than PNE, with the exception of TAC capacity (Table 2).
Lipid peroxidation, cell proliferation and neurogenesis in the hippocampus. Scopolamine injection significantly induced the lipid peroxidation in hippocampus (P , 0.01 in DG, P , 0.05 in CA3), shown as a deep red color in the dentate gyrus (DG) and cornu ammonis 3 (CA3) regions by 4HNE staining. Pretreatment with PNE significantly attenuated 4HNE-positive staining in the DG including granular cell layer (GCL), subgranular zone (SGZ) (P , 0.05 for 25 mg/kg, P , 0.01 for 100 mg/kg) and CA3 compared with the control group (P , 0.05 for both 25 and 100 mg/kg; Fig. 3A-C).
Scopolamine injection significantly inhibited the production of new granule cells in the hippocampal DG region, particularly in the SGZ (P , 0.05), as evidenced by reduced Ki67 staining. These alterations were significantly attenuated by PNE pretreatment with notable replacement of proliferating cells with Ki67-positive staining in the SGZ compared with the control group (P , 0.05 for 50 mg/kg; Fig. 4A and B). Scopolamine injection significantly suppressed the adult neurogenesis, shown as a distributed dendrites and neuron bodies in the DG region by DCX staining, particularly in the SGZ (P , 0.05). Pretreatment with PNE completely ameliorated the adult neurogenesis by enhancing immature neurons in the SGZ compared with the control group (P , 0.05 for all groups; Fig. 5A and B). In contrast, tacrine ameliorated these alterations only in DCX staining result.
Effect on AChE activity in hippocampus. AChE activity in hippocampal tissue was significantly increased by scopolamine injection compared with the naïve group (approximately 3.8-fold, P , 0.001), while pretreatment with PNE completely inhibited the hyper-activation of AChE compared with the control group (P , 0.001 for all groups; Fig. 6A). This effect was also seen with tacrine in the hippocampus.
Western blot analysis of CREB/BDNF in the hippocampus. Scopolamine injection slightly reduced the BDNF and phospho-CREB protein levels in the hippocampus. In contrast, pretreatment with PNE increased BDNF and phospho-CREB levels compared with the control group. Tacrine had an effect on the expression of BDNF similar to that of PNE (Fig. 6B).
Changes in mRNA levels in the hippocampus. The CREB1 (P , 0.001), mAChR1 (P , 0.01), BDNF (P , 0.05) and CBP (P , 0.01) mRNA levels were significantly down-regulated by scopolamine injection compared with the naïve group in the hippocampus. This down-regulation was ameliorated significantly by PNE pretreatment compared with the control group (P , 0.01 or P , 0.001 for all groups; Fig. 6C). Moreover, scopolamine injection significantly upregulated the expression of iNOS in the hippocampus compared with the naïve group (approximately 1.6-fold, P , 0.001), while this upregulation of iNOS was significantly ameliorated by pretreatment with PNE (P , 0.001 for all groups; Fig. 6C). Similar effects were observed in the tacrine group.
Discussion
Neurodegenerative disorders are becoming a major healthcare issue as they result in progressive brain dysfunction among the aging population in industrialized countries. The major pathophysiological features involve alterations of the central cholinergic system, including cholinergic neurons, neurotransmitters and their respective receptors 20 . Several inhibitors of AChE, as well as N-methyl-Daspartate (NMDA) receptor antagonists, are available currently for the treatment of Alzheimer's disease at mild and moderate stages, even though the mechanisms mediating these processes remain to be fully elucidated and sometimes theses drugs show adverse effects 21 . Multitarget-directed ligands and natural therapeutic agents are recently drawing attention as complementary and alternative medicine for neurodegenerative disorders 22 .
Herein, we investigated the neurotherapeutic effects of PNE on memory deficits in a mouse model of amnesia induced by scopolamine injection. To evaluate the effects of PNE on spatial learning and memory, a Morris water maze task was performed 23 . Following 4 days of acquisition training, the scopolamine treated group had prolonged escape latency (3-fold) and cumulative path-length (4-fold). This result was consistent with time spent in the target quadrant, which was reduced compared with the naïve group. As expected, pretreatment with PNE ameliorated delayed escape latency ( Fig. 1A-C). These findings suggest that PNE has an anti-amnesic effect in the scopolamine-induced model.
We examined the antioxidant effects of PNE in the hippocampus since oxidative stress contributes to pathogenesis and histological changes in patients with neurodegenerative disorders 24 . The excessive production of ROS triggers neurotoxic activity through thiol-and lipid-dependent mechanisms in cell membrane 25 . The scopolamineinduced memory deficit model exhibits prominent oxidative stress and memory deficits, although the mechanism of oxidative damage in brain tissue remains unclear 26,27 . The hippocampus plays a crucial role in short-and long-term memory, as well as spatial memory 28 , and is highly susceptible to oxidative stress 29 . As expected, scopolamine injection induced oxidative stress in hippocampus, as evidenced by increased levels of ROS, NO and MDA. These alterations were attenuated significantly by PNE pretreatment ( Fig. 2A-D). Previous studies showed that scopolamine treatment elevated the production of MDA in the cerebral cortex and hippocampus 30 . These results were consistent with changes in iNOS gene expression in the hippocampus (Fig. 6C).
To protect tissues against oxidative damage, cellular organisms maintaining an antioxidant system composed of non-enzymatic and enzymatic components. In our study, scopolamine injection significantly depleted antioxidant capacity of TAC, SOD, catalase and the GSH-redox system in the hippocampus, whereas these alterations were ameliorated significantly by pretreatment with PNE (Table 2). These results suggested that the antioxidant potential of PNE contributed to neuronal plasticity and memory function. Furthermore, in the DG and CA3 region of the hippocampus, scopolamine induced lipid peroxidation, shown as positively stained 4HNE cells in the GCL and SGZ. However, pretreatment with PNE completely attenuated the over-production of 4HNE (Fig 3). Increased 4HNE is a key histopathological feature of various diseases, including Alzheimer's disease, diabetes and cancer 31 . In the hippocampus, dentate granule cells are responsible for the separation of each episodic memory, such as object and place, and the CA3 pyramidal neurons receive and store auto-associative memories in their network 32 .
The new neurons are generated as neural progenitor cells in the SGZ of hippocampal DG, in the adult mammalian brain, including humans 33 . As well known, adult hippocampal neurogenesis plays a crucial role in hippocampal memory function 34 . Accordingly, enhancing hippocampal neurogenesis may be an efficient therapeutic target for Alzheimer's disease. We confirmed the significantly decreased proliferating cells and immature neurons by Ki67 and DCX staining of the scopolamine-induced mouse hippocampus, while pretreatment with PNE markedly ameliorated repression of reproducing cell and neuronal precursor cells in the SGZ (Fig. 4 and 5). These findings can predicts the production of new granule cells, maturation of neurons as well as hippocampal neurogenesis. The neurogenesis and gliogenesis are regulated by various molecular factors, such as neurotransmitters, neurotrophins, hormones and their respective signaling pathways 35 .
To investigate the role of PNE in neurogenesis, we examined cholinergic activity, neurotransmitter levels and subsequent signaling pathways. Scopolamine injection augmented AChE activity 4-fold in hippocampal tissue, whereas pretreatment with PNE completely attenuated the excessive activation of AChE (Fig. 6A). Maintenance of ACh levels is necessary for normal memory function, but excessive AChE activity leads to disruption of ACh in hippocampal cholinergic synapses 36 . In the synaptic cleft, ACh binds to post-synaptic mAChRs and the synaptic signal communicates sequentially to cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-CREB signaling pathway via G-coupled protein receptors 37 . In our result, the downregulation of mAChR-1 gene expression by scopolamine in hippocampal tissues was normalized significantly by PNE pretreatment (Fig. 6C). Previous studies have revealed that patients with Alzheimer's disease have down-regulated expression of mAChR-1 38 , which correlates with memory disruption in a mAChR-1 knockout mouse model 39 .
In contrast, the transcription factor CREB is essential for memory and synaptic plasticity in the CNS. Disturbance of phosphorylated CREB within the hippocampal region leads to the progression of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease 40 . Previous studies indicated that CREB has a neuroprotective effect against ROS-mediated cell toxicity 11 , and activation of CREB ameliorated cognitive impairment via the cholinergic system 8 . In our study, hippocampal phospho-CREB was reduced by scopolamine injection, which was increased by pretreatment with PNE (Fig. 6B). The expression of CREB1 and CBP, a co-factor of CREB, supported these results (Fig. 6C). BDNF is known to improve learning ability and neurogenesis via phospho-CREB signaling, and mice deficient in BDNF and pro-BDNF exhibit an aging process characteristic of Alzheimer's disease 41,42 . In our study, pretreatment with PNE markedly increased BDNF protein levels (Fig. 6B), which was correlated with BDNF mRNA levels in hippocampal tissue (Fig. 6C).
Pinus densiflora, also called Japanese Red Pine, grows naturally in East Asian countries and has been used traditionally as a supplement in various foods and folk medicine. Previous studies well evidenced the pine needle has potent antioxidant effects against oxidative damage and apoptosis, with anti-obesity and anti-cancer effects 17,43,44 . To date, several active compounds have been isolated from organic-solvent extractions, including flavonoids, terpenoids and tannin 45,46 . Among them, the flavonoid-derived compounds are well-known for enhancement of memory function via stimulating neurogenesis, regulating neurotrophins, and protecting neurons against oxidative and metabolic stress 47,48 . In this study we observed the chemical characters of PNE via performing HPLC analysis using four flavonoid-derived compounds including catechin, astragalin, quercetin dehydrate, and kaempferol. As a result of HPLC analysis, catechin and astragalin were detected in PNE, and we further explored the quantitative analysis of those compounds (Fig. S1A-C). It was well corresponded to the previous studies that both catechin and astragalin showed potent neuroprotective effects on the various brain damage model. Catechin showed its protective effects on the severe brain oxidative damage via enhancement of antioxidant capacities as well as especially inactivation of NF-kB in pyramidal cells of hippocampal CA1 region 49 . On the other hand, the astragalin prevented brain ischemic injury through anti-apoptotic effect and decreases of neuronal cell adhesion molecules 50 .
In conclusion, our results demonstrate that PNE has an antiamnesic effect, which could be mediated by hippocampal neurogenesis, cholinergic activity, CREB-BDNF pathway and its antioxidant properties. This suggests that the Pinus densiflora leaf may be a promising treatment for patients with neurodegenerative disorders. Further study should aim to confirm the neuroprotective effects and its corresponded mechanisms using active compounds such as catechin and astragalin.
Material and Methods
Materials. Pine needles (Pinus densiflora Sieb & Zucc.) were collected from Guryong-Mountain in Chungwon-gun, Chungbuk, Korea. Fifty grams of dried pine needles were extracted with 0.5 L of 30% ethanol at 25uC for 72 h, and suspension was filtered using a 300-mesh 50-mm filter paper (Advantec, Toyo Roshi Kaisah, Tokyo, Japan). Filtrate was concentrated in a rotary evaporator and lyophilized. The final yield of the extract was 10.46% w/w, and it was stored at 280uC. Fingerprinting analysis of PNE was measured by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS).
The dose of PNE was determined based on prescreening results. The protocol was approved by the Institutional Animal Care and Use Committee of Daejeon University (DJUARB 2013-031), and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH).
Morris water maze task. Morris water maze task was performed in a circular pool (100-cm diameter 3 50-cm height) with a circular acrylic platform (10-cm diameter 3 35-cm height). The location of platform can be discriminated by visual cue. The pool was filled with milk water (22 6 1uC) and divided into equal quadrants. A platform was placed in one of the quadrants ,1 cm below the surface as described previously 51 . Data were recorded using a video camera connected to the corresponding software (Smart Junior, Panlab SL; Barcelona, Spain).
Mice were placed on the platform for 10 s, and removed from the pool. Mice were given acquisition trial for 4 days. The escape latency and cumulative path-length were recorded during each acquisition trial. On day 5 th , all mice were subjected to probe trial without platform, and were recorded for 120 s. The time spent in the target quadrant was measured for spatial learning and memory.
Sample preparation. All mice were sacrificed under ether anesthesia after 1 h following the Morris water maze. Blood was collected by the method followed by serum. Serum was collected by centrifugation at 3000 rpm for 15 min at 4uC, and the hippocampal region from the whole brain was isolated immediately, and then samples were stored at 280uC or in RNAlater (Ambion, TX, USA). In each group, three mouse brains were fixed in 4% paraformaldehyde, and hippocampi of remaining seven mice was used for biochemical analysis, western blot and real-time PCR analysis. The part of hippocampus was homogenized on ice using RIPA buffer, and other part of hippocampus was used for isolation of RNA.
ROS levels. The levels of ROS in serum and hippocampus were determined as described previously 52 . Absorbance was measured at 505 nm using a UV spectrophotometer (Molecular Devices; Sunnyvale, CA, USA). The results were calculated using H 2 O 2 as a standard and expressed as unit/mL.
NO and MDA levels. The level of nitric oxide (NO) in hippocampus was determined using the Griess method 53 . The purple azo dye product was measured at 540 nm using a UV spectrophotometer.
The level of lipid peroxidation in hippocampus was determined by measuring malondialdehyde (MDA) using the thiobarbituric acid reactive substance (TBARS) method as described previously 54 . Absorbance at 535 and 520 nm was measured using a spectrophotometer. The concentration of TBARS was calculated using a standard TEP and the results were expressed as mmol/mg protein.
TAC capacity. Total antioxidant capacity (TAC) in hippocampus was determined as described previously 55 . The level of TAC was expressed as the gallic acid equivalent antioxidant capacity (GEAC). The absorbance at 600 nm was measured using a UV spectrophotometer.
Total GSH content, GSH-Rd and GST activities. Glutathione (GSH) content in hippocampus was determined as described previously 56 . Absorbance at 405 nm was measured using a UV spectrophotometer.
Glutathione S-transferase (GST) activity in hippocampus was determined using a GST assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer's protocol. Absorbance at 340 nm was measured using UV spectrophotometer. Enzyme activity was calculated using the following formula: Enzyme activity: (unit/mL) 5 [(DA340)/min 3 0.2 3 dilution factor]/(5.3 mM -1 cm 21 3 volume of enzyme sample tested).
SOD and catalase activities. Superoxide dismutase (SOD) activity in hippocampus was determined using a SOD assay kit (Dojindo Laboratories; Kumamoto, Japan) according to the manufacturer's protocol. Absorbance was measured at 450 nm using UV spectrophotometer. Dilutions of bovine erythrocyte SOD ranging from 0.01-50 unit/mL were used as standards.
Catalase activity in hippocampus was determined as described previously 58 . Absorbance of the purple formaldehyde product at 550 nm was measured using a UV spectrophotometer.
Immunohistochemical stain analysis. Immunohistochemical analysis was performed by assessing cell proliferation with Ki67, neurogenesis with DCX and lipid peroxidation with 4HNE staining in hippocampus. Three brain tissues of each group were immersed in the fixative solution for 4 h. The brain was cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogen, and cut into coronal frozen sections (35 mm) using a Leica CM3050 cryostat. Sections were stored under anti-freeze buffer.
AChE activity. Acetylcholinesterase (AChE) activity in hippocampus was determined using a AChE activity assay kit (AAT Bioquest; Sunnyvale, CA, USA) according to the manufacturer's protocol. Absorbance at 410 nm was measured using a UV spectrophotometer.
Western blot analysis. The expression of BDNF and CREB/phospho-CREB proteins in hippocampus was evaluated by Western blot. The proteins from homogenates were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skim milk, the membranes were probed overnight at 4uC with primary antibodies (CREB, p-CREB, BDNF and b-actin). The membranes were washed and incubated for 2 h with HRPconjugated anti-rabbit antibody. Western blots were visualized using an enhanced chemiluminescence (ECL) advanced kit.
Quantitative real-time PCR analysis. The mRNA expression of genes encoding CREB1, mAChR1, BDNF, CREB-binding protein (CBP), and inducible nitric oxide synthase (iNOS) in hippocampus was measured by real-time PCR. Total RNA was isolated from hippocampus using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) and cDNA synthesized using a High-Capacity cDNA reverse transcription kit (Ambion, Austin, TX, USA). Real-time PCR was performed using SYBRGreen PCR Master Mix (Applied Biosystems; Foster City, CA, USA) and PCR amplification was performed using a standard protocol with the IQ5 PCR Thermal Cycler (Bio-Rad, Hercules, CA, USA). Information of primers was summarized in Table 1. | v3-fos-license |
2018-12-16T03:02:46.581Z | 2018-10-21T00:00:00.000 | 55593960 | {
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} | pes2o/s2orc | Enrichment of Apple Slices with Bioactive Compounds from Pomegranate Cryoconcentrated Juice as an Osmodehydration Agent
Pomegranate juice is an important source of bioactive compounds, and cryoconcentrated juice is an interesting osmodehydration agent to enrich a vegetal matrix. is investigation aimed to incorporate bioactive compounds from pomegranate cryoconcentrated juice into apple slices using osmodehydration (OD) assisted by pulse vacuum (PV) and ohmic heating (OH). e apple slices (3× 4× 0.5 cm) were osmodehydrated using a 47 °Brix pomegranate cryoconcentrated juice at 30, 40, or 50°C for 180min with an electric eld of 6.66V/cm (50V) and a 5min pulsed vacuum. Over the time, all of the treatments applied to the apple slices increased the soluble solids and bioactive compounds compared with the fresh sample. PVOD/OH at 50°C had the highest content of total anthocyanin during processing, and the best results for the total avonoids were obtained with OD/OH at 50°C and 40°C. e osmodehydration assisted by pulse vacuum and ohmic heating using a cryoconcentrated juice is a useful combined technique to acquire enriched vegetal samples with bioactive compounds.
Introduction
As consequences of changing lifestyle and eating habits, diseases such as obesity, cardiovascular problems, diabetes type II, hypertension, and cancer among others have increased in the last decades [1,2].ese adverse e ects have developed an interest in healthy food and the consumption of functional food [3].Functional foods include bioactive compounds (BAC) that have health bene ts and nutritional value.Vitamins, polyphenols, and minerals are examples of BAC [3].
Pomegranate (Punica granatum L.) recently has gained interest in the past few years because of its nutritional and antioxidant activities [4].Pomegranate juice is a potential source of anthocyanins, avonoids, organic acids, and ellagic acid [5][6][7].Additionally, recently, pomegranate juice has been used as a functional ingredient, for example, in fermented milk and smoothies [8,9].erefore, its health bene ts are associated with those chemical characteristics.
e process of enriching vegetables to create functional products with BAC has been achieved through the combination of emerging technologies such as cryoconcentration with ohmic heating (OH) and assisted techniques such as pulsed vacuum (PV) and osmodehydration (OD).In this context, the food industry must link consumer demands and these available technologies [10].
Cryoconcentration, also called freeze concentration, as mentioned above, is an emerging technology, which consists of entirely or partially freezing the liquid food solution for later separation of the ice fraction from the liquid [11].Vitamins, as well as polyphenols, are thermolabile compounds, and by using freeze concentration, the high nutritional value and organoleptic characteristics can be protected [12].erefore, the BAC present in pomegranate juice could be protected using this technology.
OH is a thermal process, in which energy is generated by the passage of an alternating electrical current that diffuses through the food [1].PV has been widely used to obtain a rapid penetration of compounds in vegetable tissues and enriches fruits and vegetables with antioxidants, vitamins, and minerals, among others [13].
Osmotic dehydration is a process in which water is partially removed from the cellular material or product when exposed directly to a concentrated solution of solutes or hypertonic medium [14,15].It has the advantage of higher nutritional content compared with drying methods because it has a minimal effect on the components of the food matrix [15].e type of osmotic agent used and its molecular weight or ionic behavior affect the kinetics of water elimination and the gain of solids [15].e OD mass transfer occurs through a semipermeable cell membrane, which makes substantial changes to the tissue structure [16].
e OD applied to vegetables is commonly performed using sucrose or other osmotic agents such as salt; however, recently, Lech et al. [17] reported the use of a fruit juice as an alternative OD solution (commercial chokeberry juice), obtaining interesting results in the final vegetable product in terms of bioactive compounds and antioxidant activity.In addition, Cano-Lamadrid et al. [18] reported a similar use of fruit juices such as OD solutions in this case was used to improve the sensorial and antioxidant capacity of pomegranate cultivar Mollar de Elche, a popular Spanish pomegranate.
is investigation aimed to determine the incorporation of bioactive compounds from frozen concentrated pomegranate juice into osmodehydrated apple slices using pulsed vacuum and ohmic heating treatments and to evaluate the mechanical and optical properties of the enriched product.e pomegranates were cut in half, and the seeds were removed manually for juice extraction and filtered to eliminate any residue from the fruit and be ready for the experimental procedure.Apples were cut into sizes of 3 × 4 × 0.5 cm and submerged in a solution of 2% citric acid and 1% ascorbic acid for 3 min to prevent oxidation.e pomegranate (cv.Wonderful) has a slightly acid sweet flavor, a flavor that is compatible with the apple used as a vegetable matrix to be osmodehydrated.
Osmodehydration
Agent.Cryoconcentrated pomegranate juice (osmodehydration agent) was obtained as described by Orellana-Palma et al. [19] and Petzold et al. [20,21] with modifications.Samples were frozen at −20 °C for 12 h and placed in a refrigerated centrifuge (Eppendorf AG, model 5430R, Hamburg, Germany) operated at 15 °C for 15 min for the first cycle and 10 min for the second and third cycles at a speed of 1878 RCF, until the juice reached 48 °Brix (value obtained at the end of cryoconcentration cycles).e final cryoconcentrated juice had the following phenolic content: total polyphenols of 4953.64 ± 95.48 mg gallic acid equivalents/L, total anthocyanin of 117.64 ± 1.30 mg cyaniding-3-glucoside/L, and total flavonoids of 368.84 ± 4.62 mg catechin/L.For the optical parameters (CIE L * a * b * coordinates), L * 1.07 ± 0.41, a * 4.45 ± 0.24, and b * 1.05 ± 0.04.
Osmodehydration Treatments Assisted by Pulsed Vacuum and Ohmic
Heating.Osmodehydration with atmospheric pressure (OD) or pulsed vacuum (PVOD) and conventional and ohmic heating (OH) at 30, 40, and 50 °C were conducted using a thermoregulated bath, as described by Moreno et al. [22,23].Osmotic treatments were carried out in a stainless steel tank made with two concentric cylindrical electrodes (3.7 and 19 cm in diameter) and a nonconducting bottom, and the distances between them were 7.50 cm [24].Pomegranate juice was exposed to an alternating current at 60 Hz and 50 V, generating an electric field E of 6.66 V/cm, given by the following equation: e temperature during the ohmic heating treatments was controlled through a refrigeration system.
Total Phenolic Content (TPC).
e total phenolic content was determined through a colorimetric method by Folin-Ciocalteu (FC) reagent [25] using gallic acid as the standard.Treated sample extracts (100 µL) were mixed with 7900 µL of distilled water, 500 µL of FC reagent, and 1500 µL of sodium carbonate.e samples were incubated for 120 min before measurement at 760 nm in a spectrophotometer (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of gallic acid equivalents in 100 g of dry matter (mg GAE/100 g d.m.).
Total Anthocyanin Content (TAC).
Total anthocyanin content was determined spectrophotometrically by a pH differential method using two buffer solutions, potassium chloride 0.025 M at pH 1 and sodium acetate 0.4 M at pH 4.5, to treat the sample extracts following the methodology of Lee [26] with modifications.One part of the extract was mixed with one part of the buffer, and measurements were made at 510 and 700 nm in a spectrophotometer (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of cyanidin-3-glucoside in 100 g of dry matter (mg cyanidin-3glucoside/100 g d.m.).
Total Flavonoid Content (TFC).
Total flavonoid content was determined using a spectrophotometric method, using (+)-catechin as the standard.e assay was carried out as described by Dewanto [27].An aliquot (0.25 mL) of the extract was mixed with distilled water (1.25 mL), and 75 µL of sodium nitrite 5% solution was added.After 6 min, 150 µL of 10% solution of hydrated sixth aluminum chloride was added and the mixture was allowed to stand for 5 more min.Afterwards, 0.5 mL of 1 M sodium hydroxide was incorporated.Distilled water was added until it reaches a volume of 2.5 mL.Spectrophotometric measurements were made at 510 nm (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of catechin/100 g of dry matter (mg of catechin/100 g d.m.).
Sample Compositional Analysis.
Soluble solids (X ss ) and moisture content (X w ) were determined during the osmodehydration processing time, and the effects on water loss and mass, solid gain, and water activity (a w ) were examined.Moisture content was determined according to the method defined by the AOAC (Association of Official Analytical Chemist, 2000).Samples, fresh and treated, were dried at 60 °C in a vacuum oven (Lab Line Instruments Inc.) at 10 kPa.
e solute gain was determined as described by Moreno et al. [23], taking 3 g of sample (fresh or treated) and 25 mL of distilled water and grinding with Ultra-Turrax (Ika-Werke, model T25 basic, USA).
e solid content was measured using a digital refractometer (Leica Mark II, Buffalo, NY, USA).
e X ss and X w contents were determined for both fresh and treated samples, and the compositional changes were calculated through the following equations: where W 0 and W t represent the sample weights at time 0 and t, respectively.W is the weight of the distilled water and A stands for the sample °Brix.e net changes of the soluble solid and moisture contents (ΔM ss t and ΔM w t ) were calculated with the following equations: where M 0 0 and M t 0 stand for the sample weight at time 0 and t, respectively.X ss 0 , X w 0 , X ss t , and X w t are soluble solids (ss) and water (w) mass fractions at time 0 and t, correspondingly.
e water activity (aw) of samples was determined using a dewpoint hygrometer (Aqua Lab Model 4TE, Pullman, USA).
Mechanical Properties.
e firmness of the samples was evaluated with a Texture Analyzer TA-XT (Stable Microsystems, Haslemere, UK), using a slice shear blade.e mechanical parameter was considered as the maximum peak force and reported in N.
Optical Properties.
e color of apple slices, fresh and treated, was measured with a spectrocolorimeter (Konica Minolta CM -500).e CIE L * a * b * coordinates used a D 65 illuminant and 10 °observer as a system reference.e chroma (C * ab), hue (h * ab), and the color differential (ΔE) were calculated according to the following equations: 2.10.Statistical Analysis.e experiment was carried out using a randomized factorial design (4 × 3) considering the osmodehydration treatments and temperatures as the primary sources of variance (Figure 1).e results were subjected to analysis of variance (ANOVA) and least significant difference test (LSD) using Statgraphics Centurion XVI software with p ≤ 0.05.Measurements were done in triplicate, except for the mechanical properties, which were tested six times.
Total Phenolic Content (TPC).
Figure 2 shows the TPC of the apples treated over the osmodehydration time.e PVOD/OH treatment at 30 °C had a higher TPC (Figure 2(a)) over time (180 min), increasing to twice the initial value, followed by PVOD/OH at 50 °C (Figure 2(b)). is behavior is connected with that of the bioactive compounds from the cryoconcentrated pomegranate juice used as a osmodehydration agent; similar results and explanation were reported using chokeberry juice in the OD processing of carrot and zucchini [17].e samples with the lowest phenolic retention were PVOD at 40 °C and OD/OH at 50 °C (Figures 2(b)-2(c)).Furthermore, the TPC decreased for all samples after 30 min, except for PVOD/OH at 30 °C (Figure 2(a)); this decrease was attributed to the treatment combination, temperature, and time.Additionally, after 180 min, most of the treatments retained at least the initial value of TPC, except for PVOD at 30 °C and 40 °C.Such findings agree with those reported by Moreno et al. [23], in which for these osmodehydrated treatments, the initial content of polyphenols was reduced significantly during processing time compared with that of the fresh sample.
Total Anthocyanin Content (TAC). As mentioned by
Mahmoud et al. [28], anthocyanin pigments are responsible for the pomegranate red color and for the color of the osmodehydrated apples.Figure 3 shows the total anthocyanin content during processing time.As observed, PVOD/OH at 50 °C had the highest anthocyanin content, reaching a value near 117 mg of cyanidin-3-glucoside/L, very close to the anthocyanin content of the cryoconcentrated pomegranate juice (Figure 3 4 Journal of Food Quality pulsed vacuum, and electroporation, which enables the osmodehydration agent (cryoconcentrated pomegranate juice) to enter the apple matrix pores [29,30].Reports showed that anthocyanins are less a ected at 40 °C than at 30 °C and 50 °C; this is due to a combination of temperature, time, pH, enzymes, stability, and physicochemical properties of these individual pigments [31,32].In the same study conducted by Kechinski et al. [33], it was demonstrated that anthocyanins were less likely to su er from thermal degradation; the susceptibility to heat might be caused by the di erent forms of the pigments and the interactions of other components in the fruit.For the rest of the treatments, they present a decrease in the TAC after 180 min of treatment.).e avonoids had a drop at 90 min in most of the treatments.e loss of macromolecules such as avonoid during heat treatment might be caused by temperature and time [34].Wet thermal treatments may a ect the cell's structure compromising the integrity and causing the migration of components, leading to damages by leakage or breakdown by several chemical reactions [35].For the 30 °C treatment (Figure 4(a)), the OD presented better retention of avonoids during the processing time. 1 shows the compositional changes between the fresh sample and di erent treatments of osmodehydration after 180 min of processing.According to previous ndings, osmotic dehydration with concentrated juice is more signi cant compared with osmotic dehydration with sucrose [36]. is behavior is in agreement with a recent study of Lech et al. [37], who conclude that the osmodehydration process is a ected by the solution applied and the particular chemical properties of this solution.
Compositional Changes and Water Activity. Table
e combination of PVOD/OH at 40 °C and 50 °C had the highest solute gain, and PVOD at 30 °C had more signi cant water loss and solute gain due to the di usion and convection forces, both of which facilitate the mass transfer process.Application of pulsed vacuum eases the osmotic agent into the matrix pores and facilitates the water loss and has a bene cial e ect on the kinetics, reaching equilibrium easily [23,38].Additionally, the combination of OD/OH encouraged more substantial concentrated levels in the samples than OD.In this case, the mass transfer in PVOD and PVOD/OH in comparison to OD is due to the unsteadiness of particles in the juice.Likewise, inadequate dissemination of the components may occur when gradient pressure in the vegetable tissue provokes the irregular ow of the osmotic agent through the structure and consequently accumulates bioactive components in some areas [39], making the mass transfer harder in OD. 6 Journal of Food Quality e most significant water activity reduction was in PVOD/OH at 50 °C, followed by OD/OH at 50 °C in contrast to the fresh sample.
is difference is attributed to temperature, pH, and electroporation, which promotes the gain from cryoconcentrated pomegranate juice and water loss [40,41].Other treatments also presented a significant difference compared with the fresh one.
On the other hand, it is important to mention the potential use of ultrasound as a technology that in general intensifies the mass exchanges and reduces drying times.In the case of ultrasound application in combination with the apple osmotic dehydration, Ciurzyńska et al. [42] demonstrated that it is an effective technology to accelerate the solid gain when using sucrose as an osmotic solution; however, when using chokeberry juice, it does not change.
Changes in Optical and Mechanical Properties.
Table 2 shows the values obtained for the fresh and treated apple for the optical parameters: L * , a * , b * , the hue angle (h * ab), chrome (C * ab), and change in color (ΔE).Previous studies have demonstrated that L * (luminosity) increases with the osmotic treatment [40].However, samples with a combination of PVOD and PVOD/OH have reduced L * due to the concentration of anthocyanins from the pomegranate juice. is same effect was reported by Petzold et al. [21], in which the treated sample had a lower L * than the fresh one.Temperature also contributed to this effect.Samples treated at 40 °C and 50 °C have the lowest value, and ΔE was significantly higher in the treatments with lower L.
is is due to the anthocyanin pigments being present in the pomegranate cryoconcentrated juice, especially for PVOD/OH at 40 °C and 50 °C (Section 3.2).On the other hand, we observed no significant difference in C * ab of PVOD/OH treatments.For h * ab, there was a significant difference between the fresh sample and the osmotically treated samples.
In general, the sample optical changes after the osmodehydration process could be connected with the plant material used (i.e., cryoconcentrate pomegranate juice), agreeing with the previous results that used chockeberry juice as an osmodehydration solution [17,18,37].
e firmness values acquired from the mechanical test are shown in Table 2. e sample of PVOD at 30 °C had the highest firmness value, which indicates a higher resistance [43].e fresh sample differs significantly from the samples treated at 50 °C.However, the values of treated samples at 40 °C had no difference with the fresh one, as well as with that of OD/OH and PVOD at 30 °C.
Conclusion
e combination of pulsed vacuum and ohmic heating in the osmodehydration of apple slices at 30 °C affected the retention of total phenolic content positively during processing time (180 min).
e highest preservation of anthocyanin pigments was observed with PVOD/OH at 50 °C, followed by the other two temperatures with the same type of treatment.On the other hand, the best results of flavonoids were obtained with OD/OH at 50 °C and 40 °C.Osmotic dehydration combined with pulsed vacuum and ohmic heating at 50 °C and 40 °C intensified the solid gain and water loss due to the diffusion and convection forces which accelerated the mass transfer process.PVOD/OH and OD/OH at 50 °C had the most significant water activity mainly caused by the electroporation and temperature.e changes in color were significant in C * ab (chrome) and L * (lightness), for apple slices treated with PVOD and PVOD/OH, due to the retention of anthocyanins from the pomegranate cryoconcentrated juice.e highest firmness was observed in the sample of PVOD at 30 °C.erefore, our results suggest that the PVOD/OH process at 50 °C for 120 min is the optimal treatment for osmodehydrated apples with pomegranate cryoconcentrated juice to obtain enriched apple slices.Finally, a possible future work is to study the proposed technology using other pomegranate cultivars such as the Mollar cultivars of Elche or Valenciana.
Figure 4
displays the total avonoid content kinetics.Similar to the TPC and TAC, avonoids were best retained by OD/OH at 50 (Figure 4(c)) and 40 °C (Figure 4(b)
Wonderful) and apples (cv.Granny Smith) were acquired in the local market (Chillán, Chile) during the same harvesting season (March 2017) and stored under refrigeration (≈5 °C) until processing.
Table 1 :
Composition parameters of fresh and processed samples (processing time: 180 min), water mass fraction (X w ), soluble solids mass fraction (X ss ), water loss (ΔM w Di erent letters in each column indicate signi cant di erences at p ≤ 0.05, according to a LSD test.OD: osmotic dehydration at atmospheric pressure; PVOD: pulsed vacuum, both with conventional heating; OH: ohmic heating (OD/OH and PVOD/OH), electric eld intensity at 6.66 V/cm (50 V).
Table 2 :
Firmness and color determination in fresh and treated apples with three temperatures (30 °C, 40 °C, and 50 °C). | v3-fos-license |
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} | pes2o/s2orc | Charge-Shift Bonding in Xenon Hydrides: An NBO/NRT Investigation on HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CCH, CN) via H-Xe Blue-Shift Phenomena
Noble-gas bonding represents curiosity. Some xenon hydrides, such as HXeY (Y = Cl, Br, I) and their hydrogen-bonded complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH), have been identified in matrixes by observing H-Xe frequencies or its monomer-to-complex blue shifts. However, the H-Xe bonding in HXeY is not yet completely understood. Previous theoretical studies provide two answers. The first one holds that it is a classical covalent bond, based on a single ionic structure H-Xe+ Y−. The second one holds that it is resonance bonding between H-Xe+ Y− and H− Xe+-Y. This study investigates the H-Xe bonding, via unusual blue-shifted phenomena, combined with some NBO/NRT calculations for chosen hydrogen-bonded complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH). This study provides new insights into the H-Xe bonding in HXeY. The H-Xe bond in HXeY is not a classical covalent bond. It is a charge-shift (CS) bond, a new class of electron-pair bonds, which is proposed by Shaik and Hiberty et al. The unusual blue shift in studied hydrogen-bonded complexes is its H-Xe CS bonding character in IR spectroscopy. It is expected that these studies on the H-Xe bonding and its IR spectroscopic property might assist the chemical community in accepting this new-class electron-pair bond concept.
INTRODUCTION
The chemical bond is the most central concept in chemistry. The model of chemical bonding can help chemists to understand and design matter. Despite the apparent utility of the model of chemical bonding, it is incomplete. Developing bonding models is undoubtedly important to our understanding of novel molecules, such as the molecules of noble gas chemistry (Grandinetti, 2018).
The challenge to noble-gas bonding comes mainly from the inertness of noble-gas atoms. However, significant progress has been made in noble-gas chemistry during the past 20 years. Besides noble-gas hydrides , noble gas-noble metal complexes have been studied for their thermodynamic and kinetic stabilities in theory (Jana et al., 2017(Jana et al., , 2018aPan et al., 2019;Saha et al., 2019). Experimentally, about 30 noble-gas hydrides had been identified by the beginning of 1995 (Pettersson et al., 1995;Räsänen et al., 2000;Lundell et al., 2002;Feldman et al., 2003;Khriachtchev et al., 2003Khriachtchev et al., , 2009Duarte and Khriachtchev, 2017). About 10 hydrogen-bonded complexes between HNgY (Ng = Xe, Kr) and H 2 O/HCl/HBr/HI/HCCH have been well-characterized via infrared (IR) spectroscopy. Interestingly, these complexes show unusual shifts of the H-Xe stretching vibration. For example, the H-Xe stretching mode of the complex HXeI···HCCH exhibits a blue shift of 49 cm −1 , in comparison with the monomer HXeI (Zhu et al., 2015). Some other complexes such as HXeBr···H 2 O and HXeI···H 2 O are characterized by much larger experimental blue shifts of the H-Xe stretching frequency (>100 cm −1 ) (Tsuge et al., 2014). The largest blue shift (300 cm −1 ) has been reported for the H-Kr stretching mode of the complex HKrCl···HCl (Corani et al., 2009). These experimental findings provide theoretical chemistry researchers with excellent opportunities to develop a bonding model for noble-gas hydrides.
In pioneering theoretical work, Last and George put forward one simple ionic structure model H-Ng + Y − (Last and George, 1988), where H-Ng + belongs to a classical covalent bond, to be exact, an electron-sharing bond, while the interaction between H-Ng + and Y − comes from electrostatic attraction. Energy decomposition analyses (EDAs) carried out by Frenking for HArF provided a consistent bonding picture (Lein et al., 2004). Besides this ionic structure model, empirical resonance bonding models were proposed by Räsänen's group (Pettersson et al., 1999) and Alabugin's group (Alabugin et al., 2004). Recently, our group has carried out the resonance bonding analyses for noble-gas hydrides (Zhang et al., 2016) by using Natural Bond Orbital (NBO) and Natural Resonance Theory (NRT) methods (Glendening and Weinhold, 1998a,b;Glendening et al., 1998Glendening et al., , 2001Glendening et al., , 2013aWeinhold and Klein, 2014;Weinhold et al., 2016). We found that each molecule HNgY could be best described as three structures, H-Xe + Y − , H − Xe + -Y, and H ∧ Y, where the first two resonance structures mix to form hyperbonding of H-Xe + Y − ↔ H − Xe + -Y, as proposed by Weinhold and Landis (2005b). Such bonding provides a picture of resonance covalency for noble-gas hydrides Klein, 2012, 2014;Landis and Weinhold, 2013;Zhang et al., 2015Zhang et al., , 2017aJiao and Weinhold, 2019).
It is important to note that they are two variants of covalent bonding, which are related to two kinds of electron-pair bonds. They are our familiar electron-sharing bonds and dative bonds. The difference between these two types of bonds is due to the origin of the bonding electrons. In electron-sharing bonds, each fragment provides one electron. In dative bonds, both electrons come from one fragment, which donates two electrons to the vacant orbital of the other. Also note that besides our familiar electron-sharing bonds and dative bonds there is one new kind of electron-pair bonds, charge-shift (CS) bonds. The concept of charge-shift bonds was first proposed by Shaik and Hiberty et al. in 1992, to describe a new-class electron-pair bonds, such as F 2 (Shaik et al., 1992). Ten years later, they further presented experimental manifestations of CS bonding (Shaik et al., 2005(Shaik et al., , 2009. They have applied the concept of CS bonds to the understanding of bonding and stabilities of several hypervalent molecules (Braïda and Hiberty, 2013;Braïda et al., 2014), such as XeF 2 . In 2018, Grandinetti pointed out that a stabilization like HNgY is, generally, known as CS bonding (Grandinetti, 2018). Indeed, the decades following their original work saw more CS bonding molecules. Very recently, they published their latest review on CS bonds (Shaik et al., 2020).
Given that XeF 2 and HXeY are similar in geometrical and electronic structures, two obvious questions arise: (1) Is the H-Xe bond in HXeY a charge-shift bond? (2) What is the H-Xe CS bonding character in IR spectroscopy? This study explores these two questions through blue-shifted phenomena, with the help of NBO/NRT analyses. We choose HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH) as study systems. Some of them are identified in the matrix experiment. We analyze the relationship between the H-Xe bonding and H-Xe blue shifts. We aim to understand the H-Xe bonding in HXeY and to extend CS bonding concepts to noble-gas hydrides.
This paper will be organized as follows. Firstly, we summarize the computational details and discuss the geometrical structural details and H-Xe IR spectroscopic properties for our studied monomers and complexes. Secondly, we analyze the resonance bonding of HXeY, especially for the bonding between H and Xe. Thirdly, we analyze the H-Xe bond order. And we determine that the H-Xe bond order must include the contributions of two resonance structures. Fourthly, we confirm that the H-Xe in HXeY is a charge-shift bond, and further analyze its CS bonding character. Finally, we present the concluding summary, with emphasis on the H-Xe CS bonding and its bonding character.
COMPUTATIONAL DETAILS
The geometry optimization and vibrational frequency calculations were carried out with the Gaussian 09 program at the level of the second order MØller-Plesset perturbation theory (MP2) (Head-Gordon et al., 1988;Frisch et al., 2009). The def2-TZVPPD basis set was used. This basis set, taken from the EMSL basis set library (Feller, 1996;Schuchardt et al., 2007), is the triple-zeta-valence basis set augmented with two sets of polarization and diffuse basis functions. No imaginary frequencies were found in any case, which confirms that our obtained structures are true local minima on the potential energy surface. The NBO and NRT were employed to analyze the bonding of our studied systems with the NBOPro 6.0 program (Glendening and Weinhold, 1998a,b;Weinhold, 2012Weinhold, , 2013. Directed NBO analyses could provide the second order perturbation energy of a donor-acceptor interaction in the best Lewis structure. For any other resonance structure, we use $CHOOSE keylist to calculate the second order perturbation energy of a donor-acceptor interaction. It is important to attach the $NRTSTR keylist in NRT analyses, for it insures a consistent set of reference structures for NRT comparisons of studied complexes. The NBO-based natural resonance theory complements and extends NBO analyses to other resonance structures. By using $NRTSTR keylist to specify key structures as reference structures, we can obtain accurate weightings (ω I , ω II , ω III ...) of resonance structures, and NRT bond orders (b AB ) that express the strength of resonance-weighted chemical bonds between any atom pair. More importantly, the NBO/NRT-based models provide a framework for analyzing chemical bonding in terms of familiar concepts, such as Lewis structures, resonance, and donor-acceptor interactions. Herein, we use NBO/NRT methods to analyze H-Xe bonding. Besides, the NBOview 2.0 module was acquired to obtain the orbital overlap graphics.
Geometry and Blue Shifts
In general, four structures need to be considered for each of the complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH). Take HXeY···HCl, as shown in Figure 1, as one representative example. In the first two structures (A and B), the HCl moiety is closed to the halogen atom of the monomer HXeY to form a bent structure. Structure A is stabilized with the Cl-H···Y hydrogen bond, whereas Structure B is dependent on the Xe-Y···Cl halogen bond. Structure C, whose halogen atom in the moiety is collinear with the monomer HXeY, is formed by the Cl atom interacting with the H atom. Structure D is stabilized with the dihydrogen Cl-H···H-Xe bond with all atoms in line. Experimentally, it has been observed that several infrared absorption bands originate from the H-Xe stretching mode for our chosen complexes. With the aid of quantum chemical calculations, they are assigned to Structure A (Lignell et al., 2008;Tsuge et al., 2013Tsuge et al., , 2014Zhu et al., 2015). Therefore, the following analyses are restricted to Structure A.
As shown in Structure A in Figure 1, HXeY maintains its original linear structure for all the HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH) optimized geometry. At the MP2/def2-TZVPPD level, the calculated H-Xe and Xe-Y bond lengths (R H−Xe , R Xe−Y ) and vibrational frequency shifts of H-Xe stretching mode in complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH) as well as in monomers HXeY are collected in Table S1 and compared with the available experimental data. Note that Räsänen's group has computed R H−Xe and R Xe−Y as well as a variety of H-Xe frequency blue shifts for some of the complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH) at the CCSD(T) level. The difference in calculated H-Xe bond lengths at both MP2 and CCSD(T) levels is <0.060 Å, whereas for Xe-Y bond lengths the largest difference is 0.048 Å in the monomer HXeI. These comparisons show that the MP2/def2-TZVPPD is an appropriate model chemistry for this study. Thus, the following discussion will be based on the calculated data at the MP2/def2-TZVPPD level.
Data in Table S1 show that the complexation has nonnegligible influences on monomers HXeY. On the one hand, the bond lengths of H-Xe in complexes become shorter as compared with the value in the corresponding HXeY monomer. Taking HXeCl species as one example, H-Xe bond lengths R H−Xe in complexes are 1.646, 1.645, 1.644, 1.646, 1.648, and 1.655 Å for HX = H 2 O/HCl/HBr/HI/HCN/HCCH, respectively, while the H-Xe bond length of the monomer HXeCl is 1.666 Å. This indicates that the interaction between H and Xe atoms in monomers HXeY is weaker than that in corresponding complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH). On the contrary, the bond lengths of Xe-Y within complexes are slightly larger than that in the corresponding monomer HXeY, which means that the interaction between Xe and Y atoms is stronger in monomers than in corresponding complexes. It is worthwhile noting that the calculated R H−Xe ranges from 1.644 to 1.708 Å, slightly larger than the covalent limits R cov [r cov (H)+r cov (Xe)] for 1.63 Å and significantly shorter than VdW limits R vdw [r vdw (H)+r vdw (Xe)] for 3.86 Å (Pyykkö, 2015;Rahm et al., 2016). This indicates strong covalency of the H-Xe bond.
On the other hand, the complexation leads to experimentally observable blue shifts of the H-Xe stretching frequency, which seems to be normal phenomena according to Khriachtchev's group studies. The calculated and experimental vibrational frequency shifts of the H-Xe stretching mode are also collected in Table S1. Taking hydrogen-bonded complexes HXeCl···H 2 O, HXeBr···H 2 O and HXeI···H 2 O as one example group, calculations at the MP2 level predict blue shifts of 101, 116, and 139 cm −1 , respectively. The experimental data are 82, 101, and 138 cm −1 , respectively. Obviously, the calculated complexation-induced spectroscopic shift of the H-Xe stretching mode at the MP2 level is in qualitative agreement with the experimental values. These preliminary structural and blue-shifted analyses provide the backdrop for the following exploration of H-Xe bonding for Xe hydrides.
H-Xe Resonance Bonding in HXeY
Earlier studies have found that the NBO/NRT method is a helpful tool to explore the resonance bonding because NBO analyses can provide the best Natural Lewis Structure (NLS), identify donor-acceptor orbital interactions, and estimate the second-order perturbation energy [E (2) ] of each donor-acceptor orbital interaction (Glendening et al., 2013a,b;Weinhold, 2013;Weinhold et al., 2016).
Our studies begin with NBO/NRT analyses for HXeY species. The results show that each of the studied HXeY could be better described as a hybrid of the three structures (I,II,and III) as shown in Figure 2, where the H-Xe + :Y − (I) is the best NLS. Table S2 lists three types of donor-acceptor interactions and the value of the second-order perturbation energies [E (2) ] of the studied HXeY. For the best NLS H-Xe + :Y − (I), the donoracceptor interaction (n Y → σ * H−Xe ) takes place between the lone pair orbital of Y (n Y ) and the antibonding orbital of H-Xe moiety (σ * H−Xe ). Pay particular attention to such a delocalization interaction. It represents resonance mixing between H-Xe + :Y − (I) and H: − Xe + -Y (II), where the latter corresponds to the lone pair of H atom (n H ) delocalizing to the antibonding orbital of Xe-Y moiety (σ * Xe−Y ). As proposed by Weinhold et al., these two structures make up resonance bonding. Additionally, there is a non-negligible long-bonding structure H ∧ Y (III), shown in the final entry in Figure 2. It arises from the delocalization of Xe atom lone pair (n Xe ) to the antibonding orbital of H-Y moiety (σ * H−Y ). Special attention is paid to the unusual values of E (2) for n Xe → σ * H−Y and n H → σ * Xe−Y interactions. Such results show that they are no longer suitable for the description of low-order perturbative NLS limit. Even in the unavailable value of some second-order perturbation energies, the importance of these three donor-acceptor interactions can be exhibited by the orbital overlap contour diagrams or 3-D surface views. Figure 3 presents one illustrative example. These results are consistent with our previous studies for HXeY noble-gas hydrides (Y = Cl, Br, I) at the B3LYP level of theory (Zhang et al., 2016).
Once again, the importance of resonance bonding H-Xe + Y − ↔ H − Xe + -Y is emphasized. On the one hand, resonance bonding is an essential feature of H-Xe bonding in HXeY. On the other hand, we note that earlier studies on the bonding of noble-gas hydrides have already analyzed the leading resonance structure H-Xe + Y − (Pérez-Peralta et al., 2009;Juarez et al., 2011). In the next section, we will carry out detailed analyses on H − Xe + -Y, as well as H-Xe + Y − .
H-Xe Bond in HXeY Is Not a Classical Covalent Bond
HXeY is a particularly simple and interesting molecule. Its H-Xe stretching modes provide experimental probes to learn more about the bonding of noble-gas hydrides. It was reported that the H-Xe stretching vibration frequencies of HXeY species are blue shifts upon complexation. In this section, we will explore the H-Xe bonding via this complexation effect, combining with quantitative NRT analyses on chosen hydrogen-bonded complexes. Table 1 displays the weighting of three resonance structures upon complexation for Xe cases. Obviously, the moiety H 2 O/HCl/HBr/HI/HCN/HCCH complexing with HXeY has a significant influence on the weighting of three resonance structures for HXeY. To be specific, for the long-bonding structure Zhang et al., 2017bZhang et al., , 2018, its weighting always decreases upon complexation. This indicates that the complexation of the moiety H 2 O/HCl/HBr/HI/HCN/HCCH studied here is not beneficial to the stability of long-bonding in HXeY. For two other resonance structures, the ω I decreases while ω II increases relative to the corresponding monomer in studied hydrogen-bonded complexes except for the complex HXeCl···HCCH. For example, the weightings of H-Xe + :I − and H: − Xe + -I in the hydrogenbonded complex HXeI···HI are 62.8 and 19.2%, compared with 66.4 and 9.5% in the monomer HXeI. In contrast, the complex HXeCl···HCCH shows a different trend. As it is easily seen, the weightings of H-Xe + :Cl − and H: − Xe + -Cl structures in the monomer HXeCl are 74.3 and 10.9% respectively; in the complex HXeCl···HCCH they are 76.1 and 9.7%, respectively. In short, for all of our studied complexes, the HXeY complexation with small molecules always leads to a decrease of the weighting about the long-bonding structure. For weightings of these two other resonance structures, one structure always shows a decreasing trend while the other exhibits an increasing trend for all of the studied complexes. In most of the Xe cases studied here, the complexation results in an increasing weighting of the resonance structure H-Xe + :Cl − , with a decreasing weighting of the resonance structure H: − Xe + -Cl. In HXeCl···HCCH the situation is different. An opposite trend is seen for the complex HXeCl···HCCH. From preliminary NBO analyses on our studied complexes, peculiarities in HCCH complexes shown here, in Tables 1-3, and Figure 4, may be due to n Xe(d) → π * C−C donor-acceptor interaction. Table 2 lists natural bond orders of the H-Xe and H − Y bonds in the monomer HXeY and its complex. It is worthwhile noting that the weighting and the corresponding bond order are equivalent for our studied cases in NBO/NRT framework if the former is expressed as a fraction rather than a percentage. For instance, the weightings of the H-Xe + :Cl − in the monomer HXeCl and in the complex HXeCl···HCl are 74.3% and 71.8%, with corresponding bond order b H−Xe 0.743 and 0.718, respectively. Generally, the larger the bond order is, the stronger the bond is. The data obtained from current NBO programs show the decrease of H-Xe bond orders, reflecting the weakening H-Xe bonds upon complexation. Obviously, these calculated results do not reflect the experimental fact: the strengthened H-Xe bond. This disagreement confirms that the H-Xe bond in HXeY is not a classical covalent bond.
This conclusion deserves some illustration. According to the natural bond orders' definition (Weinhold and Landis, 2005a), the H-Xe bond in HXeY is regarded as a classical covalent bond. The resonance structure H: − Xe + -Y does not contribute to the H-Xe bond order at all. The H-Xe bond order is only contributed to by the resonance structure H-Xe + :Y − . Thus, the H-Xe bond order in Table 2 reflects merely the electron-sharing contribution
H-Xe Bond in HXeY Is a CS Bond
The above studies on the H-Xe bond in HXeY confirm that it is not a classical covalent bond. Then, is it a CS bond? To address this question, we first need to solve the problem of the H-Xe bond order. Let us return to the original NBO/NRT theory. In the framework of NRT theory (Weinhold and Landis, 2005a), the H-Xe total bond strength in HXeY could be written in resonanceaveraged form D(H-Xe) = ω I x D I + ω II x D II , where "ω I " and "ω II " respectively correspond to the weighting of H-Xe + Y − and H: − Xe + -Y; "D I " and "D II " represent the H-Xe bond strength in H-Xe + Y − and in H: − Xe + -Y respectively. As we know, a bond order is roughly proportional to the bond strength or the bond length. If introducing two arbitrary constants is presumed to be expressed in terms of the same factor k, D I, and D II can be written in the form, D I = k x b I and D II = k x b II . Importantly, we obtain that b(H-Xe) = ω I x b I + ω II x b II , where b I represents the H-Xe bond order in resonance structure I, as we know, b I = 1; b II refers to the H-Xe bond order in resonance structure II. Therefore, a calculation that includes two resonance structures is needed to deal with the H-Xe bond order. Note that the procedure employed to calculate the H-Xe CS bond order can be found in Supplementary Material "Explanation of the Procedure Employed to Calculate the BO of the H-Xe Bond." To obtain b II , we have to carry out some analyses on H: − Xe + -Y. For this structure, where does the H-Xe bonding originate from? Here we emphasize that the H: − Xe + -Y structure is a natural Lewis structure, in the NBO/NRT language. Consideration of the antibond of the Xe + -Y could lead to extension of the elementary Lewis structure concept to include hyperconjugative delocalization corrections in simple NBO perturbative estimates (Weinhold, 2012). Such hyperconjugative delocalization forms one starting point for our understanding of bonding about the H: − Xe + -Y structure. As shown in Figure 3, one donor-acceptor interaction n H → σ * Xe−Y exists in the H: − Xe + -Y structure. On the basis of this result, we propose that the H-Xe bonding about the structure H: − Xe + -Y is attributed to this donor-acceptor interaction. In our familiar language, it is dative bonding due to hyperconjunctive interaction.
However, the question was still left open: How do we estimate the degree of this H-Xe dative covalency? For this question, our research ideas are from the natural bond orders' definition proposed by Weinhold and Landis (2005b). If such a single dative bond order b II is defined as 1, the NRT bond order of the dative structure is equal to the corresponding fractional weighting. Taking HXeCl as one illustrative example, the dative weightings of monomer HXeCl and hydrogen-bonded complex HXeCl···HCl are 10.9 and 14.6%, respectively. This simple method lets us respectively estimate the dative bond orders: 0.109 and 0.146. The above discussion is an effort to rationalize b II . We now begin a discussion with a sum of ω I x b I, and ω II x b II . For convenience, we use b H−Xe (electron-sharing) = ω I x b I, , b H−Xe (dative) = ω II x b II . As shown in NRT theory, it is a sum of b H−Xe (dative) and b H−Xe (electron-sharing) that can reflect the H-Xe total strength in HXeY. Figure 4 shows correlation plots between the H-Xe bond order [b H−Xe (dative) + b H−Xe (electron-sharing) and the H-Xe bond length R H−Xe for our studied Xe species. One additional example is also shown in Figure 4 for Kr analogs. Good correlation shown in Figure 4 for our studied species except Y = I, provides evidence to support our estimated method. Not enough good correlation in Y = I case may be due to a larger coupling effect between n Y → o Obviously, the HXeCl complexing with the molecule HCl leads to an increase of the H-Xe bond order. The same is true for other complexes studied here. Table 3 lists the total bond order of the H-Xe bond in HXeY and in its hydrogen-bonded complexes. Note that it includes dative contribution and electron-sharing contribution of H-Xe bonding. As shown in Table 3, the HXeY complexing with the small molecule leads to an increase of the H-Xe bond order. In other words, it is an enhancement of the H-Xe bond. Such a result is consistent with experimental observations about our studied cases.
It becomes clear that the bonding between H and Xe in HXeY must meet two conditions. First, the resonance bonding between H − Xe + -Y and H-Xe + Y − is a necessary condition. Second, for the H-Xe bond order, including contribution of these two resonance structures is essential. These two conditions are, in effect, consistent with emphases in original CS bonding concept paper (Shaik et al., 1992). The mixed covalent-ionic description, such as F· ·F↔F − F + , is an essential feature of CS bonding, wherein most, if not the entire, bond energy is provided by the covalent-ionic resonance energy. And both the covalent and ionic structures must be treated explicitly and on an equal footing. Thus, we conclude that the H-Xe bond in HXeY is a chargeshift bond.
It should be noted, however, that neither the H-Xe bond length nor its bond strength can be reliable probes for addressing the question on whether the bond between H and Xe in HXeY should be classified as a CS bond or a classical covalent bond.
H-Xe CS Bonding Character
Now that the H-Xe bond in HXeY is a charge-shift bond, a new and unique form bonding, one question which arises is whether the unusual H-Xe blue shift is its CS bonding character in IR spectroscopy.
Deep analyses on the data in Table 3 show that the complexation leads to an increase of b H−Xe (dative) while b H−Xe (electron-sharing) decreases, for most Xe cases. The competition of these two opposite factors results in the strengthened H-Xe bonds, corresponding to the blue shifts. Here, we want to point out that unlike other Xe complexes, the HXeCl···HCCH complex shows a difference in the dominant factor. It is the electron-sharing factor that dominates the H-Xe frequent shifts. The overall effect of two opposing factors is still an enhancement of the H-Xe bond and a blue shift for the H-Xe stretching frequency. These analyses reflect that the monomer-to-complex blue shifts of H-Xe stretching modes for HXeY species should be attributed to the balance of dative and electron-sharing covalency in H-Xe bonds, corresponding to H − Xe + -Y and H-Xe + Y − resonance structures. In brief, the H-Xe frequent shifts in HXeY···H 2 O/HCl/HBr/HI/HCCH/HCN hydrogen-bonded complexes is controlled by a balance of two factors acting in opposite directions.
All in all, blue shifts of H-Xe vibrational frequencies are controlled by a balance of two opposing factors for dative and electron-sharing covalency. The blue shift in our studied complexes can be seen as a normal spectroscopic phenomenon. It is natural for us to conclude that the H-Xe blue shift is the H-Xe CS bonding character in IR spectroscopy.
SUMMARY AND CONCLUSIONS
The H-Xe bonding in HXeY has been a debated question in the chemical community. The usual answer is that it is classically covalent in character, or to be exact, it is electron-sharing. The unusual blue shifts of complexes HXeY···HX (Y = Cl, Br, I; X = OH, Cl, Br, I, CN, CCH) definitely reflect the unusual features of H-Xe bonding in noble gas hydrides. Via observed blue-shifted phenomena, we have computationally investigated the H-Xe bonding in HXeY from an NBO/NRT perspective.
We establish that the resonance bonding between H-Xe + Y − and H − Xe + -Y is an essential feature of the H-Xe bonding, and that the H-Xe bonding in these two resonance structures must be considered explicitly. Specifically, its bonding includes the n H → σ * Xe−Y donor-acceptor interaction in H − Xe + -Y, as well as the electron-sharing interaction in H-Xe + Y − ; the H-Xe bond order is contributed to by these two resonance structures. We confirm that the H-Xe bond in HXeY is not a classical covalent bond but a charge-shift bond, and that H-Xe blue shifts is a normal spectroscopic phenomenon.
Our conclusions are (1) the H-Xe bond in HXeY is a chargeshift bond. (2) The H-Xe blue shift in its hydrogen-complexes is its CS bonding character in IR spectroscopy.
The first conclusion is consistent with ab initio VB methods' insight into the F-Xe CS bonding in XeF 2 (Braïda and Hiberty, 2013). But we note a little difference in the understanding of CS bonding mechanism. We stress the point that the H-Xe CS bonding is due to the resonance between H-Xe + :I − and H: − Xe + -I, based on the natural Lewis structures' concept, whereas Shaik et al. (1992) think that CS bonding is due to strong mixing between the covalent structure and the ionic structure, based on Pauling-type Lewis structures' concepts.
Finally, we want to point out that H-Xe CS bonding is significantly different from some two-structure resonance bonding, such as hydrogen-bonding, although there is a formal resemblance in their resonance description. We have noticed that the bond order of hydrogen-bonding in literature (Jiao and Weinhold, 2019) is only considered a contribution from one resonance structure; the other does not contribute to bond orders of hydrogen-bonding at all. Why is CS bonding not important in hydrogen-bonding? More studies are on the way to answer such a question and to generalize CS bonding models. We believe that more surprise will be gained via NBO/NRT methods, in particular, new-type NBO-based NRT methods on analyses for larger CS bonding species (Glendening et al., 2019).
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
AUTHOR CONTRIBUTIONS
GZ, CS, and DC made contributions to the design of the study. YS, XZ, JS and LF performed the calculations. GZ and YS wrote the draft of the manuscript. All the authors contributed to the manuscript revision, read and approved the submitted version.
ACKNOWLEDGMENTS
We thank Professor Frank Weinhold of the University of Wisconsin-Madison for his continuous help. Especially his help and important suggestions during the last few months, which helped us make great improvements to our manuscript. | v3-fos-license |
2019-04-08T13:11:01.115Z | 2017-01-01T00:00:00.000 | 99072351 | {
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} | pes2o/s2orc | Numerical modelling of combined heat and mass transfer in film absorption
Heat and mass transfer during absorption on a film of lithium bromide water solution flowing by a cooled wall in the steam atmosphere is numerically investigated in this paper. The self-similar solutions are using as the initial conditions for solving the problem beyond the entrance region. The key criteria characterizing heat and mass transfer in the film absorption with uniform velocity profile and with a constant thickness have been determined. 1 Problem statement Absorption of gases or vapors by liquid takes place in various energy devices [1]. The first theoretical description of combined heat and mass transfer processes at vapor absorption in a laminar falling liquid film was made in [2-4]. The overview of works on film flows with absorption is presented in [5]. Heat and mass transfer in the processes of absorption, desorption, evaporation and condensation in the initial part of semi-infinite axisymmetric film falling under the pressure is studied in [6]. The absorption and desorption processes under the conditions, corresponding to the operating modes of thermal transformers are described in [7]. The analytical solution to the problem of conjugate heat and mass transfer in a laminar falling liquid film with a linear velocity profile is presented in [8]. Data of numerical simulation of absorption are compared with experimental data in [9]. Let us consider the two-dimensional stationary flow of a laminar liquid film (water solution of lithium bromide with constant thickness h) over a plate, inclined at angle to the horizon, as in [4]. The liquid surface contacts with stationary steam. Absorption is considered in the framework of usual assumptions [1]. The vapor phase is singlecomponent, and vapor pressure does not change during absorption. Thermal-physical properties of solution are considered constant. We believe that absorption heat is released at the interface and it is spent only for solution heating. For small ranges of concentrations and temperatures, according to [1], we will approximate the dependence of absorbed substance concentration on temperature by linear function 1 2 i i C k k T , where coefficients k1 and k2 are determined by the pressure. The values of , i i C x T x are interrelated, unknown and should be determined. * Corresponding author: [email protected] DOI: 10.1051/ , (2017) 79201061 92 matecconf/201 MATEC Web of Conferences 01061 Thermophysical Basis of Energy Technologies 2016 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Let us introduce Cartesian coordinate system with axis Оx along the flow and axis Оy normal to the plate. The velocity profile in the film takes a form 2 2 3 2 / 2 u u y h y h , where u is an average velocity of liquid motion in the film. The process of heat and mass transfer at film absorption is described by the equation of heat conductivity and diffusion: 2 2 u T x a T y , (1) 2 2 u C x D C y , (2) with the following boundary conditions. At the inlet for 0 x : 0 0 , T T C C . At the interface for y h , similarly to [1,4] there is the equilibrium solution-vapor system: 1 2 i i C k k T , 0 ( (1 )) a T y r D C C y . This equilibrium condition connects the equilibrium temperature with concentration. On the wall at 0 y , we set: 0 0 , 0 y w y T y q C y , where w q is heat flux on the wall. The heat transfer coefficient w is describing the heat transfer from the liquid film to a wall and defined from the relation ( ) w w f w q T T , where w T is a dimensional wall temperature, 1 1 0 0 f T uTdy udy is dimensional bulk temperature of solution. Let us use dimensionless variables ( ) x Peh , y h , 2 3 2 2 v u u , 0 0 ( ) ( ) e T T T T , 0 0 ( ) ( ) e C C C C . Here 1 2 0 e C k k T is equilibrium concentration, corresponding to initial temperature of solution 0 T , e T is equilibrium temperature, corresponding to initial concentration 0 C ( 0 1 2 e C k k T ), similarly to [1]. Pe uh a is Pecklet number. The system of equations (1) – (2), reduced to the dimensionless form, is written as: 2 2 v , (3) 2 2 Le v , (4) here Le / D a . The boundary conditions at the inlet for 0 : 0 , 0 . At the interface for 1 , similarly to [1]: , i i , DOI: 10.1051/ , (2017) 79201061 92 matecconf/201 MATEC Web of Conferences 01061 Thermophysical Basis of Energy Technologies 2016
Problem statement
Absorption of gases or vapors by liquid takes place in various energy devices [1]. The first theoretical description of combined heat and mass transfer processes at vapor absorption in a laminar falling liquid film was made in [2][3][4]. The overview of works on film flows with absorption is presented in [5]. Heat and mass transfer in the processes of absorption, desorption, evaporation and condensation in the initial part of semi-infinite axisymmetric film falling under the pressure is studied in [6]. The absorption and desorption processes under the conditions, corresponding to the operating modes of thermal transformers are described in [7]. The analytical solution to the problem of conjugate heat and mass transfer in a laminar falling liquid film with a linear velocity profile is presented in [8]. Data of numerical simulation of absorption are compared with experimental data in [9].
Let us consider the two-dimensional stationary flow of a laminar liquid film (water solution of lithium bromide with constant thickness h) over a plate, inclined at angle T to the horizon, as in [4]. The liquid surface contacts with stationary steam. Absorption is considered in the framework of usual assumptions [1]. The vapor phase is singlecomponent, and vapor pressure does not change during absorption. Thermal-physical properties of solution are considered constant. We believe that absorption heat is released at the interface and it is spent only for solution heating. For small ranges of concentrations and temperatures, according to [1], we will approximate the dependence of absorbed substance concentration on temperature by linear function where u is an average velocity of liquid motion in the film. The process of heat and mass transfer at film absorption is described by the equation of heat conductivity and diffusion: with the following boundary conditions. At the inlet for 0 At the interface for y h , similarly to [1,4] there is the equilibrium solution-vapor system: This equilibrium condition connects the equilibrium temperature with concentration. On the wall at 0 y , we set: 2 e C k k T ), similarly to [1]. Pe uh a is Pecklet number. The system of equations (1) -(2), reduced to the dimensionless form, is written as: , 0 where w q is dimensionless heat flux.
Numerical calculations
Numerical calculations were performed by the finite-difference method. The relationship of temperature and concentration along the interface was calculated via conjugated calculation of the modified coefficients, taking into account the conditions at the interface. Calculations were carried out at Ka = 10, Le = 0.01. Calculated distributions of dimensionless temperature and concentration along the film surface are shown in Fig. 1 for different heat fluxes on the wall w q . Dimensionless bulk temperature of solution is shown in Fig.2. Dependences of Nusselt number on longitudinal coordinate are presented in Fig. 3 for different heat fluxes along the initial region of the flow.
Conclusions
The problem of conjugated heat and mass transfer at film absorption on a cooled wall with a given constant heat flux is studied numerically. The analytical solution is used as an inlet condition for further numerical solution by the finite-difference method. The key dimensionless criteria characterizing heat and mass transfer at absorption on the falling liquid film have been defined.
This work was carried out at the Kutateladze Institute of Thermophysics SB RAS and financially supported by the Russian Science Foundation (project number 15-19-10025). | v3-fos-license |
2020-12-20T06:18:04.882Z | 2020-12-18T00:00:00.000 | 229324291 | {
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} | pes2o/s2orc | Comparative efficacy of selenate and selenium nanoparticles for improving growth, productivity, fruit quality, and postharvest longevity through modifying nutrition, metabolism, and gene expression in tomato; potential benefits and risk assessment
This study attempted to address molecular, developmental, and physiological responses of tomato plants to foliar applications of selenium nanoparticles (nSe) at 0, 3, and 10 mgl-1 or corresponding doses of sodium selenate (BSe). The BSe/nSe treatment at 3 mgl-1 increased shoot and root biomass, while at 10 mgl-1 moderately reduced biomass accumulation. Foliar application of BSe/nSe, especially the latter, at the lower dose enhanced fruit production, and postharvest longevity, while at the higher dose induced moderate toxicity and restricted fruit production. In leaves, the BSe/nSe treatments transcriptionally upregulated miR172 (mean = 3.5-folds). The Se treatments stimulated the expression of the bZIP transcription factor (mean = 9.7-folds). Carotene isomerase (CRTISO) gene was transcriptionally induced in both leaves and fruits of the nSe-treated seedlings by an average of 5.5 folds. Both BSe or nSe at the higher concentration increased proline concentrations, H2O2 accumulation, and lipid peroxidation levels, suggesting oxidative stress and impaired membrane integrity. Both BSe or nSe treatments also led to the induction of enzymatic antioxidants (catalase and peroxidase), an increase in concentrations of ascorbate, non-protein thiols, and soluble phenols, as well as a rise in the activity of phenylalanine ammonia-lyase enzyme. Supplementation at 3 mgl-1 improved the concentration of mineral nutrients (Mg, Fe, and Zn) in fruits. The bioaccumulated Se contents in the nSe-treated plants were much higher than the corresponding concentration of selenate, implying a higher efficacy of the nanoform towards biofortification programs. Se at 10 mgl-1, especially in selenate form, reduced both size and density of pollen grains, indicating its potential toxicity at the higher doses. This study provides novel molecular and physiological insights into the nSe efficacy for improving plant productivity, fruit quality, and fruit post-harvest longevity.
Introduction Nowadays, diverse attempts have been made to improve productivity, stress tolerance, and disease management in crops as well as to produce biofortified seeds or fruits containing minerals essential for humans. Zinc (Zn), iron (Fe), and selenium (Se) are the most important mineral nutrients that are often considered in biofortification programs. In this regard, the daily nutritional requirement of Se is estimated to be 55 micrograms. Taking nano-fertilizers and nanopesticides into account, nanotechnology has provided a great opportunity to improve crop productivity and crop protection and has solved some of the challenges that we face today in agriculture. It has been highlighted in recent studies that bioavailability and function of Se in plant growth and metabolism were significantly more efficient in form of nanoparticles (nSe) compared to other natural Se forms such as selenate and selenite [1][2][3][4][5][6]. On the other hand, there is evidence that nSe use could potentially compromise plant growth and development [3,5,6]. However, the potential benefit or phytotoxicity of nSe is still controversial. Therefore, more detailed investigations are necessary to fill the knowledge gaps.
In plants, microRNAs (miRNAs) are transcriptionally modulated by endogenous and surrounding environmental cues [7]. Most miRNAs are efficiently involved in adjusting the perception/signaling of phytohormones, as well as regulating plant growth and development through transcriptional and posttranscriptional regulations of a multitude of target genes [8]. Some miR-NAs play a significant role in adapting plants to different types of environmental stresses [7,8]. Moreover, the flowering phase and fruit development are rigorously controlled through orchestrated molecular strategies in which miRNAs play critical roles at transcriptional and post-transcriptional levels [7]. In this regard, microRNA172 (miR172) is one of the most important miRNAs playing critical roles in controlling developmental programs, particularly at the flowering phase, as well as in conferring stress tolerance [8,9]. To the best of our best knowledge, there has been no study investigating the role of miRNAs during plant responses to nanoparticles. In this study, we attempted to monitor transcriptional rates of miR172 following the nSe applications.
Carotenoid pigments are natural organic substances derived from an isoprene precursor. These pigments are involved in plant protection against oxidative stress and the photoinhibition process. Furthermore, these antioxidant metabolites provide numerous advantages to the health maintenance of humans owing to their great antioxidant characteristics. Therefore, investigating the effects of environmental cues, chemicals, and fertilizers on the concentrations of carotenoids is of great importance. In carotenoid production, the carotene isomerase (CRTISO) is one of the most important genes involved in the biosynthesis of carotenoids through mediating the conversion of z carotene to trans-lycopene [10][11][12]. CRTISO is widely considered in plant biotechnology programs to improve fruit quality and plant resistance to stresses [10][11][12]. Hence, the concentration of carotenoids in tomato fruit is considered an important aspect of fruit quality. Therefore, we aimed to highlight the potential alterations in fruit production, transcription of the CRTISO gene, and fruit longevity following foliar applications of nSe or selenate as a bulk (BSe).
The basic leucine zipper (bZIP) transcription factors are involved in a plethora of plant biological processes and responses, including signal transduction [6,13], tissue differentiation [13], hormone signaling [14], nutrition [15], and stress tolerance [6,16]. For this reason, evaluating the potential nSe-associated alteration in the bZIP transcription factor was also one of the aims of this study.
Within this framework, this study was carried out to address the possible responses of tomato seedlings to foliar application of BSe or nSe at different concentrations. In this manuscript, for the first time, we aimed to address transcriptional responses of miR172, CRTISO, and bZIP genes to foliar application of bulk Se (selenate) or nano counterpart (nSe) at the same concentration. Moreover, this study provides anatomical evidence highlighting the potential risk of Se or nSe on the development of pollen grains for the first time. Taking knowledge gaps into account, we attempt to present comparative comprehensive data on the Se-or nSe-mediated changes in plant growth, physiology, metabolism, the transcriptional program of genes, crop productivity, fruit quality, and fruit postharvest longevity to gain new insights into the benefits or the risk associated with Se function in agriculture. The main aims of this experiment are documenting the nSe-associated changes in (I) plant growth and flowering time (II) fruit production, quality, and postharvest life, (III) development of pollens in stamen, (IV) transcriptional responses of miR172, CRTISO, and bZIP genes, (V) enzymatic (catalase and peroxidase) and non-enzymatic (ascorbate and thiols) antioxidants, (VI) H 2 O 2 concentration and membrane stability, (VII) proline concentration (a multifunctional protective amino acid), and (VIII) nutritional status in fruits.
In soilless conditions, seeds were grown in pots containing perlite and vermiculite (3:1) and irrigated with a Hoagland nutrient solution. The 37-day-old seedlings in the treatment groups were sprayed with different doses of nSe (0, 3, and 10 mgl -1 ) dissolved in deionized water and those in the control groups with different doses of sodium selenate (bulk Se (BSe); 0, 3, and 10 mgl -1 ). Seedlings were treated six times within 42 days, with one-week intervals between treatments. Growth-related traits (shoot fresh mass and root fresh mass), flowering time, number of fruits, and fruit postharvest longevity were recorded. Furthermore, molecular and physiological responses were monitored in leaves and fruits.
Transcriptions of miR172, CRTISO, and bZIP genes
Total RNA was extracted using an RNA extraction kit (Promega-GRE#AS1500) from both leaves and fruits. The forward and reverse sequences (5'-3') of primers for miR172, bZIP, CRTISO, and GAPDH genes are displayed in Table 1. Quantitative polymerase chain reaction (qPCR) was performed using mi-1STEP RT-qPCR kit (Metabione, Co-GRE#mi-E8105), the transcription level of target genes were quantified using a PCR instrument (QIAGEN's realtime PCR cycler, Rotor-Gene Q) under the program specified by the manufacturer (reverse transcription in 50˚C for 10-15 min; initial denaturation in 95˚C for 5 min; denaturation in 95˚C for 15 sec; annealing and elongation in 60-65˚C for 1 min). Transcription rate was calculated using the equation 2 -ΔΔCT in which ΔCT was calculated by subtracting the internal control CT value from the CT amount of each target gene.
Hydrogen peroxide (H 2 O 2 ) concentration and lipid peroxidation level
H 2 O 2 concentrations in leaves of BSe/nSe-treated plants were measured according to the procedure described by Sheteiwy et al. [17]. Briefly, 0.1% trichloroacetic acid (TCA) was applied to prepare leaf extract. After that, the resulting extract was centrifuged and the supernatant was separated. The reaction mixture consisted of 1000μL KI (1 mM), 500μL of potassium phosphate buffer (10 mM, pH 7.0), and 500 μL supernatant. Next, the absorbance was spectrophotometrically recorded at 390 nm. H 2 O 2 concentration was calculated according to the equation of a standard curve. Membrane integrity was also evaluated by quantifying Malondialdehyde (MDA) contents in leaves according to the protocol described by Salah et al. [18] with a small modification. Briefly, fresh leaf tissue was grounded in 5 ml of 0.25% thiobarbituric acid (TBA) in trichloroacetic acid (TCA) (0.25 g TBA was dissolved in 100 ml of 10% TCA). The resulting homogenate was then placed at 95˚for 20 minutes. Next, the samples were quickly cooled in an ice bath and centrifuged. The supernatant absorption was spectrophotometrically measured at 600 and 532 nm. Calculation of MDA was performed using the extinction coefficient of 155 mM -1 cm and expressed in micromol per gram fresh weight (μMg -1 fw).
Activities of peroxidase, catalase, and phenylalanine ammonia-lyase (PAL) enzymes
In order to extract enzymes, phosphate buffer (0.1 M; pH of 7.2) was applied to the liquid nitrogen-grounded leaves. Next, the homogenate was centrifuged at 4˚C and the resulting supernatant was stored at −80˚C until enzyme analysis. The nSe-mediated variations in defense-related enzymes, including peroxidase [19], catalase [18], and PAL [20] were quantified in leaves. The peroxidase reaction was triggered by the addition of 100 μl enzyme extract to the reaction medium containing 100 μl guaiacol, 100 μl H 2 O 2 , and 2700 μl phosphate buffer (25 mM, pH 7.1). To estimate enzyme activity, the absorbance difference was monitored at 470 nm. Finally, the peroxidase activity was expressed in terms of Unit enzyme per gram fresh weight (UnitEg -1 fw). To determine catalase activity, the reaction mixture containing 200 μl H 2 O 2, 200 μL enzyme extract, and 2600 μl phosphate buffer (25 mM, pH 7.1) were prepared. In this assessment, a decrease in absorbance at 240 nm was recorded following the addition of H 2 O 2 . To quantify PAL activity in leaves, 200 μl enzyme extract was added to the reaction mixture containing 6 μM phenylalanine and Tris-HCl buffer (500 mM, pH8). After that, the reaction mixture was incubated at 37˚C for 1 hour. Then, the PAL reaction was stopped by the addition of 50 μl HCl (5 N). Finally, the PAL activity was determined according to the cinnamate standard curve and calculated in terms of microgram cinnamate per hour per gram fresh weight (μgCin.h −1 g −1 fw).
Characterization of Mg, Fe, Zn, and Se concentrations
Ash solution was prepared by thermal decomposition in a furnace (650˚C) and subsequently dissolving in nitric acid and hydrochloric acid (2:1). Mg, Zn, and Fe contents were assessed using Atomic Absorption Spectroscopy (AAS; Varian, Spectr AA.200). Furthermore, Se concentration was measured using a fully automatic Flame/Graphite Furnace AASAA.200; YOUNGLIN AAS 8020).
Ascorbate, non-protein thiols, proline, and soluble phenols
The concentrations of non-protein thiols were quantified in leaves according to the protocol described by Del Longo et al. [21]. The leaves were homogenized in a 5% (w/v) solution of sulfosalicylic acid. The assay reaction of non-protein thiols consisted of 500μl DTNB (1 mM), 500μl phosphate buffer (100 mM, pH 7.1), 100μl of leaf extract, and 500 μM EDTA. After incubation for 10 minutes, the absorbance amount was recorded at 412 nm. The ascorbate contents were measured in leaves according to the protocol described by De Pinto et al. [22]. Briefly, metaphosphoric acid (5%) was used to prepare the leaf extract. Then, the resulting extract was centrifuged and the supernatant was separated. The reaction mixture was a 100 μl leaf extract, 250 μl phosphate buffer (50 mM, pH 7.2) supplemented with 50μl dithiothreitol (DTT; 10 mM), and 5 mM EDTA. After that, this mixture was incubated at room temperature for 10 minutes. After incubation for 10 min at room temperature, the process was followed by the addition of 150 μl N-ethylmaleimide (0.5%). Next, 200 μl dipyridyl of 4% in 70% ethanol, 200 μl orthophosphoric acid (44%), 200μl trichloroacetic acid (TCA; 10%), and 0.3% (w/v) FeCI 3 were added and the mixture was vortexed. After incubation at 40˚C for 40 minutes, the absorbance of each sample was recorded at 525 nm. Ascorbate concentration was calculated based on the standard curve equation of ascorbic acid. The concentration of proline amino acid in leaves was determined following the protocol described by Bates et al., [23]. To prepare leaf extract for proline assay, sulfa salicylic acid (3% w/v) was utilized. The ninhydrin reagent was prepared by mixing 30 ml acetic acid and 20 ml phosphoric acid (6 M). Next, 2 ml leaf extract was added to the reaction mixture containing 2 ml ninhydrin reagent and 2ml acetic acid (glacial). This mixture was heated at a boiling water bath for one hour. Then, this reaction mixture was immediately cooled down at an ice bath. After that, the reaction was followed by the addition of toluene solvent and vigorously shaking using a vortex. Then, the absorbance of the toluene phase was spectrophotometrically recorded at 520 nm. Proline concentration was estimated by using the equation of the standard curve of proline amino acid. Finally, Folin-Ciocalteu reagent was utilized to measure soluble phenols in leaves [24]. Briefly, total soluble phenols were extracted by homogenizing leaves in an ethanol solvent of 80% (v/v) and incubating in a boiling water bath. The reaction was started by the addition of 1 ml leaf ethanolic extract to a mixture containing 1 ml Folin-Ciocalteu reagent and saturated Sodium carbonate (21%). After 10 minutes and the development of a blue color, the mixture was centrifuged. The absorbance of the resulting supernatant was read at 760 nm. The concentration of total soluble phenols was quantified based on the equation of the standard curve of tannic acid.
Histological study
The flower buds at the same developmental age were fixed in FAA fixator [6]. Longitudinal sections were made by a microtome and were stained using Hematoxylin/Eosin staining protocol. Stained sections were observed at different magnification levels and photographed using a light microscope [6].
Statistical analysis
The experimental design was completely randomized. Every experiment was independently performed in the replication of three. All data were subjected to analysis of variance (ANOVA) using GraphPad software. The mean values of the three independent replications were compared using Tukey's range test.
Growth, biomass, productivity, and fruit postharvest longevity
To understand whether the nSe effectiveness in inducing growth, flowering, and yield changes are different from selenate, several traits, including shoot biomass, yield, fruit quality, and fruit postharvest life were evaluated. Compared with controls, both BSe and nSe treatments at 3 mgl -1 significantly increased fresh biomass in the shoot by 23% and 35%, respectively (Fig 1A). While BSe and nSe treatments at 10 mgl -1 led to a significant decrease in fresh biomass in the shoot, 31% and 20%, respectively, when compared with controls ( Fig 1A). Similarly, treatment of seedlings with both BSe or nSe at 3 mgl -1 significantly improved fresh biomass in roots, by an average of 20.75%, whereas treatments at 10 mgl -1 adversely influenced root biomass by an average of 23% relative to the control (Fig 1B). However, all applied treatments accelerated the vegetative/reproductive stage switch and reduced flowering time (Fig 1C). The numbers of produced fruits were increased in response to foliar application of either BSe or nSe (especially the latter) at 3 mgl -1 by an average of 25.3% compared to the control (Fig 1D). On the other hand, the BSe or nSe treatment at 10 mgl -1 was associated with toxicity and restricted fruit production by a mean of 39.5% when compared to the control (Fig 1D). The fruit fresh mass in the seedlings treated with BSe3 or nSe at 3 mgl -1 was also higher than the control by an average of 18% (Fig 1E). The supplements at the high dose (10 mgl -1 ) not only reduced fruit production but also significantly reduced fruit fresh weight (Fig 1E). Finally, treatments at all applied concentrations, significantly improved fruit postharvest longevity by an average of 38% ( Fig 1F).
Transcriptional responses of genes miR172, bZIP transcription factor, and CRTISO
In addition to the growth-related responses, BSe or nSe-mediated changes in the expression of target genes were investigated to provide comparative molecular evidence, for the first time. In leaves, the BSe3, nSe3, BSe10, and nSe10 treatments transcriptionally upregulated miR172 by 2.4, 4.6, 3.2, and 3.9 folds, respectively (Fig 2A). likewise, the BSe or nSe treatments at the lower concentration (3 mgl -1 ) moderately stimulated the expression of the bZIP gene (mean = 5.7 folds) ( Fig 2B); while, the supplements at the higher dose (10 mgl -1 ), mediated drastic transcriptional upregulation in the bZIP gene (mean = 13.7 folds) (Fig 2B). Similarly, the transcription of the CRTISO gene was upregulated in leaves of the seedlings treated with BSe or nSe (mean = 4.5 folds); the highest expression was observed in seedlings treated with BSe or nSe at 10 mgl -1 (Fig 2C). In fruit, the CRTISO gene was also found to be transcriptionally upregulated in response to the supplements by an average of 5.9 folds (Fig 2D).
Physiological responses
To evaluate the occurrence of oxidative stress, activation of the defense system, and changes in metabolism in response to BSe or nSe utilization, several physiological traits were measured. In this regard, H 2 O 2 concentration and MDA content (membrane integrity index) were monitored to estimate the BSe-or nSe-associated risk of oxidative stress. To monitor the activation of the defense system, the BSe-or nSe-mediated changes in enzymatic antioxidants (catalase and peroxidase), non-enzymatic antioxidants (ascorbate and non-protein thiols), and proline concentration were assayed. PAL activity and concentration of total soluble phenols were measured to address the BSe-or nSe-associated alteration in secondary metabolism. The BSe or nSe treatments at 10 mgl -1 led to a drastic rise, of about two folds, in the H 2 O 2 contents of leaves ( Fig 3A). The BSe or nSe treatments at 3 mgl -1 did not make a statistically significant change in membrane integrity (Fig 3B) but made a slight increase in leaf proline concentration (Fig 3C). Additionally, the BSe or nSe treatments at a high dose (10 mgl -1 ) drastically increased lipid peroxidation level as well as in the leaf proline content (Fig 3B and 3C). Catalase enzyme activity was significantly higher in all seedlings treated with BSe or nSe, by a mean of 2.2 folds when compared to the controls at all applied concentrations ( Fig 3D); similarly, peroxidase activity and ascorbate concentration were higher in leaves, by an average of 30% and 36%, respectively (Fig 3E and 3F). With increasing the BSe or nSe concentration, the non-protein thiols were also increased linearly by an average of 27% (Fig 3G). Furthermore, treatments with the BSe3 (26.8%), nSe3 (52%), BSe10 (39.6%), and nSe10 (75.3%) treatments significantly stimulated the PAL activity in leaves (Fig 3H). Likewise, Se treatment, particularly the nSe treatment, significantly increased concentrations of the soluble phenols by an average of 39% (Fig 3I).
Nutritional status in fruit
Fruit quality is an important determining factor in terms of fruit longevity and human nutrition. In this regard, the BSe or nSe-mediated changes in concentration of several important minerals were monitored. The Se supplementation altered concentrations of minerals in fruits in a dose-dependent manner (Fig 4A-4C). At 3 mgl -1 , the BSe or nSe, especially the latter, significantly increased Mg, Fe, and Zn concentrations in fruits by averages of 29.8%, 27.6%, and 21%, respectively, when compared to the control (Fig 4A-4C); while supplementation at high dose (10 mgl -1 ) moderately decreased Mg, Fe, and Zn contents in comparison to the control (Fig 4A-4C). Furthermore, the foliar applications of both BSe or nSe led to Se-bioaccumulation in tomato fruits ( Fig 4D); with nSe treatments leading to a higher amount of bioaccumulated Se in fruits compared with the bulk treatments ( Fig 4D).
The density of pollen grains in the pollen sac
As mentioned above, our results showed that a high concentration of BSe or nSe significantly reduced crop yield. Hence, the BSe-or nSe-associated toxicity on the pollen grain development was evaluated for the first time. Assessment of the longitudinal sections of the flower buds indicated that the size and density of pollen grains were affected by the BSe/nSe treatments in a dose-and material type-dependent manners (Fig 5A-5F). The BSe or nSe treatments at all concentrations were associated with a decreased density of pollen grains in pollen sacs. However, the size of pollen grains increased with supplementation at 3 mgl -1 . It is important to note that Se treatment at 10 mgl -1 , especially in form of BSe, reduced both the size and the density of pollen grains, highlighting its potential toxicity at the higher dose (Fig 5D-5F).
Discussion
According to our findings, the potential benefits of nSe utilization toward improving plant growth, biomass accumulation, yield, nutrition, flowering time, fruit quality, and postharvest longevity were much higher than the use of selenate, and at the same time, the potential nSeassociated risks are fewer. These results are consistent with several recent reports [5,6,25,26].
The following mechanisms appear to mediate partly differential responses to nSe relative to the selenate; (I) variations in uptake kinetics (nSe inactive influx through aquaporins vs secondary active influx mechanism of selenate through symporter) [2], (II) differences in their interactions with biomolecules and metabolism into the organic forms [2,26], (III) differential physicochemical properties of nanoparticles by which a nSe interaction with biomolecules can be different from that of selenate [2,5,6,26], (IV) variation in a nSe-mediated change in phytohormones [3,4,27], and (V) epigenetic modification [5,6]. However, the involved mechanisms are still poorly characterized which further emphasizes the need for further studies. This study provides the first comparative evidence on the Se-or nSe-mediated variations in miRNAs as key regulatory checkpoints. The observed selenate/nSe-associated changes in developmental vegetative/reproductive switch, fruit-related characteristics, and plant physiology may be attributed to the miR172 involvements. However, future transcriptome and proteome studies can improve our knowledge, fill knowledge gaps, and validate or disprove this hypothesis. Earlier reports suggested this hypothesis that miR172 acts a critical role in modulating plant reproduction, developmental programs, and conferring stress tolerance [9,28,29]. Moreover, this experiment provides an important molecular mechanism on how Se or nSe application can improve photosynthesis performance, confer stress tolerance, and increase fruit quality through a transcriptional up-regulation in CRTISO. Carotenoid concentration closely correlates with the expression of the CRTISO gene [10][11][12], and with conferring stress tolerance [12]. Several studies showed cytoprotective effects of Se [30][31][32][33] and nSe [1,25,27] at low doses during stress conditions in plants. We concluded that the bZIP transcription factor is more responsive to selenate whereas CRTISO and miR-172 are more responsive to the nSe supplementation. Recently, Sotoodehnia-Korani et al. [6] reported that the bZIP transcription factor is a responsive gene to nSe. However, they did not monitor the plant's response to selenate. The observed transcriptional responses can be explained by Se or nSe-associated variations in redox status [25], phytohormones [4,27], and epigenetic modification [5,6]. An almost similar trend in the expression of the target genes (CRTISO, bZIP, and miR-172) and the physiological traits suggests the involvement of similar and convergent signaling pathways in response to selenate and nSe. However, significant differences in terms of transcription rates in the nSe-treated plants relative to selenate can be a good sign of partly differential signal perception, transduction, and signaling cascades. Several recent studies have addressed transcriptional responses to nSe, namely, HSFA4A in wheat [34], RAS and HPPR in Melissa officinalis [3], a bZIP transcription factor in pepper [6], DREB1A, PAL, HCT1, and HQT1 genes in chicory [25], and WRKY1, PAL, and 4CL in bitter melon [5]. However, these studies have only reported a response to the nano form. This research can, therefore, provide a new perspective in terms of comparing molecular responses to selenate and nSe.
According to earlier literature, there is a knowledge gap in the field of potential changes in signaling molecules and redox-based regulation of cellular transcription program in response to Se, nSe, and other kinds of nanoparticles. In this study, we monitored H 2 O 2 only once which is not good enough for addressing H 2 O 2 fluctuation. Owing to the close relationship of H 2 O 2 with other signaling molecules, the monitoring of potential fluctuations in nitric oxide, H 2 O 2 , and H 2 S is, therefore, recommended in designing future studies. Fig 6 displays schematics of the potential molecular mechanisms contributing to the plant responses to exogenously applied nSe. This study provides the first comparative data in terms of physiological responses to selenate and nSe. According to the H 2 O 2 accumulation and MDA level, Se or nSe at the low dose did not mediate oxidative stress. It seems that Se or nSe signaling at low concentration occurs through a slight transient increase in the H 2 O 2 concentration which is subsequently detoxified by an increase in enzymatic antioxidants. H 2 O 2 signaling triggers the transcriptional changes in downstream genes and activation of the defense system, including antioxidants and secondary metabolism. These physiological responses along with the observed molecular changes suggest mechanisms by which Se or nSe utilization, especially the nanoform, can improve crop tolerance to environmental stresses. However, it should be warned that these supplements at the high dose were associated with induction in the oxidative burst, impaired membrane integrity, disrupted metabolism, and growth disorders. The lower concentration of H 2 O 2 and MDA along with higher content of proline in the nSe10-treated group is another reason for the lower toxicity of the nano form relative to the selenate counterpart. The nSe-mediated changes in proline, a multifunctional protective amino acid, have been attributed to the modified nitrogen assimilation metabolism [24], and activation of the defense system [5,6]. Se or nSe supplementation, especially the latter, stimulated enzymatic antioxidants, non-enzymatic antioxidant compounds, and secondary metabolism. In agreement with our results, the nSe treatment improved enzymatic antioxidants [5,6,25], non-enzymatic antioxidants [25], and accumulation of secondary metabolites [3]. However, previous reports have only reported physiological responses to nSe. To the best of our knowledge, this study, for the first time, showed that nSe is more efficient than selenate in activating the antioxidant system and stimulating secondary metabolism.
The results confirmed a higher efficacy of the nano type for application in biofortification programs. Our results on Se-associated modification in nutritional status are in line with the findings of Amirabad et al., [33] in radish, Babajani et al. [3] in lemon balm, Alam et al. [32] in mung bean, and Zahedi et al. [4] in strawberry. Along with these reports, the nSe-associated modifications in vascular conducting tissues (xylem and phloem) [5,6] may be responsible for the alterations in plant nutritional status. The higher fruit longevity, an economically important index, in the nSe-supplemented plants can be explained by a higher efficiency of nSe in enhancing the antioxidant system, improving minerals, and the antimicrobial properties of nSe when compared to that of selenate.
The results highlight how Se or nSe-mediated changes in vegetative growth and metabolism are coordinated with alteration in plant productivity at a reproductive stage. Herein, the provided anatomical evidence, for the first time, underlines the risk of Se at high concentration in restricting crop fertility through disrupting the development of pollen grains. It has been recently reported in pepper [6] and bitter melon [5] plants that in vitro toxicity of nSe is associated with impaired tissue differentiation and abnormalities in stem's apical meristem. Further anatomical and developmental investigations of Phyto-toxicological concerns of nanomaterials are required to improve our knowledge of plant responses to nanoproducts, especially nano-pesticides and nano-fertilizers [5,[35][36][37].
Conclusion
Taken together, this study highlights the considerable efficacy of Se or nSe, especially the nano form, for modifying growth, yield, primary and secondary metabolism, nutrition, defense system, and transcription program. The Se-or nSe-associated transcriptional changes in miR172, CRTISO, and bZIP are introduced as key potential molecular mechanisms through which Se application may confer stress tolerance. Moreover, this study provided anatomical evidence displaying potential phytotoxicity of Se or nSe in terms of disrupting the development of pollen grains. Taking knowledge gaps into account, these comparative comprehensive data can be helpful to gain novel insights into the benefits or the risk associated with Se or nSe function in agriculture. This study underlines the necessity of including anatomical, developmental, and molecular assessments in future phyto-toxicological investigations of nanomaterials. | v3-fos-license |
2018-12-22T05:30:41.585Z | 2016-06-28T00:00:00.000 | 84832794 | {
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} | pes2o/s2orc | Conformational changes in C methyl -resorcinarene pyridine N -oxide inclusion complexes in the solid state †
Aromatic N -oxides interact with C methyl -resorcinarene resulting in marked changes in the conformation of the host resorcinarene. In the solid state, 2-and 3-methylpyridine N -oxides form pseudo-capsular 2:2 endo host – guest complexes with C methyl -resorcinarene stabilized by C – H ⋯ π interactions. The C methyl - resorcinarene · 2-methylpyridine N -oxide complex has a C 4v crown conformation, while the C methyl - resorcinarene · 3-methylpyridine N -oxide complex has a slightly open C 2v boat conformation. On the contrary, other para -substituted and benzo-fused pyridine N -oxides form only exo complexes with C methyl -resorcinarene. In the exo complexes, the asymmetry of the guest, conformational flexibility and preferred inter-host hydrogen bonding of C methyl -resorcinarenes exclude endo complexation. All the exo complexes form robust 1-D hydrogen bonded chains between the host hydroxyl groups assisted by the guest N – O groups, resulting in a C 2v boat conformation.
Introduction
With flexible host molecules, several factors such as substituents, size and electronic properties of the guest, solvents, temperature or even reaction conditions can induce conformational changes in the host. 1 The understanding of the interplay of host-guest interactions, both in the solid state and in solution, is a very important research area in supramolecular chemistry due to its major impact on the potential applications of host-guest systems as functional materials. 2 Many host and receptor molecules adopt specific geometries and conformations. Amongst these compounds are resorcinarenes and pyrogallarenes which natively occur in the crown C 4v conformation, and this favourable conformation allows them to act as hosts for a variety of guest molecules. 2 The versatility of these host systems originates from the synthetically easy modifications either at the upper or lower rim of these macrocycles. 2 However, it is known that core (unsubstituted) resorcinarene conformations are quite easily modulated by the reaction conditions when the host is synthesized. The most common resorcinarene and pyrogallarene conformations are crown, boat, chair, diamond and saddle. 3 Other unusual conformations such as the rarely observed rcct-diamond C methyl -resorcinarene, 3 the rcct-boat C methyl -2nitroresorcinarene 4 and the rcct-crown C 4t -pyrogallarene (4t = tert-butyl) 5 have been reported. In the C 4v conformation, intramolecular circular hydrogen bonds (HB) between adjacent phenolic hydroxyl groups preserve the crown structure. 5 In this conformation, the π-rich cavity is suitable for inclusion of spherical and planar guests such as ammonium and phosphonium cations, the main complexation force being the cation⋯π interactions. 6 With resorcinarenes, these cationic guests play a key role in numerous solid-state architectures such as open inclusion complexes, 7 dimers, 8 hexamers, 9 and tubular assemblies. 10 Though inclusion complexes of resorcinarenes with cationic compounds are prevalent, there are several reports of complexes with neutral N-heteroaromatic five-and sixmembered planar guests as well. 11 N-Aromatic compounds such as 2,2′-and 4,4′-bipyridines, due to their size and strong hydrogen bond acceptor properties, have been particularly effective in inducing conformational changes in resorcinarene complexes. 12 A Cambridge Structural Database (CSD) 13 search using C methyl -resorcinarene 1 as the host revealed 34 crystal structures with 4,4′-bipyridine as the guest molecule. 14,12a-c Aromatic N-oxides are well-known synthetic intermediates for functionalization of the pyridine ring in organic synthesis, 15 and as ligands in supramolecular chemistry. 16 The oxygen in the N-oxide group can exhibit hyper-dentate coordination behaviour with metal cations, resulting in interesting supramolecular architectures. 16 Most recently, pyridine N-oxides (PyNOs) have been shown to be excellent halogen bond acceptors and thus form very strong halogen bonds. 17 The N-oxide, viz. + N-O − , group makes the aromatic ring electron deficient but the electron deficiency can be modulated by the substituents on the aromatic ring, and due to this, aromatic N-oxides can act as guest molecules with calixarenes and cavitands. 18 Recently, we have reported dimeric pseudocapsular assemblies with aromatic N-oxides using C ethyl -2methylresorcinarene as the host. 19 The C-H⋯π and π⋯π interactions 20 have proven to be the governing factors in the recognition of electron-deficient N-oxides by π-rich host systems.
In addition, halogen bond breaking using C ethyl -2methylresorcinarene in halo-substituted pyridine N-oxide systems was an unexpected result of the conformational change in the host. 21 While our previous work has focused on C ethyl -2-methylresorcinarene, 19,21 there exists one study with 4,4′bipyridine N,N′-dioxide and C methyl -resorcinarene as the host. 22 In this work, we report solid-state complexes with C methyl -resorcinarene 1 as the host and seven PyNOs (Fig. 1) as the guests, and the conformational behaviour of the host upon complexation.
forms a similar dimeric pseudo-capsular 2 : 2 complex to 2-MePyNO (Fig. 2c). These dimers are hydrogenbonded to each other by bridging exo 3-MePyNO molecules as shown in Fig. 2d. In complex 3-MePyNO·1, the cavity of host 1 is elongated so that the centroid-to-centroid distances are 5.8 and 7.5 Å to accommodate the slightly bigger 3-MePyNO molecule, as shown in Fig. 3d. The endo guest in 3-MePyNO·1 is situated 3.2 Å from the centroid of the lower rim carbon atoms, exactly the same distance as that in 2-MePyNO·1 and previously studied 3-MePyNO·C ethyl -2methylresorcinarene. 21 In 3-MePyNO·1, the guest is situated near the center of the cavity between the benzene rings with no apparent aromatic π⋯π interaction, but manifesting a weak methyl C-H⋯O hydrogen bond to the host O-H group (C-H⋯O distance of 2.51 Å) and C-H⋯π interactions from the hydrogen atoms meta to the N-O group (Fig. 3c). These C-H⋯π distances range from 2.8 Å to 2.9 Å. The increased conformational flexibility of host 1 and the larger guest size in the endo complexation via the C-H⋯π interactions between the host and the guest distort the parent C 4v crown conformation to a nearly ideal C 2v boat conformation (Fig. 3d). With our previous work on resorcinarene endo PyNO complexes, 21 the PyNOs located deep in the cavity, viz. <2.9 Å from the centroid of the lower rim carbon atoms, usually show short C-H⋯π(centroid) contacts. However in 3-MePyNO·1, where the 3-MePyNO guest is situated higher in the cavity (3.2 Å), the shortest C-H⋯π(centroid) distance of ca. 2.5 Å was observed. Comparing the host-guest chemistry of 2-MePyNO·1 and 3-MePyNO·1 with that of C ethyl -2methylresorcinarene, 21 both host systems show a similar encapsulation profile to 2-MePyNO and 3-MePyNO. In general, the formation of endo complexes with host 1 is considerably more difficult than that with conformationally more rigid host molecules due to the stronger influence of the intermolecular hydrogen bonding between the hosts, which can lead to the conformational change and subsequent formation of host-to-host 1-D, 2-D or 3-D networks.
The conformational flexibility of resorcinarenes is due to their lower rim R chains. The methyl chain is smaller, thus allowing more different conformations for the resorcinarene skeleton. The ethyl chain is sterically more bulky and thus results in conformationally less flexible resorcinarenes. 2,5 Sterically suitable PyNOs like 2-MePyNO and 3-MePyNO which do form endo complexes with conformationally flexible host systems are also proven to form endo complexes with more rigid systems; this we have recently shown with C ethyl -2methylresorcinarene. 21 The question arises if the larger PyNOs 21 would also behave similarly to the more flexible host 1. As such, larger PyNOs were utilized to probe their behaviour. O] manifest only exo complexes with a prominent feature being the formation of 1-D tubular hydrogen-bonded chains with a C 2v boat conformation of the host (Fig. 3f and g). The asymmetric structure and larger size of these PyNOs prevent the formation of endo complexes and the ease of intermolecular hydrogen bond formation leads to the formation of host-to-host 1-D tubular chains in all complexes, as shown in Fig. 4. During crystallization, the host interactions with themselves, the guest and the solvent molecules (methanol and water) change the conformation from the C 4v crown to C 2v boat and provide possibilities for better intermolecular hydrogen bonding between the hydroxyl groups of the hosts. Two types of 1-D chains were observed, only PyNOs (type 1) and a combination of PyNOs and solvent molecules (type 2). The type 1 situation appears in complexes 4-MeOPyNO·1 and 4-PhPyNO·1, while type 2 is observed in complexes 2-MeNPyNO·1 and IsQNO·1 ( Fig. 5 and ESI, † Fig. S6).
In complex Fig. S2). These interdigitated 1-D chains are stabilized by C-H⋯π and π⋯π interactions. During interdigitation, the aromatic rings of host 1, and the hydrogen atoms of the methoxy group in 4-MeOPyNO, and the para hydrogen atoms of 4-PhPyNO have short C-H⋯π contacts with distances of ca. 2.8 Å and 2.7 Å, respectively (see the ESI, † Fig. S2 and S3).
The host molecules in complex 2-MeNPyNO·1 also form a 1-D hydrogen bonded chain as shown in Fig. 5d. However, the hydrogen bonding via the hydroxyl groups of the host and the crystal packing differ due to interactions with solvent molecules and a second 2-MeNPyNO molecule in the asymmetric unit. Two methanol molecules, a water molecule and a 2-MeNPyNO molecule hydrogen bond to three host hydroxyl groups of the 1-D tubular chain in a continuous fashion (see the ESI, † Fig. S4a). Also, the second 2-MeNPyNO molecule hydrogen bonds to water and resides close to the host aromatic ring at centroid-to-centroid distances of ca. 3.7 Å. The crystal packing of 2-MeNPyNO·1 shows that the 1-D chains are connected by two methanol molecules to give a 2-D sheet motif (see the ESI, † Fig. S4c).
The mode of action of the host 1 hydroxyl groups in the 1-D polymeric chain of complex IsQNO·1 (Fig. 5e)
Solution studies
Solution studies between host 1 and PyNO, 2-MePyNO, 2,QNO and IsQNO as guests were conducted via 1 H NMR experiments in CD 3 OD at 293 K. In these experiments, the host and guests were mixed in a 1 : 1 (6.6 mM on both molecules) ratio in CD 3 OD; the 1 H NMR spectra were measured and the results were compared with the free host (6.6 mM) and free guests (6.6 mM).
Complexation-induced shielding of the guest proton resonances was observed in all cases except with 4-PhPyNO. The shielding effects of the aromatic rings of the bowl-shaped host cavity upon addition of the guest are responsible for this upfield shift and indicate that guest exchange is fast on the NMR time scale. Taking the 1 : 1 mixture between 1 and 3-MePyNO as an example (Fig. 6, I), the aromatic protons (g, f) are the most shielded (0.16 ppm and 0.17 ppm). These shift changes confirm the orientation of the guest deep in the host cavity, supporting the X-ray crystal structure (Fig. 3). Analysis of the 1 : 1 mixture between 1 and QNO (Fig. 6, II) reveals the aromatic protons (f, g) to be the most shielded (0.14-0.15 ppm), once again suggesting the localization of QNO within the host cavity. The analyses of the 1 H NMR results between the host and the other guests (PyNO, 2-MePyNO, 4-MePyNO, 2-MeNPyNO, 2,6-DiMePyNO, 4-MeOPyNO, and IsQNO) also indicate the localization of the guest in the host cavity ( Fig. S7-S16 †). No shift changes were observed with 4-PhPyNO, implying that in solution no host-guest complex is formed (Fig. S14 †).
Variable temperature (233-333 K) 1 H NMR experiments were done to probe the conformational changes of host 1 in the presence of the different guests ( Fig. S17-S18 †). No obvious changes in the host 1 signals were observed, implying that the conformational changes observed in the solid state with the N-oxide guests are caused by multiple intermolecular interactions existing only in the solid state.
Conclusions
The X-ray structures of seven PyNO complexes led to the conclusion that there is either endo complexation via C-H⋯π interactions or exo complexation via (N-O)⋯IJH-O) host hydrogen bonds between the aromatic N-oxides and C methyl -resorcinarene. Due to the rarity of C methyl -resorcinarene·pyridine N-oxide complexes, the endo host-guest complexes 2-MePyNO·1 and 3-MePyNO·1 were compared with our previous results with C ethyl -2-methylresorcinarene, 21 which allowed us to probe factors such as the conformational flexibility of the cavity, the strength of C-H⋯π interactions and the localization of the guest molecule in the cavity. Despite the enhanced conformational flexibility, C methyl -resorcinarene forms a pseudocapsular 2 : 2 endo complex with 2-MePyNO and 3-MePyNO stabilized by C-H⋯π interactions in the solid state. The larger overall size of 3-MePyNO forces the C 4v crown conformation to open, very close to the ideal C 2v boat conformation, but still having the guest inside the cavity. Additionally, the strong C-H⋯π interactions found in the endo complex 3-MePyNO·1 hint that the guest similar to 3-MePyNO with C methyl -resorcinarene could show interesting host-guest interactions. The larger guests 4-MePyNO, 2-MeNPyNO, 4-MeOPyNO, 4-PhPyNO and IsQNO do not form endo complexes, as the exo complexes are preferred due to the strong intermolecular hydrogen bonding between the host 1 molecules leading to 1-D chains. All exo complexes show very similar overall crystal packings. The complementary solution studies show that host-guest complexes between host 1 and all the N-oxide molecules, except 4-PhPyNO, are formed in solution. Furthermore, the variable temperature 1 H NMR measurements proved that the conformational changes in host 1, observed in the solid state upon complexation, were not observed in solution. | v3-fos-license |
2020-11-19T09:13:02.806Z | 2020-11-01T00:00:00.000 | 227101483 | {
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} | pes2o/s2orc | Oxidative Stress in Endurance Cycling Is Reduced Dose-Dependently after One Month of Re-Esterified DHA Supplementation
Docosahexaenoic acid (DHA) supplementation can reduce exercise-induced oxidative stress generated during long aerobic exercise, with the minimum dose yet to be elucidated for physically active subjects. In this study, we performed a dose finding with re-esterified DHA in triglyceride form in a randomized double-blind parallel trial at different doses (350, 1050, 1750, and 2450 mg a day) for 4 weeks in males engaged in regular cycling (n = 100, 7.6 ± 3.7 h/week). The endogenous antioxidant capacity of DHA was quantified as a reduction in the levels of the oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) recollected in 24-h urine samples after 90 min of constant load cycling before and after intervention. To ascertain incorporation of DHA, erythrocyte polyunsaturated fatty acid (PUFA) composition was compared along groups. We found a dose-dependent antioxidant capacity of DHA from 1050 mg with a trend to neutralization for the highest dose of 2450 mg (placebo: n = 13, F = 0.041; 350 mg: n = 10, F = 0.268; 1050 mg: n = 11, F = 7.112; 1750 mg: n = 12, F = 9.681; 2450 mg: n = 10, F = 15.230). In the erythrocyte membrane, the re-esterified DHA increased DHA and omega-3 percentage and decreased omega 6 and the omega-6 to omega-3 ratio, while Eicosapentaenoic acid (EPA) and PUFA remained unchanged. Supplementation of re-esterified DHA exerts a dose-dependent endogenous antioxidant property against moderate-intensity long-duration aerobic exercise in physically active subjects when provided at least 1050 mg a day for 4 weeks.
Introduction
Omega-3 have been studied for its health benefits during the last decades [1,2]. They represent a broad family of polyunsaturated fatty acids (PUFAs), with the most active compounds probably being the double bond molecules EPA (Eicosapentaenoic acid, C20:5n-3) and DHA (Docosahexaenoic acid, C22:6n-3), which exhibit different biochemical behaviors when administered [3][4][5], thus they should not be interpreted as the same intervention treatment [6,7]. There is some controversy about the prooxidant/antioxidant capacity of DHA since its six double bonds are susceptible to oxidation and in fact, it showed a dual effect both as a pro-oxidant and antioxidant when given at different doses [8]. Oxidation by-products of DHA contribute to increased oxidative stress in the organism, but on the other side, they contribute to the expression of endogenous enzymatic antioxidant systems, like catalase, superoxide dismutase, and glutathione peroxidase [9][10][11][12][13][14].
The minimum DHA supplementation to elicit this effect remains inconclusive, partly due to the diversity of protocols employed in the literature, finding no variation in redox status in some population samples [15][16][17], and with positive results for other samples studied [18][19][20]. Additionally, clinical trials employed different fish oil products, which are not equally bioavailable [21], and dosing strategies that involve different cumulative doses and intervention times. Aerobic exercise inevitably augments oxygen consumption and increases acute oxidative stress [22,23] whose magnitude is dependent on the intensity and duration, as well as the training status of the subject whose antioxidant defense system is more adapted [24]. Thus, a model that employs modest-intensity and long-duration aerobic exercise in trained subjects can serve as a model to assess the antioxidant capacity of DHA at different doses. One gap we found in the literature is that the impact of DHA on oxidative stress in healthy physically active people has not been studied before, thus results from this assessment would serve as a reference to healthy subjects engaged in regular exercise.
In the present study, we conducted a dose finding of a high content (>70% of total fatty acids) of re-esterified DHA (rDHA) to assess the endogenous antioxidant effect in moderate-intensity exercise-induced oxidative stress in healthy active people. We hypothesized a response in a dose-dependent manner, with a greater effect for higher doses.
Trial Design
A double-blind, parallel, unicentric, controlled, and randomized experimental study was employed, with five different study arms to test the effect of DHA supplementation (rDHA at four different doses: 350, 1050, 1750, or 2450 mg) or placebo (PLA) on the oxidative stress generated by a moderate-intensity long-duration cycling test. Randomization was performed by a scientist not participating in the study, using software (Epidat 4.2, 2016) that generated random codes, which were assigned to participants. An initial incremental exercise test to exhaustion (IETE) was carried out to make an initial assessment of the physical conditioning of each participant. Then, the supplementation protocol of each group (rDHA or PLA) commenced for a period of 4 weeks, after which cyclists underwent the same exercise protocol to measure the intervention effect.
Subjects
Inclusion criteria were as follows: (1) Healthy male adults engaged in regular cycling of at least 5 h per week. Exclusion criteria were as follows: (1) serious clinical pathology or antecedents; (2) regular smoker; (3) regular consumer of alcoholic beverages; (4) supplementation with omega 3 fatty acids or food enriched in omega-3 fatty acids in the last 4 weeks; (5) subjects who had undergone ionizing radiation for accidental, diagnostic, or therapeutic reasons during the trial or in the month prior to it; and (6) allergy to fish or any of its by-products. Participants were informed (verbally and written) of the purpose of this study, the characteristics of the product used for supplementation, its effects, as well as any possible risk and side effects resulting from the supplement and the procedures of the study. Subjects were informed of their right to quit the study at any time, without the need to provide any reason. Participants gave written consent before the study was started. The study protocol and informed consent were approved by the Ethics Committee of the Catholic University of Murcia (UCAM protocol number 21052008) and were in agreement with the Declaration of Helsinki. A total of 75 subjects were finally recruited for the study.
Supplementation Protocol and Dietary Assessment
The study population was divided into five groups, based on daily re-esterified triacylglycerol (rTAG) rDHA (BRUDY PLUS ® , BRUDYTECHNOLOGY, Barcelona, Spain) consumption or PLA (refined sunflower oil) provided by the same manufacturer. Re-esterified triacyclglycerols are concentrated fish oils in which the natural TAGs are transferred to ethanol-forming etyl-esters and, after removal of undesired FAs, reconverted into triacylglycerols (thus re-esterified triacyclglycerols: rTAG). The composition per soft-gel of rDHA was: DHA (Tridocosahexaenoine-AOX ® ) 350 mg, EPA 40 mg, total Omega-3 content 405 mg, and a total fatty acid content of 500 mg, consumed in a single dose in the morning before breakfast, for 4 weeks. Participants were allocated to one of the following five groups: (1) PLA (3 softgels); or rDHA: (2) 350 mg/day (1 soft-gel); (3) 1050 mg/day (3 soft-gels); (4) 1750 mg/day (5 soft-gels); or (5) 2450 mg/day (7 soft-gels). Both products were identical in appearance: soft-gel oval, with transparent gelatin and yellow-colored oil, inside a plastic container, properly labeled and identified to preserve blinding. The batch number was the only difference between them. Sunflower oil is not a true placebo since it contains tocopherols with antioxidant properties [25]. Refined sunflower oil has a decreased amount of tocopherols by up to a third [26] but can still exert a biological effect. For simplicity, we will refer to it as a placebo in the paper, even if this is not an accurate description. Participants were asked to return the empty packs to ensure compliance by counting the left-over soft-gels. For the follow-up, participants were reminded verbally to consume the product.
Dietary assessment was performed through a qualitative and quantitative questionnaire that subjects had to fill out during the study period, which was analyzed by a nutritionist to determine dietary intake using software (Cronometer Software Inc., https://cronometer.com). Additionally, subjects were instructed in products that should not be consumed at breakfast prior to the physical test (products with antioxidant properties) (see Appendix A), which could interfere with the reading of the primary outcome.
Exercise Test
Each subject performed an initial physical assessment to determine the physical condition of the subjects and the external load of the second test. The initial assessment consisted of an incremental exercise cycling test to exhaustion (IETE) while the main test consisted of a square-wave endurance exercise test (SWEET) to induce exercise oxidative stress. Both tests were spaced 1 week apart to allow full physical recovery. A second SWEET test was performed after intervention to quantify the intervention effect in each group. In every occasion, they had to arrive after a 2-h fast and bring their own bike. All tests were conducted in the morning at the same time, and they were instructed not to change their physical activity habits during the study and to avoid physical exercise 48 h before the initial assessment, and 24 h before the main tests. Environmental conditions (room temperature and humidity) were replicated in every exercise test for optimal conditions using a room air conditioning system.
Initial Physical Assessment: Aerobic Capacity and Health Assessment
A preliminary IETE test was performed to assess the training status of volunteers at baseline and establish the load to be employed in the SWEET. Volunteers performed an incremental exercise to exhaustion using their own personal bike mounted on an electrically braked ergometer. The starting load was 12 km/h with load increases of 2 km/h every minute, maintaining a constant slope of 2%. A self-selected cadence between 60 and 100 revolutions per minute had to be maintained during the whole test. The following measures were employed to ensure repeatability: (1) Front-rear slope-ratio was corrected to zero (using a front wheel riser); (2) bike configuration (gear set, saddle and handlebars) must be kept during the study; (3) Bike fitting (seat-post height and angle, handlebar reach, height and grip position) must be the same; and (4) the preferred pedaling system (use of cycling shoes and type of clip/cleat) should be consistent. Exhaustion was deemed to occur when the subject decided to stop, when pedal cadence dropped 20 RPM below the minimum cadence established (i.e., 40 RPM), or when power output could not be maintained. During the test, volunteers were verbally encouraged by the staff to exert maximal effort.
During the entire test, subjects were monitored using an electrocardiograph, with expired gases determined by an automated breath-by-breath system (Jaeger Oxycon ProTM, CareFusion, Höchberg, Germany). The parameters evaluated were: maximum/peak absolute and relative oxygen consumption (VO2 max), maximum heart rate and heart rate at ventilatory threshold 2 (VT2) using the model described by Skinner and Mclellan [27] for VT2 calculation. All measures were analyzed using software (LABManager 5.3.0.4, VIASYS Healthcare GmbH, Höchberg, Germany) and were stored in a personal computer for later recall.
Exercise-Induced Oxidative Stress Cycling Protocol
In this test, subjects performed a SWEET test on the same device but with a constant load equivalent to a speed corresponding to 75% of its relative VO2 max calculated in the preliminary IETE test maintaining the same 2% slope. Subjects whose heart rate at 75% VO2 max exceeded the heart rate at VT2 (which means the exercise at this intensity would be more anaerobic and could be too exhausting to be maintained for 90 min) performed the trial 5 bmp lower than their heart rate at VT2. The duration of the test was 90 min, with ad libitum water consumption during the test. Subjects were weighed immediately before and after the test. The water consumption protocol was as follows: At the beginning of the test, the subject was given a bottle with 500 mL of mineral water (without gas) filled with a ml graduated cylinder. Subjects were provided as many bottles as requested. At the end of the test, the supplied bottles were counted by subtracting the water that was left in the last unit after measuring it with the graduated probe. Heart rate was monitored and recorded at 30, 60, and 90 min.
Endogenous Antioxidant Effect of rDHA
The endogenous antioxidant effect of rDHA was determined by the difference of the oxidative stress as 8-hydroxy-2 -deoxyguanosine (8-OHdG) levels in 24-h urine samples in relation to weight by each cycling test before and after supplementation. Oxidative stress from each physical test was quantified by subtracting the resting (basal) values of 8-OHdG from the values after the SWEET on each occasion. Subjects collected a 24-h urine sample on the day before the cycling test and on the same day starting after the SWEET. If the sample was considered incomplete (some urination not collected), it was discarded from the study. After quantification of the total volume of urine excreted, a sample of 15 mL of each container was obtained and frozen at −80 • C for later analysis. The method used for this determination was the enzyme-linked immunosorbent assay (ELISA) with a kit called DNA Damage ELISA Kit (Catalog number #K059-H1, Assay Designs, Inc., Ann Arbor, MI, USA). The linearity of the kit was of R 2 = 0.9956 and tests were performed thrice. Results were discarded and the assay repeated if the coefficient of variation (CV) between three tests was higher than 10%. This is a brief description of the method of the kit (for a full description, see [28]): A standard curve was generated with the 8-hydroxy-2 -deoxyguanosine (8-OHdG) stock solution provided. An 8-hydroxyguanosine conjugate was added to the standards and samples in each well, and binding initiated by peroxidase-labeled mouse monoclonal antibody. After a 2-h incubation, the plate was washed, and substrate added, which reacts with the peroxidase-labeled antibody that reacted with the bound conjugate. After a short incubation, the reaction was stopped, and the intensity of the generated color was detected in a microtiter plate reader capable of measuring a 450-nm wavelength. The concentration of the 8-hydroxy-2 -deoxyguanosine in the sample was calculated, after making suitable correction for the dilution of the sample.
Although more than 20 DNA damage of different bases are currently identified, only a small number of them were thoroughly studied, and 8-OHdG is the most certain considered. 8-OHdG appears in the DNA as a result of the damage by free radicals and can be extracted by repairing enzymes (8-hydroxy-2 -deoxyguanosine). If such repair does not occur, a base change (guanine for thymine) is induced during DNA replication. The cleaved 8-OHdG is then excreted in the urine [29]. However, not all 8-OHdG excreted in eliminated urine comes from oxidative stress to DNA and its subsequent repair; other factors like diet and cell death can modify such excretion. In the absence of these confounding factors, the urinary excretion of this metabolite should be attributed entirely to the repair of damaged DNA, so 8-OHdG can be considered as a marker for endogenous oxidative stress to DNA, a factor of initiation and promotion of carcinogenesis, and a risk factor for many diseases [30].
DHA and PUFA Incorporation to Erythrocyte
Analysis of the fatty acid profile in the erythrocyte membrane was analyzed as follows: 10-mL blood samples were taken from the antecubital vein 10 min before the physical tests in tubes with tripotassium EDTA (ethylenediamine tetraacetic acid) washed with isotonic saline and purged with nitrogen gas and centrifuged at 2000 rpm for 30 min at 4 • C. The supernatant was collected and frozen at −80 • C and the cell phase (erythrocytes) was frozen at −80 • C for fatty acid analysis. Erythrocyte lipids were determined as methyl esters after methylation reaction, using the method of Lepage and Roy [31]. GC analysis was performed on a Shimadzu GCMS-QP2010 Plus gas chromatograph/mass spectrometer (Shimadzu, Kyoto, Japan). Fatty acid methyl esters (FAMEs) were identified through mass spectrometry and through comparison of the elution pattern and relative retention times of FAMEs with the reference FAME mixture (GLC-744 Nu-Chek Prep. Inc., Elysian, MN, USA). The results were expressed in relative amounts (percentage molar of total fatty acids) [32]. The fatty acid methyl esters are analyzed by capillary gas chromatography with an ionized flame detector [33].
Statistical Analysis
Quantitative variables are described as the mean with standard deviation. This description was made for the total sample and was stratified by the randomized treatment arm. Qualitative variables are presented in tabular form, including the relative and absolute frequencies for the treatment groups and the global sample. Data were checked prior to analysis; in all cases, the Kolmogorov-Smirnov test was employed to test for normal distribution and Levene s test was used to test for homoscedasticity. The evolution of these quantitative variables was analyzed by parametric tests: a two-way repeated measures ANOVA test with one within-subject factor (product) and one between-subject factor (time) for the variables obtained in the SWEET endurance exercise test. For the post hoc group comparison, the Bonferroni test was employed. Statistical analysis was performed using SPSS software (v21.0, Chicago, IL, USA) and p values are reported for every group and group × time interaction; p < 0.05 is considered statistically significant.
Sample size was calculated according to the primary variable of 8-OHdG in urine. Taking into consideration the standard deviation of 105 ng of a previous study [34] and a desired precision of 100 ng, with an alpha risk of 5% and a statistical power of 80%, we required 14 participants in each group. We estimated a 10% dropout in each group, so a total of 15 participants per study arm was required.
Participant Flow Diagram and Baseline Characteristics
Fifty-six males were included in the final analysis (Figure 1). From the 75 participants randomized and balanced throughout the 5 groups (n = 15), a total of 19 participants dropped out: nine decided to voluntarily quit the study (no reason was provided), eight were lost in the follow-up, and one presented an adverse event (reflux), which was expected for the consumption of fish oils. Subjects completing the study according to the treatment group were 13 subjects (placebo), 10 subjects (350 mg/day rDHA), 11 subjects (1050 mg/day rDHA), 12 subjects (1750 mg/day rDHA), and 10 subjects (2450 mg/day rDHA).
Demographic characteristics of subjects are presented in Table 1. Either age (38.6 ± 9.8 years; mean ± SE), weekly training time and cycling (p = 0.961 and p = 0.518 respectively), training conditioning as per their estimated relative maximum oxygen consumption (p = 0.536), and weight (p = 0.867) were similar between groups. Therefore, we assumed homogeneity of the different study groups. Detailed information about the absolute and relative oxygen consumption can be found in Appendix A.
Physical Tests
All groups significantly increased the excretion of 8-OHdG after the first cycling test (placebo p < 0.001; 350 mg p = 0.008; 1050 mg p = 0.002; 1750 mg p < 0.001; 2450 mg p = 0.003), thus we assumed an effective exercise-induced oxidative stress among all groups by this physical test.
Environmental conditions (humidity and temperature) were similar between the two occasions (p = 0.985 for humidity and p = 0.265 for temperature) and along the different groups (p = 0.967 for humidity and p = 0.863 for temperature). Therefore, we assumed that environmental conditions were homogeneous over time between all groups.
The hydration assessment showed no significant differences on water consumption during the SWEET on each occasion (p = 0.454, for numerical data, see Appendix A). Not surprisingly, subjects in all groups suffered weight loss due to sweating after the test (p = 0.002) on each occasion, with a mean weight reduction of 0.9%. Additional information about the course of the heart rate during the trial can be found in Appendix A.
Nutritional Assessment
All groups had a similar intake for omega-3 (p = 0.651), omega-6 (p = 0.256), and PUFA (p = 0.770). The antioxidant micronutrients vitamin C (p = 0.905) and E (p = 0.794) and zinc (p = 0.927) as well as energy intake (p = 0.745) were also not different among groups. More details for each group can be found in Table 2.
Endogenous Antioxidant Effect of rDHA
Data shows a dose-response effect from rDHA against exercise-induced oxidative stress (Table 3
Main Finding: rDHA Reduces Constant-Load Medium-Intensity Aerobic Exercise-Induced Oxidative Stress Dose-Dependently
In the present study, we found that supplementation for 4 weeks (28 days) of a re-esterified DHA counteracted exercise-induced oxidative stress (generated by a constant-load aerobic exercise) from 1050 mg a day, in a dose-dependent manner, with a trend towards neutralization at the highest dose of 2450 mg. This finding can be useful to prescribe supplementation strategies that aim to decrease oxidative stress, in which a dose-dependent response is expected to occur. With this purpose, we also suggest taking into account the following factors: (a) Total cumulative dose (total grams during the treatment); (b) standardization (purity and total DHA content); (c) chemical structure and configuration of the product (triglyceride, etyl-esther or re-esterified form); and (d) days of treatment (since some adaptations may require structural conformation, which might not be accelerated by a higher daily dose).
To the best of our knowledge, this is the first trial in humans examining the dose-response effect of a re-esterified DHA against moderate-intensity exercise-induced oxidation. Contrary to our results, a previous study found that supplementation with omega-3 fish oil (400 mg of DHA and 2000 mg EPA/day for 6 weeks) increased exercise-induced oxidative stress plasma markers (as F2-isoprostanes [35]) in people engaged in routine cycling (about 1 h/day) after a 3-h cycling bout (at 57% of maximum watts) for three consecutive days [36]. In contrast, urinary F2-isoprostanes were reduced by omega-3 consumption in combination with aerobic exercise [20] either as fish oil (4 g/day of purified DHA or EPA, for 6 weeks) in mildly hyperlipemic overweight men without exercise prescription [37] or as dietary fish meals (3.6 g ω-3/day for 8 weeks) in type 2 diabetes mellitus, engaged in moderate (55-65% VO 2 max) or light (heart rate <100 bpm) exercise [38]. However, F2-isoprostanes derive solely from the oxidation of arachidonate [39], which are lipid peroxidation by-products, and therefore not a general marker of oxidative stress to cells (like 8-oxo-dG), as was the case in this experiment. Additionally, F2-isoprostanes along with other oxidation by-products of DHA promise further research due to its presumably anticancer properties [40], so interpretation of this marker as an undesirable outcome from oxidative stress should be carefully considered.
The product employed in this trial is different from regular fish oils, for instance, because it is predominantly DHA over EPA. One possible advantage of DHA as an antioxidant and compared to EPA is that it preferentially accumulates in mitochondrial cardiolipin [41] and into phospholipids of the cell membrane, where they can exert antioxidant properties, different to dietary antioxidants, which act as a barrier to external oxidation, which interacts with endogenous antioxidants to form a cooperative network of cellular antioxidants [42], thus providing a different antioxidant defense system. Apart from modifying tissue composition, DHA is capable of stimulating endogenous antioxidant systems by the activation of signaling pathways to upregulate intracellular endogenous antioxidants (MnSOD or SOD2: a mitochondrial form of SOD), probably as a side mechanism to protect the new cell's membrane composition more prone to oxidation due to double chains in PUFAs [43], which may also be a mechanism that contributes to an organism's antioxidant defense system. Achieving such a dose of DHA daily by dietary intake of fish products can be more difficult than taking a DHA food supplement regularly, especially in areas with low availability of fish products, and to a broader population if they are too expensive for them. Regular fish oils represent an alternative but usually lack standardization in their active components like DHA, which are subject to the natural variability of food by-products. To this respect, the re-esterified DHA product employed in this study can assure homogeneity in their composition and guarantee proper administration.
One limitation of the study was the low number of participants that were finally analyzed. This was due to a higher drop-out rate than the 10% we expected, which perhaps was too optimistic. Another limitation was the blinding of subjects since subjects receiving one softgel (350 mg group) would guess they belonged to the low-dose group, and those receiving seven softgels (2450 mg group) would guess they belonged to the high-dose group. Subjects did not belong to the same social group (i.e., same cycling team) so communication between participants to guess the number of softgels in the intermediate-and high-dose groups as well as the control product was partially impeded during the study. To overcome this problem, pill count-matched controls would be employed but would inevitably increase study cost.
Secondary Finding: DHA Is Incorporated to Erythrocytes Dose-Dependently
As a secondary finding, we found dose-dependent incorporation of rDHA to erythrocyte cells in all groups (starting from 350 mg/day) after 4 weeks of supplementation, which was paralleled by an increase in the overall omega-3 percentage, and a change in the omega-6/omega-3 ratio. This is in line with previous studies [44], which might be favored by the modified triacylglycerol composition of the product employed (re-esterified (rTAG), instead of the etyl-ester forms, more widely employed in the literature) partly due to placement of DHA in the second position (sn-2) of the triacylglycerol [45], which is the monoglyceride position preserved after pancreatic lipase action during the digestion process [21]. To this respect, a previous clinical trial, divided in two studies [46,47], found that 3 and 6 months of supplementation with rTAG increased the DHA and omega-3 index of the FA composition of erythrocytes in a more pronounced manner in the rTAG group compared to the etyl-ester (EE) group (and both compared to placebo). In the present study, we found this outcome with a shorter supplementation time (4 weeks). One explanation can be that our product was richer in DHA while the previous trial employed primarily EPA, which requires transformation to DHA by the enzyme omega-3 desaturase (which adds the additional double bond required to the C15) which showed variability in its conversion to DHA in previous clinical trials [48]. Previous studies found that DHA incorporation to other tissues is paralleled by incorporation of DHA to erythrocyte tissues [49,50], thus we assumed that the product employed effectively reached target tissues to exert antioxidant properties.
Contextualization of the Findings
The capacity of DHA to act as an antioxidant must be discussed in a broader sense to understand the current findings. As discussed earlier, blood plasma contains circulating compounds (like vitamins and polyphenols), which can exert antioxidant properties, acting as first barrier agents. In this study, we controlled and quantified dietary intake of these compounds, but as a limitation of the study, we did not measure plasma oxidation biomarkers or endogenous antioxidant enzymes to ascertain whether the antioxidant property of rDHA was due to a barrier effect in plasma or due to an enhancement of the intracellular endogenous antioxidant defense, which is the expected antioxidant effect of DHA [43]. Therefore, we examined the capacity of DHA to prevent oxidative stress to cells nonspecifically and should not be considered as the only strategy to increase antioxidant defenses against exercise-induced oxidation. Likewise, the model employed showed decreased oxidative stress in a specific condition with dietary control (limiting antioxidant and PUFA intake), in a narrow population sample (males engaged in regular cycling) subject to regular exercise-induced oxidative stress (with an adaptation of their endogenous oxidative defense system). The generalizability of these findings is limited and should not be assumed to be reproduced in a general population without control of dietary antioxidants or previous adaptation to exercise-induced oxidative stress by regular physical training. However, we think that the dose-response effect is expected to be reproduced in a broader population.
In a broader context, employment of exogenous antioxidants can interfere with exercise training-induced adaptations, which require signaling activation mediated by exercise-induced oxidative stress [51,52], to the point that some authors consider they have more harm than good in health and sports [53,54]. On the other hand, oxidative stress is increased without any benefit in some chronic diseases or physiopathological conditions, or as a consequence of their concomitant pharmacological treatment in which adjuvant treatment with exogenous antioxidants may be beneficial [55]. They conform a broad family of substances and molecules like vitamins [56] and polyphenols [57,58], which usually act as first barrier agents, while DHA seems to act as a pro-oxidant, which can increase endogenous enzymatic antioxidant defenses [9,10,14]. Both kinds of exogenous and endogenous antioxidant systems interact in a cooperative network of cellular antioxidants [42], with different underpinning antioxidative mechanisms acting at different sites in the body and cell. Therefore, recommendations about the employment of exogenous antioxidants and/or increasing of endogenous antioxidant systems and overall prevention of oxidative stress in sport and health require a thorough study in each circumstance, the discussion of which is extensive and out of the scope of this work.
Conclusions
Supplementation of re-esterified DHA exerts a dose-dependent antioxidant property against moderate-intensity long-duration aerobic exercise in physically active subjects when provided at a dose of at least 1050 mg a day for 4 weeks. Acknowledgments: A special dedication to José Antonio Villegas-García who passed away on 4 August 2020. He pioneered the research of DHA in our department and inspired the team to continue with the investigation until today. Special thanks to Antonio Martínez Garrido for his technical and administrative support. Thanks to all the staff of the Department of Exercise Physiology of the UCAM for their professional and technical support.
Conflicts of Interest:
The authors declare no conflict of interest. Table A2. Maximum oxygen consumption and oxygen consumption at ventilatory threshold 2 (absolute and relative to weight) for each of the study groups, expressed both as absolute (mL per minute) and relative to weight (mL per minute and kg). Values are presented as mean ± standard deviation. | v3-fos-license |
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} | pes2o/s2orc | Gcd Gene Diversity of Quinoprotein Glucose Dehydrogenase in the Sediment of Sancha Lake and Its Response to the Environment
Quinoprotein glucose dehydrogenase (GDH) is the most important enzyme of inorganic phosphorus-dissolving metabolism, catalyzing the oxidation of glucose to gluconic acid. The insoluble phosphate in the sediment is converted into soluble phosphate, facilitating mass reproduction of algae. Therefore, studying the diversity of gcd genes which encode GDH is beneficial to reveal the microbial group that has a significant influence on the eutrophication of water. Taking the eutrophic Sancha Lake sediments as the research object, we acquired samples from six sites in the spring and autumn. A total of 219,778 high-quality sequences were obtained by DNA extraction of microbial groups in sediments, PCR amplification of the gcd gene, and high-throughput sequencing. Six phyla, nine classes, 15 orders, 29 families, 46 genera, and 610 operational taxonomic units (OTUs) were determined, suggesting the high genetic diversity of gcd. Gcd genes came mainly from the genera of Rhizobium (1.63–77.99%), Ensifer (0.13–56.95%), Shinella (0.32–25.49%), and Sinorhizobium (0.16–11.88%) in the phylum of Proteobacteria (25.10–98.85%). The abundance of these dominant gcd-harboring bacteria was higher in the spring than in autumn, suggesting that they have an important effect on the eutrophication of the Sancha Lake. The alpha and beta diversity of gcd genes presented spatial and temporal differences due to different sampling site types and sampling seasons. Pearson correlation analysis and canonical correlation analysis (CCA) showed that the diversity and abundance of gcd genes were significantly correlated with environmental factors such as dissolved oxygen (DO), phosphorus hydrochloride (HCl–P), and dissolved total phosphorus (DTP). OTU composition was significantly correlated with DO, total organic carbon (TOC), and DTP. GDH encoded by gcd genes transformed insoluble phosphate into dissolved phosphate, resulting in the eutrophication of Sancha Lake. The results suggest that gcd genes encoding GDH may play an important role in lake eutrophication.
Introduction
In the process of growth and reproduction of phosphate-solubilizing microorganisms in water environments, some organic acids are produced and secreted, reducing the environmental pH, transforming insoluble phosphate into dissolved phosphate, promoting the growth and development of cyanobacteria, and causing eutrophication of water [1]. Among the dissolving phosphoric acids produced by microorganisms, gluconic acid (GA) is the most important and main
Site Description and Sample Collection
Situated in Tianfu New District of Chengdu, Sichuan Province, Sancha Lake is located at 104 • 11 16 to 104 • 17 16 east longitude and 30 • 13 08 to 30 • 19 56 north latitude, with an average depth of 8.3 m and a maximum depth of 32.5 m. This region is in the subtropical humid monsoon climate zone, with an average annual temperature of 15.2-16.9 • C and average annual precipitation of 786.5 mm. The water source of Sancha Lake is mainly from the Min River, accounting for about 80% of the total water volume of the reservoir, and about 20% comes from rainfall and two creeks. The area of the basin above the dam site is 161.25 km 2 . Sancha Lake is a vital source of drinking water. According to the monitoring results over years, the inflow of COD Cr and BOD 5 into Sancha Lake shows a downward trend year by year for the control of external pollutants. However, the total nitrogen (TN) and TP of the water body has been on an upward trend. The corresponding chlorophyll increases on a yearly basis, while the transparency decreases year by year, and the water has been eutrophicated [10]. Sancha Lake eutrophication might be attributed to insoluble phosphate transformed to soluble phosphate by gcd-harboring bacterial communities.
According to the characteristics of sediment distribution and eutrophication of Sancha Lake, six sampling sites were selected, as shown in Figure 1. The longitude and latitude of the sampling sites were determined by GPS. The characteristics information of all sampling sites is presented in Table 1. At each sampling site, there were three sampling points. In April (spring) and November (autumn) 2017, a claw-like Peterson dredge was used to capture surface sediments at each sampling point ( Figure 2). The surface (0-5 cm) sediment was collected by a plexiglass column and put into a clean, sealed polythene bag [11]. Three parallel samples were collected at each sample point and mixed as the representative sample of the sample point. Some sediments were stored at 4 • C for physical and chemical analysis (within 24 h), and some sediment samples were stored at −80 • C for DNA extraction. At the same time, an air-tight water sampler was used to collect the overlying water above the sediment layer at each sampling point for the water environment index analysis [11]. were determined by GPS. The characteristics information of all sampling sites is presented in Table 1. At each sampling site, there were three sampling points. In April (spring) and November (autumn) 2017, a claw-like Peterson dredge was used to capture surface sediments at each sampling point ( Figure 2). The surface (0-5 cm) sediment was collected by a plexiglass column and put into a clean, sealed polythene bag [11]. Three parallel samples were collected at each sample point and mixed as the representative sample of the sample point. Some sediments were stored at 4 °C for physical and chemical analysis (within 24 h), and some sediment samples were stored at −80 °C for DNA extraction. At the same time, an air-tight water sampler was used to collect the overlying water above the sediment layer at each sampling point for the water environment index analysis [11].
Characterization of Sediment Physicochemical Properties
Several morphological grading experiments of phosphorus in sediments were performed using the Standards Measurements and Testing Program of the European Commission (SMT) [12,13]. Determination of TP, inorganic phosphorus (IP), organic phosphorus (OP), phosphonium hydroxide (NaOH-P), and HCl-P in sediments was conducted, and the content of different forms of phosphorus was determined by ammonium molybdate spectrophotometry [14]. Total organic carbon in sediments (determination of total organic carbon in sedimentary rocks) was determined by the Chinese method GB/T 19145-2003 [15]. The determination of total nitrogen was determined by alkaline potassium persulfate digestion by UV spectrophotometry [16]. Ammonium nitrogen content was measured with the extraction of potassium chloride solution spectrophotometric method [17]. The determination method of overlying water DTP was as follows: the content of phosphorus was determined by the ammonium molybdate spectrophotometric method [14] after the water was filtered by a 0.45-μm removal filter membrane, the pH value and temperature was measured by the portable multiparameter temperature meter HI991301, and the dissolved oxygen was gauged by the HQ3OD portable dissolved oxygen meter.
DNA Extraction, PCR, and Illumina MiSeq Sequencing
The total DNA was extracted after centrifugation to remove excess water from the sediment. The centrifugal sediment samples were then added to a Power Bead Tube following the steps according to the manufacturer with adjustment only for shock duration. The concentration and purity of DNA was analyzed with NanoDrop2000. The DNA extracts were purified with a Wizard DNA Clean-Up System (Axygen Biosciences, Union City, CA, USA), as recommended by the manufacturer. The DNA was stored at −80 °C until analysis. The GDH-encoding gene gcd was amplified by PCR from DNA using gcd-FWCGGCGTCATCCGGGSITIYRAYRT and gcd-RW
Characterization of Sediment Physicochemical Properties
Several morphological grading experiments of phosphorus in sediments were performed using the Standards Measurements and Testing Program of the European Commission (SMT) [12,13]. Determination of TP, inorganic phosphorus (IP), organic phosphorus (OP), phosphonium hydroxide (NaOH-P), and HCl-P in sediments was conducted, and the content of different forms of phosphorus was determined by ammonium molybdate spectrophotometry [14]. Total organic carbon in sediments (determination of total organic carbon in sedimentary rocks) was determined by the Chinese method GB/T 19145-2003 [15]. The determination of total nitrogen was determined by alkaline potassium persulfate digestion by UV spectrophotometry [16]. Ammonium nitrogen content was measured with the extraction of potassium chloride solution spectrophotometric method [17]. The determination method of overlying water DTP was as follows: the content of phosphorus was determined by the ammonium molybdate spectrophotometric method [14] after the water was filtered by a 0.45-µm removal filter membrane, the pH value and temperature was measured by the portable multiparameter temperature meter HI991301, and the dissolved oxygen was gauged by the HQ3OD portable dissolved oxygen meter.
DNA Extraction, PCR, and Illumina MiSeq Sequencing
The total DNA was extracted after centrifugation to remove excess water from the sediment. The centrifugal sediment samples were then added to a Power Bead Tube following the steps according to the manufacturer with adjustment only for shock duration. The concentration and purity of DNA was analyzed with NanoDrop2000. The DNA extracts were purified with a Wizard DNA Clean-Up System (Axygen Biosciences, Union City, CA, USA), as recommended by the manufacturer. The DNA was stored at −80 • C until analysis. The GDH-encoding gene gcd was amplified by PCR from DNA using gcd-FWCGGCGTCATCCGGGSITIYRAYRT and gcd-RW GGGCATGTCCATGTCCCAIADRTCRTG [9]. The amplicon size was 330 bp. Each sample involved three technical replicates for gcd gene amplification, which was carried out in TaKaRa Taq™ Hot Start Version (New England Biolabs, Ipswich, MA, USA). Indexing PCR was performed in 25-µL reactions containing: 2.5 µL buffer (10×), 2 µL 2.5 mMdNTPs, 2.5 µL forward primer (5 µM), 2.5 µL reverse primer (5 µM), 0.3 µL rTaq polymerase, 10 ng template DNA, and ddH 2 O added to bring to volume. The amplification procedure included an initial denaturation step (95 • C; 12 min), 10 cycles of denaturation (95 • C, 30 s; 65 • C, 1 min; 72 • C, 30 s), 40 cycles of annealing (95 • C, 30 s; 54 • C, 1 min; 72 • C, 30 s), and elongation (72 • C; 5 min).
PCR products were recovered using 2% agarose gel and purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Then, Tris-HCl elution and 2% agarose gel electrophoresis were conducted. Quantitative detection was performed by QuantiFluor™-ST (Promega, Madison, Wisconsin, USA). The purified amplified fragments were constructed according to the standard operating procedures of Illumina MiSeq platform (Illumina, San Diego, CA, USA). Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) was used for the sequencing using an Illumina MiSeq platform PE300.
The Diversity and Composition of gcd-Harboring Bacterial Communities Were Assessed Using High-Throughput Sequencing Technique
The original sequencing sequence used Trimmomatic software for quality control and adopted FLASH (version 2.7, http://ccb.jhu.edu/software/FLASH/, Center for Bioinformatics and Computational Biology, Iowa City, IA, USA) for sequence assembly. Operational taxonomic unit (OTU) clustering was performed at 97% similarity by UPARSE software (version 7.1 http://drive5.com/uparse/, Edgar, R.C., Tiburon, CA, USA), and the single sequence and chimera were removed in the process of clustering. The sequence with the highest abundance in each OTU was annotated for species classification in the NCBI database (http://www.ncbi.nlm.nih.gov/) by RDP classifier (http://rdp.cme.msu.edu/). The comparison threshold was 70%. R software was used to conduct analysis and mapping of the community structure according to the taxonomic information corresponding to each OTU.
Based on the results of OTU clustering analysis, the coverage, Chao1, and Shannon's diversity index [18] were calculated for each sample by using QIIME software (version 2.0, http://qiime.org/, Rob Knight Lab, Boulder, CO, USA). According to the total sequence number in the OTU abundance matrix, the rarefaction curve [19] was plotted using QIIME software, to compare the richness of the microbial community in the sediment samples.
To investigate the similarity of community structure among different samples, the community data structure was naturally decomposed by using QIIME software and R software through principal coordinates analysis (PCoA) [20] and the unweighted pair group method with arithmetic mean (UPGMA) [21]. The sample ordination was sequenced, to clarify the time and space differences in diversity and composition of gcd in sediment samples.
Statistical Analysis
Variance inflation factor (VIF) was used to screen the environmental factors to obtain those factors uncorrelated with each other [22]. Pearson correlation analysis was performed to determine whether significant correlations existed between gcd gene richness and the abovementioned environmental factors, and between gcd diversity and the abovementioned environmental factors. Analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA) for Windows. Significance levels were set at p = 0.05 and extreme significance levels at p = 0.01 in all statistical analyses. Canonical correlation analysis (CCA) was applied to evaluate the effect of environmental factors on the gcd-harboring bacterial community structure and was carried out with R language vegan package [23].
Physicochemical Properties of the Sediments and Overlying Water
The determination of physical and chemical factors of the sediments and overlying water in the spring and summer are shown in Table 2. As can be seen from Table 2, the pH of the overlying water showed little change among different seasons and different sampling locations. The temperature of the overlying water showed little difference between spring and summer for water stratification in Sancha Lake. However, the temperature was higher in L6, at the intake water area; L2, at the tail water area; and L4, near the area with intense human activity. DO was higher in the spring than in summer for all locations. In space, DO was higher in shallow water areas such as L6, L1, L2, and L4. DO in the dam was the lowest for deep water. The change trend of DTP was consistent with DO. TOC content in sediments showed little difference between spring and autumn. TN content in the spring was a little higher than in autumn, but the change of NH 3 -N content was the opposite. The contents of TP, IP, OP, HCl-P, and NaOH-P were all higher in the spring than in autumn. In space, all environmental factors except NH 3 -N were related with the intensity of human activity. The contents of TP, IP, OP, HCl-P, and NaOH-P were the highest at L1 and L3, in highly dense enclosure cultures; second highest at L5, in a dense enclose culture; and at L4, near an intense human activity area. The lowest was at L6 in the incoming water area. Table 2. Physicochemical properties of the sediments and overlying water in spring and autumn.
Season
Location
OTU Classification and Determination and Alpha Diversity of the gcd Gene in Sediments
Total DNA was extracted from the microorganisms in sediment samples, and gcd gene sequences were acquired by PCR amplification. A total of 219,778 high-quality sequences were obtained by high-throughput sequencing, with an average length of 319 bp. The rarefaction curve of the sediment samples tended to be flat, indicating that the sequencing data volume was reasonable. Figure 3 shows the rarefaction curve diagram. Divided by 97% similarity, 610 OTUs were obtained. Table 3 shows that the coverage of each sample library was 99.58-99.94%, suggesting that the probability of gcd gene sequence detection in the sediment was very high. The sequencing results can represent different types of gcd genes and population types in the sediment samples of Sancha Lake.
OTU Classification and Determination and Alpha Diversity of the gcd Gene in Sediments
Total DNA was extracted from the microorganisms in sediment samples, and gcd gene sequences were acquired by PCR amplification. A total of 219,778 high-quality sequences were obtained by high-throughput sequencing, with an average length of 319 bp. The rarefaction curve of the sediment samples tended to be flat, indicating that the sequencing data volume was reasonable. Figure 3 shows the rarefaction curve diagram. Divided by 97% similarity, 610 OTUs were obtained. Table 3 shows that the coverage of each sample library was 99.58-99.94%, suggesting that the probability of gcd gene sequence detection in the sediment was very high. The sequencing results can represent different types of gcd genes and population types in the sediment samples of Sancha Lake. The Chao1 index was used to estimate the total number of OTUs contained in the samples, reflecting the abundance of the gcd-harboring bacterial community. The larger Chao1, the higher abundance of the gcd-harboring bacterial community. Shannon's index reflects the alpha diversity index of the bacterial community. The larger the Shannon value, the higher diversity of the bacterial community. There were spatial and temporal differences in the abundance and diversity of the gene gcd in the sediment of Sancha Lake ( Table 3). The gcd-harboring bacterial community abundance (OTUs, Chao1) and diversity index (Shannon) were both higher in the spring than in autumn. The abundance of gcd-harboring communities was not exactly consistent with the sequence of diversity in the sampling sites in the spring and autumn. The highest abundance and diversity of gcd was at L6, in the incoming water area, and the lowest was at L3, in the center of the lake. In general, from the lake center to the lake dam and lake end, the biodiversity index and abundance of gcd showed an upward trend. The biodiversity and abundance of gcd at seriously polluted sampling points were even lower. The gcd biodiversity index in the spring was higher than that in the autumn. The Chao1 index was used to estimate the total number of OTUs contained in the samples, reflecting the abundance of the gcd-harboring bacterial community. The larger Chao1, the higher abundance of the gcd-harboring bacterial community. Shannon's index reflects the alpha diversity index of the bacterial community. The larger the Shannon value, the higher diversity of the bacterial community. There were spatial and temporal differences in the abundance and diversity of the gene gcd in the sediment of Sancha Lake ( Table 3). The gcd-harboring bacterial community abundance (OTUs, Chao1) and diversity index (Shannon) were both higher in the spring than in autumn. The abundance of gcd-harboring communities was not exactly consistent with the sequence of diversity in the sampling sites in the spring and autumn. The highest abundance and diversity of gcd was at L6, in the incoming water area, and the lowest was at L3, in the center of the lake. In general, from the lake center to the lake dam and lake end, the biodiversity index and abundance of gcd showed an upward trend. The biodiversity and abundance of gcd at seriously polluted sampling points were even lower. The gcd biodiversity index in the spring was higher than that in the autumn. RDP classifier was used to annotate the sequence with the highest abundance in each OTU, and the corresponding taxonomic information of each OTU was obtained by comparison with the NCBI database. In the sediment samples collected from Sancha Lake, a total of 46 genera of the gcd community with confirmed classification information were detected, which were subordinate to 29 families, 15 orders, nine classes, and six phyla. Compared with the NCBI database, there were still about 30,000 sequences that had no clear classification information, and about 20% of the gcd-harboring bacterial communities could not be identified. Therefore, the diversity of gcd-harboring bacterial communities in Sancha Lake sediments may actually be higher, containing many new species which require further exploration.
Bergkemper [9] et al. obtained six phyla, 17 orders, and 24 families by amplifying gcd from German forest soil samples. Rhizobiales, Rhodospirillales, and Burkholderiales (4%) were the dominant orders. Rodriguez found that Burkholderiales is an important inorganic phosphorus-soluble phosphate bacterial group [24]. In this study, six phyla, 15 orders, and 29 families were obtained. Rhizobiales, Oceanospirillales, and Burkholderiales (11.08%) were the dominant orders. At the family classification level, the functional bacteria of Burkholderiales, with the function of dissolving mineral phosphate, were more abundant and played an important role in the Sancha Lake eutrophication.
Comparison of gcd-Harboring Bacterial Community Composition Based on Classification Level
Six phyla of gcd-harboring bacterial communities were identified including Proteobacteria, Acidobacteria, Gemmatimonadetes, Planctomycetes, Bacteroidetes, and Verrucomicrobia. The relative abundance ratio greater than 1% was Proteobacteria (98.85-25.10%) and Acidobacteria (0-3.99%). Most reads came from Proteobacteria, which was the dominant group of gcd gene sources in sediments of the Sancha Lake. The reads of Proteobacteria were detected in each sample in two seasons and the number of ordinal reads in the spring was higher than that in autumn. Acidobacteria was the second dominant group, but was detected only at L1, L2, and L6 in the spring. Gcd-harboring bacterial community composition and the relative abundance in phylum level are shown in Figure 4. Please note that there were two unidentified taxa at the phylum level in the sediment samples. The abundance of one of them was 1.15-71.26%, and the number of reads was only second to Proteobacteria. It was detected in each sample in the spring and autumn and the number of reads in autumn was higher than that in spring. It was the dominant phylum but could not be identified. The other unidentified one was detected only at L6 in the spring, with an abundance of 0.35%. These two unidentified taxa indicated that there were many new species in the bacterial community of Sancha Lake sediments.
Twenty-one of 46 identified genera had more than 1% relative abundance. The distribution of relative abundance of more than 1% at the level of genera is shown in Figure 5. Rhizobium (1.63-77.99%), Ensifer (0.13-56.95%), Shinella (0.32-25.49%), and Sinorhizobium (0.16-11.88%) were detected in each sample from all locations both in spring and autumn. Their relative abundance was higher in the spring than in autumn and higher at L1, L2, and L4, where eutrophication was heavy. It is supposed that these bacteria have great effect on lake eutrophication. The relative abundance of the four genera was significantly different at all sites. Agrobacterium (0-72.92%) was detected at all sites except L3, which was in a deep-water area both in the spring and autumn, and its relative abundance was high. Enterobacter (0-1.12%) was detected at all sites only in the spring, and Paracoccus (0-1.15%) was detected at all sites only in autumn.
The distribution of gcd-harboring bacterial communities was different in different seasons and different sediment types. The diversity of community composition from phyla to genera increased gradually, but the difference of population from phyla to genera was not significant. The dominant bacterial communities with higher relative abundance could be detected in different seasons and sediment types. The abundance of gcd-harboring bacteria was higher at heavy eutrophication sites. Some bacterial communities were unique in time or space. Twenty-one of 46 identified genera had more than 1% relative abundance. The distribution of relative abundance of more than 1% at the level of genera is shown in Figure 5. Rhizobium (1.63-77.99%), Ensifer (0.13-56.95%), Shinella (0.32-25.49%), and Sinorhizobium (0. .88%) were detected in each sample from all locations both in spring and autumn. Their relative abundance was higher in the spring than in autumn and higher at L1, L2, and L4, where eutrophication was heavy. It is supposed that these bacteria have great effect on lake eutrophication. The relative abundance of the four genera was significantly different at all sites. Agrobacterium (0-72.92%) was detected at all sites except L3, which was in a deep-water area both in the spring and autumn, and its relative abundance was high. Enterobacter (0-1.12%) was detected at all sites only in the spring, and Paracoccus (0-1.15%) was detected at all sites only in autumn. The distribution of gcd-harboring bacterial communities was different in different seasons and different sediment types. The diversity of community composition from phyla to genera increased gradually, but the difference of population from phyla to genera was not significant. The dominant bacterial communities with higher relative abundance could be detected in different seasons and sediment types. The abundance of gcd-harboring bacteria was higher at heavy eutrophication sites. Some bacterial communities were unique in time or space.
Analysis of the Composition of gcd-Harboring Bacterial Communities Based on OTU Level
Clustering and sequencing were performed according to the abundance distribution of OTU or
Analysis of the Composition of gcd-Harboring Bacterial Communities Based on OTU Level
Clustering and sequencing were performed according to the abundance distribution of OTU or the similarity between samples. R software was used for cluster analysis and drawing a heatmap diagram of the top 30 OTUs. By clustering, high-and low-abundance OTUs could be distinguished. The similarity and difference of community composition between samples is reflected by the color gradient. Red indicates high out abundance, while blue indicates low OTU abundance, which directly reflects the similarity and difference in the composition of the bacterial community with high abundance in the sediment of Sancha Lake.
Heatmap analysis of gcd-harboring bacterial community composition is shown in Figure 6. It can be seen from the gradient change of color that the high-abundance gcd-harboring bacterial communities from L1, L2, L3, and L5 in spring and autumn are in one group, indicating that the seasonal change had little influence on the high-abundance gcd-harboring bacterial communities. However, gcd-harboring bacterial communities from L4 and L6 in the spring and autumn did not gather in the same group, and the composition of the high-abundance gcd-harboring bacterial community had obvious seasonal variation. This suggests that seasonal variation has a great impact on the distribution of the high-abundance gcd-harboring bacterial community of L4 and L6. In space, it can be seen that the highly enriched gcd-harboring bacterial communities in the sediments of the six sampling sites did not cluster with each other. The spatial differences of the bacterial community showed significant changes, indicating that the sampling site type had a great influence on the high abundance of gcd-harboring bacterial communities, resulting in the heterogeneous degree of Sancha Lake eutrophication.
Comparative Analysis of gcd-Harboring Bacterial Community Composition Based on Beta Diversity
The distance matrices of weighted and unweighted UniFrac were calculated by QIIME, and UPGMA and PCoA were analyzed, using R language, as a picture tree to further evaluate the similarity and difference of the composition of gcd-harboring bacterial communities in different seasons and sample sites of sediment in Sancha Lake. As can be seen from Figure 7 and 8, for seasonal change, the gcd-harboring bacterial communities from L1 to L3 and L5 in the spring and autumn have their branches or gather in the same quadrant. Regardless of the season, they all came together, indicating that seasonal variation had little effect on the difference of the composition of gcd-harboring bacterial communities in these four sampling sites.
The gcd-harboring bacterial communities from L4 and L6 changed greatly in the spring and autumn, forming different branches or not being in the same quadrant, respectively. Seasonal variation had a great influence on the difference in the composition of the gcd-harboring bacterial communities in L4 and L6. This may be attributed to the varying intensity of human activity. L6 was in the incoming water area and L4 was near an area of intense human activity. In the process of spatial change of each sampling site, three groups formed. Among them, L2 formed a group, L1 and L3 constituted a group, and L5 made up another group. As the seasons changed, L4 and L6 were
Comparative Analysis of gcd-Harboring Bacterial Community Composition Based on Beta Diversity
The distance matrices of weighted and unweighted UniFrac were calculated by QIIME, and UPGMA and PCoA were analyzed, using R language, as a picture tree to further evaluate the similarity and difference of the composition of gcd-harboring bacterial communities in different seasons and sample sites of sediment in Sancha Lake. As can be seen from Figures 7 and 8, for seasonal change, the gcd-harboring bacterial communities from L1 to L3 and L5 in the spring and autumn have their branches or gather in the same quadrant. Regardless of the season, they all came together, indicating that seasonal variation had little effect on the difference of the composition of gcd-harboring bacterial communities in these four sampling sites.
The gcd-harboring bacterial communities from L4 and L6 changed greatly in the spring and autumn, forming different branches or not being in the same quadrant, respectively. Seasonal variation had a great influence on the difference in the composition of the gcd-harboring bacterial communities in L4 and L6. This may be attributed to the varying intensity of human activity. L6 was in the incoming water area and L4 was near an area of intense human activity. In the process of spatial change of each sampling site, three groups formed. Among them, L2 formed a group, L1 and L3 constituted a group, and L5 made up another group. As the seasons changed, L4 and L6 were located in them, which was consistent with the characterization of the relatively high-abundance heatmap.
Based on the analysis of the composition of gcd-harboring bacterial communities from the classification level, OTU level, and beta diversity, it was found that the diversity and composition of gcd-harboring bacterial communities in the sediments of Sancha Lake presented obvious temporal and spatial changes. The diversity and abundance of the gcd-harboring bacterial communities in spring were higher than that in autumn, but the difference of the same sample site was different with seasonal changes. In space, because L6 was in the main water entry area of the lake, the water was highly fluid. By contrast, L1 and L3 were in the relatively concentrated area of fenced breeding. L2 was in the tail water area of the reservoir, L5 was in the dense area of cage breeding, and L4 was in the adjacent area of intensive human activities. The variations of time and place of feeding in the Sancha Lake resulted in different pollution zones at the bottom of the lake. Furthermore, the difference in the degree of human activity interference and water flow affected the difference in the composition of gcd-harboring bacterial communities. The environmental factors with VIF greater than 10 were filtered out by the VIF method, and multiple screenings were conducted until all the corresponding VIF values of the selected environmental factors were less than 10. Pearson correlation analysis was performed for environmental factors and showed small interactions after screening; the correlation analysis between the two factors is shown in Table 4. The results showed that the Simpson index of gcd genes was significantly negatively correlated with the concentration of TOC, TP, and HCl-P (p < 0.05) and was extremely significantly positively correlated with the concentration of DTP (p < 0.01). The Chao1 index was significantly negatively correlated with the concentration of TOC, TN, TP, and HCl-P (p < 0.05) and was positively correlated with DTP concentration (p < 0.05). Changes in effective reads were significantly positively correlated with DTP concentration (p < 0.05). The number of observed OTUs had a significant negative correlation with the change of T (p < 0.05) and showed a significant positive correlation with DTP concentration (p < 0.01). In general, the diversity and abundance of gcd genes were not strongly correlated with pH and T indicators in water and were closely related to physical and chemical factors such as the concentrations of DO, TOC, TN, TP, and DTP.
Correlation between Spatial Distribution Characteristics of gcd-Harboring Bacterial Communities and Environmental Conditions
Based on the OTU level, the correlation between the spatial distribution of gcd genes in sediments and environmental conditions was analyzed by RDA/CCA using rda or cca in the vegan package of R language. In the discriminant component analysis (DCA) by species-sample database, the length of the first axis of lengths of gradient was greater than 4.0. Thus, CCA (Jari, k., University of Oulu at Oulu, Finland) was selected to analyze the correlation between the gcd group and environmental factors, to clarify the correlation between the spatial and temporal distribution of the gcd-harboring bacterial communities in the sediments of Sancha Lake and environmental factors. Environmental factors were screened by the VIF method, and CCA analysis was conducted at the OTU level. The analysis results are shown in Figure 9.
The resolution of the X-axis and Y-axis were 15.83% and 13.72%, respectively. The physical and chemical factors of the sediments are represented by a line with an arrow, and the length of the ray represents the degree of influence of the factor on the community OTU. The quadrant in which the arrow is located represents the positive and negative correlations between the factor and the sorting axis, and the angle between the arrow line and the sorting axis represents the correlation between the factor and the sorting axis. When the angle between physicochemical factors of sediment is acute, it indicates that the two environmental factors are positively correlated, while a pure angle means they are negatively correlated.
Among all samples in two seasons, the distribution of samples was relatively dispersed, and the influence of environmental factors did not make the spatial and temporal distribution of sample sites show obvious regularity. However, in terms of the overall relationship between OTU and environmental factors, both TOC and HCl-P had long on-lines on the one and two sequencing axes, and the results show that the influence of sediment TOC and HCl-P on the OTU of the whole gcd-harboring bacterial community was very significant, and they had an extremely significantly negative correlation (p < 0.01). The change of the overlying water DTP content was positively correlated with the OTU of the whole gcd-harboring community.
Zeng Qingwei et al. found that the phosphorus-soluble activity of the inorganic phosphorus-soluble phosphor bacteria and the content of soluble phosphorus (DTP) in the environment influence each other [7] and thus speculated that the change of DTP content in the overlying water of Sancha Lake was partly caused by the secretion of organic acids by the gcd-harboring bacterial community to dissolve inorganic phosphorus. The release of DTP in the overlying water indicates that the gcd-harboring bacterial community in sediments has a certain effect on the eutrophication of water. HCl-P and NaOH-P were negatively correlated with OTUs of gcd-harboring bacterial communities. Compared with NaOH-P and HCl-P, HCl-P was more significantly correlated, indicating that the gcd-harboring bacterial community may give priority to HCl-P. Among the three physical factors of DO, T, and pH, DO had a significantly positive correlation with OTU spatial and temporal distribution of the gcd-harboring bacterial community, while T and pH had no significant correlation. effect on the eutrophication of water. HCl-P and NaOH-P were negatively correlated with OTUs of gcd-harboring bacterial communities. Compared with NaOH-P and HCl-P, HCl-P was more significantly correlated, indicating that the gcd-harboring bacterial community may give priority to HCl-P. Among the three physical factors of DO, T, and pH, DO had a significantly positive correlation with OTU spatial and temporal distribution of the gcd-harboring bacterial community, while T and pH had no significant correlation.
Conclusions
A total of 219,778 high-quality sequences were obtained from sediment samples of Sancha Lake by DNA extraction, gene gcd sequence amplification with PCR, and Illumina MiSeq platform sequencing. The gcd-harboring bacterial communities with confirmed classification information were composed of six phyla, nine classes, 15 orders, 29 families, and 46 genera. The gcd genes had a higher diversity. About 20% of the gcd-harboring bacterial communities could not be identified. Therefore, the genetic gcd diversity in the sediments of Sancha Lake may actually be higher and many new species may exist. From the center of the lake to the dam, and then to the end of the lake, the biodiversity index and abundance showed an upward trend. The gcd diversity index and
Conclusions
A total of 219,778 high-quality sequences were obtained from sediment samples of Sancha Lake by DNA extraction, gene gcd sequence amplification with PCR, and Illumina MiSeq platform sequencing. The gcd-harboring bacterial communities with confirmed classification information were composed of six phyla, nine classes, 15 orders, 29 families, and 46 genera. The gcd genes had a higher diversity. About 20% of the gcd-harboring bacterial communities could not be identified. Therefore, the genetic gcd diversity in the sediments of Sancha Lake may actually be higher and many new species may exist. From the center of the lake to the dam, and then to the end of the lake, the biodiversity index and abundance showed an upward trend. The gcd diversity index and abundance in the seriously polluted sampling sites were even lower. The bacterial diversity index of gcd in the spring was higher than that in autumn. The abundance of these dominant gcd-harboring bacteria, such as Rhizobium, Ensifer, Shinella, and Sinorhizobium, were higher in the spring than in autumn, suggesting that they have an important effect on the eutrophication of Sancha Lake. The major environmental factors affecting gcd gene diversity and gcd-harboring bacterial community composition were determined to be DO, TOC, TN, TP, HCl-P, and DTP through Pearson correlation analysis and CCA analysis. The results indicated that GDH encoded by gcd genes catalyzed the oxidation of glucose into gluconic acid, resulting in insoluble phosphate transformation into soluble phosphate in the sediments. DTP concentration in overlying water increased and accelerated the eutrophication of Sancha Lake. Thus, gcd genes encoding GDH play an important role in lake eutrophication.
Author Contributions: Y.L. conceived and designed the experiments and wrote the paper; J.Z. and W.X. performed the experiments and contributed reagents and analysis tools; Z.G. and Z.M. analyzed the data.
Funding: This research was funded by the Sichuan science and technology support project (2018GZ0416). | v3-fos-license |
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} | pes2o/s2orc | Metabolism the Difficile Way: The Key to the Success of the Pathogen Clostridioides difficile
Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios.
CLOSTRIDIOIDES (CLOSTRIDIUM) DIFFICILE
Clostridioides (Clostridium) difficile (Lawson et al., 2016) was discovered in 1935 as a commensal of healthy newborns (Hall and O'Toole, 1935). It was only in the late 1970s that C. difficile was recognized as a severe pathogen, responsible for antibiotic-related pseudomembranous colitis (Bartlett et al., 1978). In the last 20 years, an emerging number of nosocomial and communityacquired infections with symptoms ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon was documented (Bartlett, 2006;Rupnik et al., 2009;Knight et al., 2015;Lessa et al., 2015). Major risk factors are antibiotic therapy, age and immunosuppression (Bignardi, 1998). The symptoms including severe intestinal damage are believed to be mainly caused by the two large clostridial toxins A (TcdA) and B (TcdB) and the binary toxin Cdt (Carter et al., 2010;Carman et al., 2011). In vitro, the toxins are predominantly produced in the stationary phase. Toxin production directly depends on the metabolic state of C. difficile (Karlsson et al., 2008;Neumann-Schaal et al., 2015;Hofmann et al., 2018). The number of genome-sequenced C. difficile strains with short-read sequences has increased up to 7000 (https://enterobase.warwick.ac.uk/, Alikhan et al., 2018), while the number of closed genomes remains at about 50 (e.g. Sebaihia et al., 2006;He et al., 2010;Riedel et al., 2017). Currently, several high quality annotations of the C. difficile genome are available, which serve as a solid basis for a systematic investigation of the transcriptome, proteome, metabolome, and for the construction of genome-scale metabolic models (Sebaihia et al., 2006;Monot et al., 2011;Larocque et al., 2014;Pettit et al., 2014;Dannheim et al., 2017a,b;Jenior et al., 2017;Kashaf et al., 2017).
FERMENTATION PATHWAYS Stickland Metabolism
C. difficile harbors multiple pathways to utilize amino acids and sugars as energy sources (Mead, 1971;Elsden et al., 1976;Elsden and Hilton, 1979). The fermentation of amino acids via the so-called Stickland pathway occurs in three stages that ultimately couple the oxidation and reduction of amino acids to the formation of ATP (Stickland, 1934;Nisman, 1954). The first step is the transamination of an amino acid to its corresponding 2-oxo-acid (O'Neil and DeMoss, 1968;Barker, 1981) which yields NADH once coupled to the glutamate dehydrogenase reaction (Figure 1). In addition, serine, threonine, methionine, and cysteine are subject to deamination (Hofmeister et al., 1993;Morozova et al., 2013). The second part is either an oxidative or a reductive pathway. In the oxidative pathway, the formed 2-oxo acid gets oxidized by ferredoxin with the formation of a CoA thioester and the release of CO 2 (Mai and Adams, 1994;Heider et al., 1996;Lin et al., 2015). The final steps of the pathway (encoded by vorCBA, iorBA) include the cleavage of the CoA thioester with subsequent ATP formation (Nisman, 1954;Valentine and Wolfe, 1960;Cary et al., 1988;Wiesenborn et al., 1989;Musfeldt and Schönheit, 2002). In the reductive pathway, the 2-oxo acid is reduced employing NADH with the formation of a 2-hydroxy acid (Hetzel et al., 2003;Martins et al., 2005;Kim et al., 2006). The dehydratation to enoyl-CoA (Dickert et al., 2002;Kim et al., 2004Kim et al., , 2005Kim et al., , 2008 in a CoA transferase reaction follows this (Kim et al., 2006). The reduction of the enoyl-CoA to acyl-CoA is catalyzed by an electron bifurcating acyl-CoA dehydrogenase (see below). The final step of the pathway is the transfer of the coenzyme A to the 2-hydroxy acid of the reductive path releasing the carboxylic acid (Kim et al., 2006) (Figure 1A). Genes of the reductive path are organized in the hadAIBC-acdB-etfBA operon with the exception of the ldhA gene which is localized upstream of the operon in the opposite direction. Interestingly, C. difficile revealed only a limited spectrum of amino acids utilized in the reductive pathway, while multiple amino acids can be used in the oxidative pathway ( Figure 1B) (Elsden et al., 1976;Hilton, 1978, 1979;Neumann-Schaal et al., 2015;Rees et al., 2016;Dannheim et al., 2017b;Riedel et al., 2017). Some amino acids are degraded by a modified Stickland pathway like the reduction of proline and glycine or the degradation of arginine via ornithine (Schmidt et al., 1952;Mitruka and Costilow, 1967;Hodgins and Abeles, 1969;Turner and Stadtman, 1973;Cone et al., 1976;Kabisch et al., 1999;Jackson et al., 2006;Fonknechten et al., 2009).
Central Carbon Metabolism-Associated Fermentation
Besides the branched-chain and aromatic products of the Stickland reactions, C. difficile produces a number of straight-chain organic acids including acetate, lactate, propionate and butyrate (Neumann-Schaal et al., 2015;Rees et al., 2016;Dannheim et al., 2017b). Key metabolites for their formation are pyruvate and acetyl-CoA. With the exception of acetate, reducing equivalents are oxidized during the formation of the organic acids.
Pyruvate, derived from carbohydrates and amino acids, is a key metabolite in both fermentation and the central carbon metabolism. It is fermented in two ways in C. difficile. It can be transformed to propionate via the reductive Stickland pathway as was observed in Clostridium propionicum (Hetzel et al., 2003;Schweiger und Buckel, 1984;Selmer et al., 2002). Or, it can be degraded to acetyl-CoA and produce butyrate via acetoacetyl-CoA and crotonyl-CoA (Figure 2). Clostridia typically use NADH to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA (von Hugo et al., 1972;Sliwkowski and Hartmanis, 1984;Aboulnaga et al., 2013). The second reduction step of crotonyl-CoA to butyryl-CoA includes an electron bifurcating step (Aboulnaga et al., 2013) (see below). The enzymes of butyrate fermentation are organized in two operons (bcd-etfBA-crt2-hbd-thlA and ptb1buk). Other products such as valerate and 5-methylhexanoate can be formed combining acetyl-CoA with propionyl-CoA or isovaleryl-CoA via the identical set of enzymes (Dannheim et al., 2017b). These reactions play a major role as a sink for reducing equivalents when favored substrates such as proline and leucine are not available (Neumann-Schaal et al., 2015).
Electron Bifurcation and the Rnf Complex
Beside substrate-level phosphorylation, C. difficile couples several of the described fermentation pathways to the generation of a sodium/proton gradient using electron bifurcation in combination with the membrane spanning Rnf complex ( Figure 1A). The Rnf complex was originally discovered in Rhodobacter capsulatus and catalyzes the reduction of NAD + by ferredoxin (Schmehl et al., 1993;Biegel and Müller, 2010). Reduced ferredoxins can be produced through several ways. For instance, via ferredoxindependent oxidoreductases of the oxidative Stickland pathway or via dehydrogenases coupled to an electron bifurcation complex. Electron bifurcation couples the NADH-dependent reduction of a substrate (often CoA-derivatives) to the reduction of ferredoxin (Bertsch et al., 2013). This unique coupling is possible as the redox potential of enoyl-CoA (around 0 mV) is significantly higher than that of NAD + (−280 mV) and ferredoxin (−500 mV) (Buckel and Thauer, 2013). Two electrons derived from NADH are distributed to two different electron acceptors, here an enoyl-CoA and ferredoxin. In C. difficile, electron bifurcating enzymes are found in several pathways including the reductive Stickland pathways ( Figure 1A) and the butyrate/propionate fermentation pathways (butyryl-CoA und acryloyl-CoA Frontiers in Microbiology | www.frontiersin.org dehydrogenases, Figure 2) (Hetzel et al., 2003;Aboulnaga et al., 2013;Bertsch et al., 2013). Finally, the free energy resulting from redox potential difference between ferredoxin (−500 mV) and NAD + (−280 mV) is used to transport ions across the membrane (Buckel and Thauer, 2018). The nature of transported ions has not been studied in C. difficile. However, the transport of protons was observed for other clostridia (Biegel and Müller, 2010;Tremblay et al., 2012;Hess et al., 2013;Mock et al., 2015). Already in the 1980s, it was shown that also proline reduction is coupled to proton motive force generation (Lovitt et al., 1986), most likely via a direct interaction of the proline reductase with the Rnf complex. The generated ion gradient is used for either transport processes, motility, or for ATP generation via ATP synthase ( Figure 1A). What is the ATP recovery of the overall process? Usually organic acids are secreted in a protonated state. ATP synthase requires four ions for the generation of one molecule ATP (Dannheim et al., 2017b). The oxidative path reduces two molecules of NAD + to NADH and phosphorylates 1.5 ADP. The reductive pathway regenerates one molecule NAD + and produces 0.5 ATP. For leucine as substrate, the redox balance requires the reduction of two molecules leucine per one molecule oxidized leucine (Britz and Wilkinson, 1982;Kim et al., 2005). Overall, this leads to a production of 0.83 molecules ATP per molecule amino acid. Under the same conditions, the formation of acetate from acetyl-CoA yields 1.25 molecules ATP per molecule acetyl-CoA. The fermentation of two molecules acetyl-CoA to butyrate yields 1.75 molecules ATP and regenerates two reducing equivalents (Dannheim et al., 2017b).
Glycolysis and Gluconeogenesis and the Incomplete TCA Cycle
Pyruvate and acetyl-CoA are key metabolites used for a variety of different metabolic reactions in C. difficile. Pyruvate is produced via glycolysis and amino acid degradation (e.g. cysteine or alanine). Interestingly, cysteine and also pyruvate inhibit toxin production in C. difficile (Bouillaut et al., 2015;Dubois et al., 2016) emphasizing the tight connection of metabolism and pathogenicity. While glycolysis and gluconeogenesis follow classical pathways, acetyl-CoA can be produced via the Wood-Ljungdahl-pathway, via pyruvate synthase and via pyruvate formate-lyase.
For Clostridium acetobutylicum an incomplete TCA cycle was described (Amador-Noguez et al., 2011;Crown et al., 2011;Au et al., 2014). Based on genome annotation, C. difficile might also possess a truncated TCA cycle which is still sufficient for the production of biomass precursors and the degradation of nutrients (Dannheim et al., 2017b) (Figure 2). In Clostridium kluyveri, a citrate-(Re)-synthase is catalyzing the acetylation of oxaloacetate to form citrate replacing the common citrate-(Si)-synthase (Li et al., 2007). Citrate is further metabolized to 2-oxoglutarate (α-ketoglutarate), the precursor of the glutamate metabolism. Further oxidation of 2-oxoglutarate to succinyl-CoA is impaired as the 2-oxoglutarate synthase is missing. The reductive path is already interrupted at the level of oxaloacetate since a non-decarboxylating malate dehydrogenase is missing in C. difficile (Dannheim et al., 2017b). However, oxaloacetate is connected to fumarate via pyruvate and malate or aspartate. Aspartate serves as ammonium donor for arginineand purine biosynthesis with the formation of fumarate. Fumarate can be degraded to pyruvate to refill the pyruvate pool or it can serve as electron acceptor for aspartate oxidase to produce iminosuccinate as a precursor of NAD biosynthesis (Senger and Papoutsakis, 2008;Dannheim et al., 2017b). The resulting succinate is a substrate of succinyl-CoA:acetate CoA transferase. The succinyl-CoA formed is used for methionine biosynthesis or is degraded to butyrate via crotonyl-CoA (Amador-Noguez et al., 2011;Crown et al., 2011;Au et al., 2014). In summary, the only reaction that contributes to the production of NADH in the truncated TCA cycle is the oxidation of isocitrate to 2-oxoglutarate (Figure 2). Other bacteria harboring a complete TCA cycle produce 3 NADH per acetyl-CoA, which is used for proton gradient formation and ATP generation. Since C. difficile is missing the classical electron transport chains, the TCA cycle is mainly used for the production and degradation of various metabolically important intermediates (Sebaihia et al., 2006).
The Wood-Ljungdahl Pathway
Beside the already described reductive Stickland reactions and the butyrate fermentation, the Wood-Ljungdahl pathway also allows re-oxidation of NADH in C. difficile. In this pathway, which is also known as the reductive acetyl-CoA pathway, two molecules CO 2 are used as terminal electron acceptors and reduced to acetate (Ragsdale, 1997). First CO 2 gets reduced with NADPH to formate or directly into a formyl group by formate dehydrogenase. In a second step catalyzed by the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex the formyl group is reduced to a methyl group and combined with CO and coenzyme A to acetyl-CoA (Ragsdale, 1997). Köpke et al. (2013) showed that the pathway is present in all 28 sequenced C. difficile strains available at that time. Moreover, they showed that the clinical isolate C. difficile 630 and closely related strains are capable of growing autotrophically on CO 2 + H 2 . However, only slight growth was observed probably due to the lack of tryptophan biosynthesis (Sebaihia et al., 2006). Compared to true acetogens like Clostridium ljungdahlii (Köpke et al., 2010), Moorella thermoacetica (Pierce et al., 2008), and Acetobacterium woodii (Poehlein et al., 2012), C. difficile genomes only harbor an orphan acetate kinase gene. No obvious gene for a phosphotransacetylase was detected. However, this reaction might be catalyzed by the phosphotransbutyrylase (Köpke et al., 2013). In summary, fixation of the glycolysisderived CO 2 via the Wood-Ljungdahl pathway might be also a metabolic advantage for C. difficile in the human gut (Köpke et al., 2013).
Pyruvate Utilization via Pyruvate
Formate-lyase C. difficile is utilizing pyruvate via the radical enzyme pyruvate formate-lyase, which forms the products acetyl-CoA and formate in the presence of coenzyme A (Figure 2). Pyruvate formate-lyase (PflD) requires an [4Fe-4S] cluster containing activating enzyme (PflC) for the formation of the catalytic glycyl radical (Crain and Broderick, 2014). The formate generated gets subsequently oxidized to CO 2 and an electron by the formate dehydrogenase, a MoCo-containing selenoprotein (Pinske and Sawers, 2016). The electrons formed are transferred to a [NiFe] hydrogenase (Shafaat et al., 2013;Pinske and Sawers, 2016). Overall, the central metabolism in C. difficile mainly serves as an anabolic and catabolic hub and for CO 2 fixation, avoiding the generation of NADH due to the lack of classical respiratory chains.
REGULATION OF C. DIFFICILE ENERGY METABOLISM
Currently, only a partial view of the regulation of the C. difficile energy metabolism at the transcriptional and post-transcriptional level is available (Bouillaut et al., 2015). Clearly, the major regulator is the catabolite control regulator CcpA (Antunes et al., 2012). In Bacillus subtilis the LacI/GalR type regulator forms a complex with phosphorylated form of Hpr or Crh, which in turn is generated in the presence of high cellular glucose or fructose-1,6-bisphosphate concentrations (Fujita, 2009). In C. difficile about 140 genes are directly controlled by CcpA including genes of glycolysis, proline reduction, glycine reduction, butanol and butyrate formation (Figure 2; Antunes et al., 2012). In parallel, CcpA controls the expression of the toxin genes tcdA and tcdB in response to fructose-1,6-bisphosphate without phosphorylated Hpr, providing a strong link between metabolism and toxin production (Antunes et al., 2011). The proline-dependent regulator PrdR activates genes for proline reductase and represses the genes for glycine reductase (Figure 2; Bouillaut et al., 2013). The global regulator CodY provides a link to sporulation and another connection of the metabolism to toxin production (Dineen et al., 2007;Nawrocki et al., 2016;Ransom et al., 2018).
In close cooperativity with the SinR and SinR' proteins and the corresponding genes, CodY controls the toxin off state during the exponential growth phase (Girinathan et al., 2018;Ransom et al., 2018). Interestingly, culture heterogeneity caused by a bistable switch was observed (Ransom et al., 2018). CodY regulates also the energy metabolism via binding to promoters of genes involved in glycogen formation, the pyruvate formate-lyase path to hydrogen and butanol/butyrate generation (Figure 2; Dineen et al., 2010). The sigma factor SigH, controlling the genes of the glycogen metabolism and for formate dehydrogenase, represents another connection of the metabolism with sporulation (Saujet et al., 2011). The function of the NADH/NAD + -responsive regulator Rex in the fermentative metabolism of C. acetobutylicum was described before (Wietzke and Bahl, 2012). Participation in the regulation of butanol and butyrate formation in C. difficile was proposed (Figure 2; Bouillaut et al., 2015). The ferric uptake regulator Fur also directly influences glycine reduction and hydrogen formation from pyruvate in response to low iron condition (Figure 2; Ho and Ellermeier, 2015;Berges et al., 2018). Finally, the small regulatory RNA CsrA serves as carbon storage regulator influencing the genes of glycogen mobilization (Figure 2; Gu et al., 2018). Obviously, a complex regulatory network headed by the pleiotropic regulators CcpA and CodY co-regulates metabolism and toxin production. Similarly, CodY and SigH connect sporulation with the metabolism at the transcriptional level (Martin-Verstraete et al., 2016).
CONCLUSION AND FUTURE PERSPECTIVES
In Western countries, hypertoxic C. difficile strains are causing several thousand deaths per year especially after antibiotic treatments. In this context, the mystery of the ecological success of this pathogenic bacterium is closely related to its unique and highly adaptive metabolism. Amino acids as building blocks of proteins are integral parts of our nutrition and thus available in access in our gut. Similarly, sugars from sugar polymers like starch constitute the carbohydrate part of our food. Both are the major energy sources of C. difficile. The versatile organism possesses multiple pathways for amino acid fermentation. However, normal substrate level phosphorylation suffers from very low ATP recoveries and the need to utilize parts of this ATP for ion gradient formation via a reverse ATPase reaction. Thus, smart C. difficile couples amino acid fermentation via electron bifurcation to membrane potential generating processes at the Rnf complex. Similarly, the central metabolism was modified to prevent unnecessary NADH generation, which usually has to be re-oxidized via energetically cost-intensive reactions. The organism uses an incomplete TCA cycle, generating one instead of three NADH. Furthermore, pyruvate formate-lyase instead of pyruvate dehydrogenase produces acetyl-CoA and formate, which gets transformed into protons by formate dehydrogenase and finally to hydrogen by a hydrogenase. Nevertheless, major metabolic fluxes have to be determined. Most likely, additional principles of energy generation will be uncovered. We are at the beginning of an exciting period of systems biology, allowing the integration of the different levels of cellular control represented by transcriptional control, RNA stability, translational control, metabolic control and coordinated degradation. Currently, we know that this complex metabolism is controlled by a network of regulatory proteins, which directly connects it to toxin formation and sporulation. Major players are the catabolite regulator CcpA, the sporulation sigma factor SigH, the pleiotropic transcription factor CodY, the proline regulator PrdR, the iron responsive Fur and potentially the NADH/NAD + -ratio measuring Rex. Here, SigW and CodY are important players during the onset of sporulation. Similarly, CcpA and CodY regulate toxin gene transcription. A first small regulatory RNA (CsrA) was found involved in flagella formation, toxin production and host cell adherence. Most likely, this is only a small part of the yet unknown regulatory network underlying the efficient adaptation of the metabolism to changing environmental conditions. Novel regulatory principles including new regulators, novel small regulatory RNA and proteins, unknown changes in the protein-protein network with controlled proteolysis, direct metabolic regulation, control of RNA stability to name a few, have still to be elucidated. | v3-fos-license |
2018-11-15T16:40:35.277Z | 2018-11-01T00:00:00.000 | 53874124 | {
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} | pes2o/s2orc | Macrophage Inhibitory Factor-1 (MIF-1) controls the plasticity of multiple myeloma tumor cells
Multiple Myeloma (MM) is the second most common hematological malignancy with a median survival of 5–10 years. While current treatments initially cause remission, relapse almost always occurs, leading to the hypothesis that a chemotherapy-resistant cancer stem cell (CSC) remains dormant, and undergoes self-renewal and differentiation to reestablish disease. Our finding is that the mature cancer cell (CD138+, rapidly proliferating and chemosensitive) has developmental plasticity; namely, the ability to dedifferentiate back into its own chemoresistant CSC progenitor, the CD138–, quiescent pre-plasma cell. We observe multiple cycles of differentiation and dedifferentiation in the absence of niche or supportive accessory cells, suggesting that soluble cytokines secreted by the MM cells themselves are responsible for this bidirectional interconversion and that stemness and chemoresistance are dynamic characteristics that can be acquired or lost and thus may be targetable. By examining cytokine secretion of CD138- and CD138+ RPMI-8226 cells, we identified that concomitant with interconversion, Macrophage Migration Inhibitory Factor (MIF-1) is secreted. The addition of a small molecule MIF-1 inhibitor (4-IPP) or MIF-1 neutralizing antibodies to CD138+ cells accelerated dedifferentiation back into the CD138- progenitor, while addition of recombinant MIF-1 drove cells towards CD138+ differentiation. A similar increase in the CD138- population is seen when MM tumor cells isolated from primary bone marrow aspirates are cultured in the presence of 4-IPP. As the CD138+ MM cell is chemosensitive, targeting MIF-1 and/or the pathways that it regulates could be a viable way to modulate stemness and chemosensitivity, which could in turn transform the treatment of MM.
Introduction
Multiple myeloma (MM) is the second most prevalent hematological malignancy in the United States, with a survival rate of 47% within the first five years post diagnosis [1,2]. It is a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 characterized by the proliferation of clonal plasma cells within the bone marrow, which can interfere with normal blood cell production and also secrete non-functioning monoclonal antibodies called M proteins [3][4][5]. The tumor cells colonize the bone marrow, and symptomatically, this results in anemia, lytic bone lesions, and hypercalcemia, in addition to renal failure [6,7]. Together with autologous stem cell transplantation and the introduction of novel therapies such as proteasome inhibitors and immunomodulatory drugs, patients frequently show improvement, even to the point of remission. However, relapse is usually occurs, suggesting that there exists a subset of MM tumor cells that are able to evade cytotoxic insult and eventually recapitulate tumor burden [8][9][10]. This has lead to the hypothesis that a population of chemoresistant cancer stem cells (CSC) persists and remains dormant until a time posttreatment when they reinitiate growth and re-establish tumor burden [10][11][12].
In addition to cell surface markers, which have been used to identify CSC populations, "stemness" can be phenotypically defined. Similar to the hematopoietic stem cell (HSC), the CSC: 1) exists in a quiescent, slowly proliferating state, 2) possesses the ability to differentiate into more mature progeny, 3) is resistant to chemotherapy, and 4) has the ability to undergo long-term self-renewal in order to maintain the CSC population itself [13][14][15]. The CSC may be derived from a hematopoietic stem cell that has acquired oncogenic mutation(s) allowing it to become tumorigenic while maintaining stemness OR alternatively from a differentiated tumor plasma cell that dedifferentiates and reacquires stemness properties (i.e. self-renewal, differentiation, quiescence, chemoresistance). MM tumor cells are clonal plasma cells, which have isotype switched, suggesting that the progenitor arises from a post-germinal center B cell lineage, such as a plasma cell precursor or plasma cell itself [5,16]. A marker distinguishing an immature plasma cell (prePC) and a mature plasma cell [17] is the acquisition of CD138 and it is generally believed that the MM CSC population is CD138-as are pre-PCs. Matsui, et al. found that both primary tumor cells isolated from MM patients and MM cell lines are composed of a heterogeneous mixture of CD138-and CD138+ populations [4,16], but only the CD138-cells were able to engraft in the bone marrow of NOD/SCID (non-obese diabetic/ severe compromised immunodeficient) mice [18]. Upon analysis of bone marrow aspirates from these mice, they found that the cells were all CD138+ and expressed the same light chain restriction as the initial patient material, suggesting that the CD138-cells were the CSC population that with time gave rise to the differentiated CD138+ cells. Other laboratories, however, have shown that both CD138-and CD138+ cells can engraft the bone marrow when a different mouse model, NOD/SCID γ (NSG), is used [17], and the authors suggested that the recipient bone marrow niche might play a role in altering stemness. Chaidos et al. showed that patientderived purified CD138+ plasma cells could produce tumors in animals, but they detected both CD138-and CD138+ cells in the BM of mice, suggesting that the precursor CD138-cells were derived from or were the progeny of the CD138+ as a result of dedifferentiation [17]. These findings imply that a functional plasticity between differentiated progeny and dedifferentiated precursor cells might exist and reconcile the difficulties in clearly defining the CSC population. If interconversion provides a way to continually renew the chemoresistant CSC population, this would contribute to the eventual relapse seen in this disease.
To investigate this, we examined multiple MM cell lines to identify and isolate the CSC population. Our finding is that the mature myeloma cell (CD138+, rapidly proliferating and chemosensitive) has developmental plasticity; namely, the ability to dedifferentiate back into its own chemoresistant CSC progenitor, the CD138 -, quiescent plasma cell that possesses stemness potential. As these cells are grown in the absence of accessory cells, this suggests that soluble cytokines secreted by the MM cells themselves are responsible for this bidirectional interconversion. We identified MIF-1 (macrophage migration inhibitory factor-1) as a secreted cytokine that controls this interconversion in MM cell lines, and show that by manipulating the activity or levels of MIF-1 present in the culture media we can shift the set point towards more differentiation or more dedifferentiation.
CD138-cells have the characteristics of stemness
RPMI 8226 cells were stained with CD138 and CD38 antibodies, and sorted by flow cytometry. Unstained populations and single staining were used to identify doubly stained populations ( Fig 1A). Three distinct populations were consistently detected ( Fig 1B, dashed boxes, i-iii, panel A in S1 Fig). Each was recovered by sorting with high efficiency, and only population i (hereafter referred to as CD138+CD38+) and ii (CD138-CD38+) were shown to be viable ( Fig 1B, inset). The non-viable population iii was gated out of all future analysis. The CD138+ and CD138-populations (i and ii) were consistently detected in a~9:1 ratio:~90% of cells were CD138+ and~10% of cells were CD138- (Fig 1B). The viability of either the CD138+ or CD138-cells does not change with time, as measured by staining at time points post plating of pure populations (panels E,F in S1 Fig). Previous studies have suggested that these CD138cells were the MM CSC population, and we analyzed these pure, sorted populations to further characterize "stemness" by assessing: cell cycle phase (Fig 1C), long-term self-renewal/clonogenic potential (Fig 1D, 1E, 1G and 1H), chemoresistance ( Fig 1F) and expression of differentiation specific markers (Fig 1I and 1J). Purified CD138-and CD138+ populations were stained with Vybrant Green to measure DNA content ( Fig 1C). The CD138 + CD38 + cells were rapidly proliferating with only 56% in G0/G1 phase, and 20% and 24% of cells in S and G2/M phase, respectively (Fig 1C, top). In contrast, 88% of the CD138 -CD38 + cells were present in G0/G1 phase, demonstrating that these cells were quiescent or only very slowly proliferating (Fig 1C, bottom). The shift to the left seen in the CD138-cells in the presence of Vybrant Green suggests a more tightly compacted DNA, suggesting that these cells may be in G0 rather than G1 phase. To test the self-renewal capacity of each population, 10 4 sorted CD138 + or CD138cells were plated in methylcellulose and the formation of colonies was assessed after 14 days. CD138 + cells were able to form significantly more colonies than CD138cells, suggesting that more cells were able to initiate proliferation ( Fig 1D). However, these colonies were smaller than those derived from the CD138precursors ( Fig 1E, 1G and 1H). This suggests that fewer CD138-cells entered cycle, but those that did were able to undergo more duplication events than the CD138+ cells, resulting in larger colony width, suggesting that they could proliferate long after the CD138+ cells exhausted their ability to do so. To compare chemotherapeutic response, sorted CD138+ or CD138-populations were treated and mock treated for 24 h. with 0.01μM Bortezomib, a proteasome inhibitor used as frontline therapy in MM treatment. Viability was measured by trypan blue exclusion and chemosensitivity was defined as the difference in viability between mock and Bortezomib treated cells (Fig 1F). CD138+ cells were more sensitive, with a greater loss of viability seen with Bortezomib treatment, than the CD138cells, where Bortezomib had a reduced effect. Finally, we measured the expression of two transcription factors, Blimp1 and IRF4, which have been shown to be responsible for the differentiation of B cells into plasma cells [3,19,20]. CD138+ cells have higher expression of Blimp1, as detected by intracellular staining (Fig 1I, blue) and RT-PCR analysis (Fig 1J). IRF4 is also expressed preferentially in the CD138+ cells (Fig 1J), suggesting that these are the differentiated progeny, and that the CD138-cells are more immature. CD138 expression is regulated at least in part at the RNA level, as reduced expression of CD138 (SDC1) is detected in the CD138-cells (Fig 1J), although we can't rule out post-transcriptional regulation. Together, these experiments suggest that the CD138-cells meet the qualifications of stemness: they are
Cells undergo interconversion, exhibiting plasticity
An additional characteristic of stemness is the ability to undergo differentiation to produce mature progeny. To examine this, pure CD138+ and CD138-RPMI 8226 populations were replated (Fig 2A and 2B) and followed for 13 days. CD138+ cells proliferated faster than CD138cells (panel A in S2 Fig, an increase of 2.4 for CD138+ cells as compared to 1.5 for CD138cells during the first 5 days), and no significant loss of viability was seen in the populations over the 13 day time course, suggesting that the changes seen in CD138 expression were not reflective of cell death or dying (panels D,E in S2 Fig). Cells were harvested at different time points, and CD138 expression on 10,000 cells was measured by flow cytometry (Fig 2A, 2B and 2C). Within 5 days post plating, pure CD138cells were able to differentiate and give rise to differentiated CD138 + cells: 50% of the total cells were CD138+ by day 5 (Fig 2A, red). The cells continued to differentiate and by day 13, the bulk of the culture was CD138+, reestablishing the 9:1 ratio of CD138 + :CD138seen in unsorted cultures (Fig 2A, pink). The mean +/-SE for three experiments are presented in Fig 2C. However, when pure CD138 + cells were replated, they gave rise to CD138-cells, suggesting that these cells were able to de-differentiate ( Fig 2B). Only 40% of the cells remained CD138+ by day 5 (Fig 2B, red). By day 10, the culture started to shift back and 87% of the cells now were CD138+, and by day 13, the 9:1 ratio of CD138 + :CD138cells seen in unsorted cultures had been reestablished (Fig 2B, pink). Given the doubling rate of CD138-cells, the emergence of CD138-cells from the pure CD138+ population cannot be due to a small, undetectable, pre-existing CD138-population emerging. Thus, the CD138-cells could differentiate (Fig 2A), while the CD138+ cells could de-differentiate ( Fig 2B), suggesting that the cells could interconvert in culture.
To analyze whether this plasticity was intrinsic to the RPMI8226 cell line, or was a quality of other human MM cell lines, NCI-H929 and U266-B1 cells were sorted into CD138 + populations (panels B,C,F-I, J-Q in S2 Fig When pure populations of NCI-H929 and U266-B1 CD138+ cells were re-plated, with time, CD138cells were detected ( Fig 2C). By day 13, the cultures had reestablished the~9:1 CD138 +:CD138-ratio, suggesting that plasticity was a shared characteristic of MM cell lines.
To further investigate the interconversion of the CD138-and CD138+ populations, we synchronized cells to follow their differentiation or dedifferentiation. Unsorted RPMI8226 cells were treated with PD0332991 (Palbociclib), a specific inhibitor of cyclin-dependent kinase 4/6, to arrest cells in G1 phase. After 72 h. of PD0332991 treatment, cell cycle profiles were measured using the DNA stain Vybrant Green, in mock and PD0332991 treated cells, and PD0332991 treatment dramatically increased the G1 phase content, demonstrating arrest ( Fig 2D, -PD: 46.9%, +PD, 85.2%). Cells were stained with CD138, and we found that PD0332991 treatment caused a change in the CD138+:CD138-ratio, with an increase in the CD138-pool detected (Fig 2E, red), suggesting that G1 phase arrest and CD138 status might be linked. difference in viability (trypan blue) before and after treatment. (n = 4) � p<0.001. G,H) Representative colonies formed within methylcellulose by CD138+ and CD138-sorted cells (10 4 cells/ml). I) Intracellular staining of Blimp1 expression in sorted CD138+ (blue) and CD138-(red) cells as measured by flow cytometry, isotype control (purple). J) RT-PCR to measure the relative levels of Blimp1 (n = 4) and IRF4 (n = 3) and SDC1 (CD138) (n = 3). Levels in CD138+ set to 100, plotted as AU. � p<0.05, �� p<0.001, Student t-test. Error bars, mean ± SD.
Fig 2. Plasticity of cells. A, B)
Sorted CD138-CD38+ and CD138+CD38+ populations were plated, aliquots (10,000 cells) were analyzed for CD138 expression at days 5, 10, and 13. Purple line is CD138 content at time of plating (Representative of N = 5). Flow cytometry will not measure expansion of the cultures. C) Sorted CD138-CD38+ and CD138+CD38+ cells from U266-B1 and NCI-H929 MM lines plated and analyzed for CD138 status at days 5, 10 and 13. (N = 3, mean+/-SD) D) Cell Cycle status of RPMI8226 cells before (left) and after (right) treatment with 5μM PD0332991 (PD). E) CD138 expression of RPMI8226 cells before (purple) and after (red) PD treatment for 3 days. F) Cells were treated with 5μM PD for 3 days, stained with CFSE, washed, and then followed in culture for 6 days. Aliquots were analyzed for CFSE and CD138 over this time course. Dashed boxes are of non-viable cells (population iii) were not included in the overall percentages of each quadrant. PD0332991 arrested cells were then subsequently stained with the fluorescent cell permeable dye CFSE. This dye is retained in the cytoplasm, and decreases its fluorescent intensity by half upon each cycle of cell division. This allows us to track cell proliferation as measured by loss of CFSE of both the CD138+ and CD138-populations in tandem with their differentiation status, as measured by CD138 expression. PD0332991 arrested, CFSE stained cells were sorted into two pure populations: CD138 + CD38 + CFSE hi and CD138 -CD38 + CFSE hi (Fig 2F, Day 0). PD0332991 was removed from the culture through sequential washing with fresh media and the labeled cells were replated. Cells were harvested every two days and analyzed for CFSE and stained for CD138. CD138cells (bottom panel) were able to divide (reducing CFSE content) and differentiate (increasing CD138 content) and within two days, 17.5% of the population had become CD138 + CFSE lo (Fig 2F, bottom panel, Day 2). 75% of the population did not divide, and while there was a slight increase in CD138 expression when compared to Day 0, these cells with high CFSE remained within the CD138-gate. By days 4 and 6, the bulk of the culture had divided to become CD138 + CFSE lo , while a small pool of cells remained CFSE+ CD138and had not divided ( Fig 2F, When sorted, arrested, CD138 + CFSE hi cells were replated and released from arrest (Fig 2F, top panel), by day 2, the bulk of the cells are CFSE mid (between 10 1 −10 2 ) but remain CD138+, suggesting that they are starting to divide but maintain the CD138 marker (top quadrants). However, a new population, CD138 mid CFSE mid , is also detected, suggesting that some cells are losing expression of CD138 as they divide. This CD138 mid CFSE mid population continues to decrease in CD138 expression through the last two time points, becoming CD138 -CFSE mid , without a further decrease in CFSE fluorescence, supporting that they are dividing slowly and dedifferentiating. By day 6, three populations are visible: 85.4% are CD138 + CFSE lo (CD138+ cells that divided into CD138+ cells); 2.3% are CD138 -CFSE mid (dedifferentiated CD138-cells that may have divided once but then stopped and has been maintained since day 2); 11.7% are CD138 -CFSE lo (CD138+ cells that dedifferentiated and then divided into CD138-cells). All of these populations are viable and are outside of the non-viable population iii gate (dashed box) from Fig 1. Again, the small contaminating population of CD138-cells (0.17% or 425 cells) is not responsible for the increase seen in this population (13,2000 cells or 3.3% of the total) (panels R-U in S2 Fig). Thus, this suggests that not only are CD138 + cells able to proliferate but are also able to de-differentiate, giving rise to the CD138precursor.
Dedifferentiated CD138-cells exhibit characteristics of stemness
To determine whether MM cells could undergo multiple cycles of differentiation and de-differentiation and remain phenotypically the same as CD138-or CD138+ cells isolated from the parent RPMI8226 cell line, pure CD138 + cells (S3 Fig) were re-plated and maintained for 4 weeks (Fig 3A). During this time, the 9:1 ratio of CD138 + :CD138cells was reestablished ( Fig 3A), and the ratio of non-viable population iii was similar, suggesting that significant cell death had not occurred during this time period. Pure populations (CD138 +� and CD138 -� ) were recovered from these cells and tested by the stemness assays described in Fig 1. CD138 +� and CD138 -� cells were plated in methylcellulose and the formation of colonies was assessed. As seen with cells isolated from the parent cell line, the CD138 +� cells formed significantly more colonies than the CD138-� cells (Fig 3B), but the CD138 -� population formed Authenticity of newly dedifferentiated RPMI 8226 CD138-cells. A) Sorted CD138+CD38+ populations were plated for four weeks, and then re-sorted based on CD138 status, to yield newly differentiated CD138+ � and dedifferentiated CD138-� populations. Representative of N = 3. Dashed boxes is non-viable (population iii). B,C) These sorted CD138+ � and CD138-� cells were plated in methylcellulose, colonies were counted and measured (N = 3) � p<0.05. D) CD138+ � and CD138-� cells were plated and treated with Bortezomib for 24 h. Chemosensitivity was measured as the difference in viability before and after treatment (N = 3). E) RT-PCR on CD138+ � and CD138-� populations to assess the levels of Blimp1, IRF4, and SDC1 (CD138) (N = 3). The levels seen in CD138+ cells was set to 100, plotted as AU, � p<0.05. Error bars, mean ± SD. F,G) Sorted CD138+ � and CD138-� populations were plated, and analyzed for CD138 by flow cytometry at days 5, 10, and 13. Purple line is CD138 content at time of plating (Representative of N = 3). significantly larger colonies (Fig 3C). Both populations were treated with Bortezomib or mock treated for 24 h. and similar to cells isolated from the parent line, CD138 +� cells showed increased sensitivity to chemotherapeutic treatment as indicated by the greater change in viability as compared to the more chemoresistant CD138 -� cells (Fig 3D). The expression of Blimp1, IRF4 and SDC1 (CD138) in the doubly sorted CD138+ � and CD138-� cells was measured by RT-PCR (Fig 3E), and as seen with cells isolated from the parent cell line, CD138 -� cells had lower levels of Blimp1 and SDC1 RNA (Fig 3E), while IRF4 levels between the populations were more similar (Fig 3E). The doubly sorted CD138 -� cells were replated and followed in the differentiation assay for up to 13 days ( Fig 3F). These cells were able to differentiate again, with kinetics similar to those seen in cells freshly isolated from the parent line, re-establishing the~9:1 ratio of CD138 + :CD138cells by day 13 (Fig 3G). Doubly sorted CD138 +� cells were able to de-differentiate by day 5 and re-establish the~9:1 ratio of CD138 + :CD138sorted cells by day 13 (Fig 3F). The kinetics of dedifferentiation of the CD138 +� cells was different in that there was no large CD138population by day 5 as seen with the parent CD138 + cells (Fig 2B). However, in general, both of the doubly sorted populations maintained plasticity and could differentiate and/or dedifferentiate respectively.
Modulation of MIF-1 activity alters interconversion
While interconversion of MM cells has been reported before [17,21], this is the first time that plasticity has been reported to occur in 2D culture in the absence of accessory cells. This suggests that the MM cells themselves controlled their differentiation or dedifferentiation status and we hypothesized that the cells might secrete cytokines into the culture media that influence interconversion. To examine cytokine secretion directly, CD138 + and CD138-cells were sorted, plated, and conditioned media was harvested at days 1 and 2, and a cytokine array was used to detect the presence of 36 cytokines simultaneously (Fig 4A). The cytokine values detected at time 0 (plating) and in the media alone were subtracted to generate the change in expression, and resultant levels were plotted as AU ( Fig 4C, S4 Fig). While several different cytokines were modestly increased in supernatant from both CD138+ and CD138-cells, the expression of Macrophage Inhibitory Factor 1 (MIF-1) increased rapidly and significantly in both populations. MIF-1 is an inflammatory cytokine, secreted by many cell types, and shown to increase survival and proliferation of several stem cell lineages [22][23][24][25]. We hypothesized that the burst of MIF-1 secreted into the media might promote interconversion of the individual populations, and that blocking MIF-1 activity with 4-IPP, a small molecule inhibitor of MIF-1, might alter the kinetics of dedifferentiation or differentiation. Pure populations of CD138+ cells were treated with different concentrations of 4-IPP and viability was followed by PI staining (Fig 4B) Because treatment with 4-IPP decreased cell viability with time and concentration, we only analyzed viable cells, as measured by PI staining, for CD138 status in future experiments (Fig 4C, 4D, 4G and 4H). When pure CD138+ cells were plated and then mock treated, roughly 60% of CD138+ cells remained CD138+ at 24 h., suggesting that 40% had dedifferentiated during this time frame (Fig 4C, compare T = 0 to [0]). However, when treated with 30 or 60 micromolar 4-IPP, only 50 or 35% remained CD138+ respectively, suggesting that blocking MIF-1 activity increased dedifferentiation (50 to 65% became CD138-during the same time period). When CD138populations were plated and treated for 24 h. (Fig 4D). This suggests that blocking MIF-1 increased the pool of dedifferentiated cells.
The loss of viability seen in the CD138+ cells in the presence of 4-IPP (Fig 4B) was due to apoptosis, as measured by increased Annexin V staining after 24 h with increasing concentrations of drug (Fig 4E). Annexin V staining of untreated CD138+ cells did not change by the 24 h. timepoint, demonstrating the stability of untreated cells. Treatment of unsorted RPMI8226 cells with increasing concentrations of 4-IPP also caused a significant increase in the G0/G1 phase, as measured by Vybrant Green staining (Fig 4F). While unsorted cells had a G0/G1 content of~50%, 4-IPP treatment increased this to~60%. As 4-IPP promotes increases in the CD138-pool, this increase in quiescence is consistent.
To validate the specificity of 4-IPP to promote the dedifferentiation of CD138 + cells by targeting MIF-1, we used a monoclonal neutralizing antibody (NAb) to block MIF-1 (Fig 4G). Increasing concentrations of MIF NAb were added to pure sorted CD138+ cells for 24 h., and then viable cells, as measured by PI staining, were analyzed for CD138 status (Fig 4G). While approximately 50% of the mock treated CD138+ cells remained CD138+ (50% had dedifferentiated), only 30% of the NAb treated cells remained CD138+ (70% dedifferentiated into CD138-cells within the same time frame). Just like 4-IPP treatment, the viability of the NAb treated cells was reduced. Pure CD138+ cells were plated in the absence or increasing concentrations of MIF NAb for 24 h, stained with PI, and analyzed by flow cytometry (Fig 4H). A similar 20% decrease in viability was seen with NAb addition, suggesting that death might be a physiological response to forced, excessive dedifferentiation or that MIF-1 is a survival factor.
4-IPP or NAb treated cells behave like dedifferentiated CD138-cells
Blocking MIF-1 activity appeared to drive or maintain dedifferentiation status in both the CD138+ and CD138-populations (Fig 4C-4H) and we wanted to determine whether CD138and CD138+ cells generated by blocking MIF-1 activity behaved like untreated CD138-and CD138+ cells, in terms of chemotherapeutic response. Sorted parent CD138 + cells were treated with 60 μM 4-IPP for 24 h to promote dedifferentiation, and 62.5% of these cells became CD138- (Fig 5A). These cells were doubly stained with CD138 and PI to isolate viable CD138 + and CD138populations (Fig 5A, right), which were then mock treated or treated with Bortezomib for 24 h. Chemosensitivity was measured as the difference in viability between the mock and Bortezomib treated populations (Fig 5B). The 4-IPP derived CD138-cells were significantly less sensitive to chemotherapeutic treatment than the CD138+ cells, similar to what was seen with the parent derived CD138-populations (Fig1F). CD138+ cells treated with 4-IPP for 48 h. (to become CD138-) (Fig 5C, red), were replated without drug and analyzed for CD138 expression at 48 (green) and 96 (pink) h. Whereas 62% of 4-IPP treated cells were CD138-(red) at the time of replating, by 48 h. without drug, a large CD138+ population appeared, and by 96 h, 73% were CD138+. Thus, the CD138-cells derived by 4-IPP treatment could recover and regained characteristics of stemness: they were chemoresistant and could differentiate. with CD138 and PI and expression of CD138 in viable (PI negative) cells is plotted (N = 3). T = 0 is CD138 content at time of plating before 4-IPP treatment. E) Sorted CD138+ cells were plated, treated with increasing concentrations of 4-IPP for 24 h. and then stained with Annexin V, followed by flow cytometric (N = 3) Test of trend, p = 0.008. F) Unsorted cells were plated, treated with increasing concentrations of 4-IPP for 24 h., and then stained with Vybrant Green, followed by cell cycle analysis (N = 3). G) Sorted CD138+ cells were plated, treated with increasing concentrations of MIF Nab for 24 h. Cells were stained with CD138 and PI and expression of CD138 in viable (PI negative) cells is plotted (N = 3). T = 0 is CD138 content at time of plating. H) Unsorted cells were plated, treated with increasing concentrations of MIF NAb for 24 h. Viability was measured by PI staining (N = 3). � p<0.05. Error bars, mean ± SD. https://doi.org/10.1371/journal.pone.0206368.g004
MIF-1 regulates interconversion of cancer stem cells
MIF NAb treatment also increased the pool of CD138-cells ( Fig 4G) and we wanted to determine whether this treatment also increased chemoresistance in these CD138-cells ( Fig 5D). Sorted CD138 + were treated +/-NAB for 24 h., and then were treated with Bortezomib for an additional 24 h to measure viability using the MTT assay. CD138+ cells are sensitive to Bortezomib, and their viability decreases to 48% following treatment (lane 2). CD138+ cells pre-treated with NAb, however, were less sensitive to Bortezomib and their viability was only reduced 17% with treatment (lane 4). Consistent with what was seen with 4-IPP treatment, rMIF promotes differentiation into CD138 + cells. Because blocking MIF-1 activity with either 4-IPP or MIF NAb promoted dedifferentiation, we hypothesized that an excess of rMIF might promote differentiation into CD138+ cells. Sorted CD138 + cells were treated with rMIF at increasing concentrations, and PI and CD138 status was measured after 24 h (Fig 6A). Cell viability was not affected by rMIF treatment. While approximately 60% of mock treated CD138+ cells remained CD138+ (40% had dedifferentiated to CD138-cells, Fig 6A, compare T = 0 to [0]), in the presence of 50 ng/ml rMIF, almost 90% of cells remained CD138+, suggesting that rMIF prevented dedifferentiation and allowed the maintenance of the CD138 + population (Fig 6A, compare T = 0 to [50] rMIF). When sorted, pure CD138cells were mock treated, 10% of cells differentiated and became CD138+ within 24 h. (90% remained CD138-, Fig 6B, compare T = 0 to [0] rMIF), but when treated with different concentrations of rMIF, an increase in CD138+ cells was also seen. This suggests that the presence of rMIF helped to maintain and/or generate a CD138+ population by promoting differentiation.
To determine whether the CD138+ cells generated by rMIF treatment were more sensitive to chemotherapeutic agents, CD138cells were treated +/-rMIF for 24 h., and then treated with Bortezomib for 24 h to measure viability in the MTT assay ( Fig 6C). CD138-cells are relatively resistant to Bortezomib, and viability of treated cells was only reduced by 20% (lane 2). CD138-cells treated with rMIF, however, were more sensitive to Bortezomib and viability was now reduced 46% by treatment (lane 4).
CD138+ cells secrete more MIF-1 than CD138-cells.
We had seen that NCI-H929 and U266-B1 cells also underwent differentiation and dedifferentiation when plated as pure populations (Fig 2C). Similar to the RPMI 8226 line, when U266-B1 CD138+ cells were treated with increasing concentrations of 4-IPP, they also exhibited a marked decrease in the level of CD138+ cells (and an increase in dedifferentiated CD138-cells) (Fig 7A). 4-IPP did not increase dedifferentiation of the NCI-H929 cells within this 48h. timeframe (Fig 7B). Thus, MIF-1 activity appeared to regulate interconversion in at least two MM cell lines. As we had previously seen interconversion after 5 days, 48 hrs may have been too short a timeframe to see an effect of 4-IPP on NCI-H929 cells. However, as the viability of cells exposed to 4-IPP decreases at times greater than 24 h., we could not assay for increased time.
The cytokine array assay had demonstrated that MIF-1 was secreted from both pure CD138-and CD138+ cells (Fig 4A). We examined RNA levels in both CD138+ and CD138-RPMI 8226 populations using RT-PCR and did not find any statistically significant differences (Fig 7C). Using a quantitative MIF-1 ELISA kit, we were able to measure MIF-1 cytokine secretion at 1, 4, 12 and 24 h. post plating of pure populations of all three cell lines (Fig 7D, 7E and 7F). CD138+ cells secreted statistically significant more MIF-1 than CD138-cells in the RPMI8226 and the NCI-H929 cell lines (Fig 7D and 7F), while no significant differences were detected for U266-B1 cells (Fig 7E), although the trends were the same.
MIF-1 interconversion in primary MM cells.
To examine whether MIF-1 regulates interconversion in vivo, bone marrow aspirates from MM patients were obtained with informed consent (patient characteristics described in S5 Fig). MM tumor cells were purified using the Rosette Step MM purification kit, which removes other contaminating hematopoietic cells that contain CD2, CD14, CD33, CD41, CD45RA, CD66b markers and glycophorin A on red blood cells (RBCs). Viability was always >75% as measured by trypan blue staining. This material was either stained immediately with CD138 and CD38 antibodies (Fig 7G and 7H, T = 0) or plated in tissue culture in the absence or presence of 4-IPP for 24 h. (Fig 7H). The ratio of CD138+:CD138-cells varied among patients, with several patients having very high CD138content (Fig 7G, patient E, F, H). Variability in the CD138 content is consistent with previous reports [26]. Primary material was replated +/-4-IPP, cells were harvested at 24 h., stained with CD138 and CD38 antibodies and analyzed by flow cytometry (Fig 7H). Viability as measured by trypan blue staining was unchanged. In 5 out of 6 patients, after 24 h. in culture, cells differentiated into CD138+ (Fig 7H, patients B, E, F, I, H). This was most obvious in the patients with high CD138-content at the time of harvest (Fig 7H, patients E, F, H), suggesting that replating promotes differentiation. However, when plated in the presence of 4-IPP, the CD138-population either increased relative to T = 0 (patients B, I) or did not decrease as significantly as seen in the absence of 4-IPP (patients E, F, H), consistent with the role of 4-IPP on the cell lines. In patient A, plating +/-4-IPP appeared to increase the CD138-population, and the significance of this is unclear.
Discussion
While there has been some debate in the field regarding the nature of the MM CSC, our data and that of others strongly suggest that the CD138-cell meets the definition of stemness, based on phenotypic characteristics: quiescence, chemotherapeutic resistance, the ability to selfrenew and differentiate. We and others have shown that the CD138-population is consistently detected in MM cell lines and material derived from patient BM, suggesting that these are a mixture of the mature, rapidly dividing CD138+ cells, and immature, slowly proliferating CD138-cells [4,[16][17][18]21]. The fact that these populations are maintained at a near constant ratio within the tissue culture system suggested that there had to be a mechanism to maintain this set point, because otherwise the quiescent CD138-cells should be lost from the culture during passaging. We show that the mature MM cancer cell detected in tissue culture has developmental plasticity and can dedifferentiate back into its own chemoresistant, CD138-CSC progenitor. These newly born CSC regain all of the characteristics of the CSC demonstrating that stemness is a trait that is epigenetically controlled. Thus, instead of a CSC, we suggest that stemness is a dynamic, autoregulated phenotype that can be acquired or lost depending on the interaction with surrounding cells.
This interconversion occurs in the absence of support or accessory cells, suggesting that the MM cells themselves secrete cytokines or factors that mediate this plasticity, and in essence sense and make their own niche. The pure CD138-and C138+ populations secreted MIF-1, although to different levels. The secretion of several other cytokines increased but to a much more modest extent, and by blocking or increasing MIF-1 activity, we were able to show that MIF-1 could alter interconversion, and more importantly chemosensitivity. Blocking MIF-1 drives cells to dedifferentiate into chemoresistant CD138-cells, and the addition of rMIF-1 drives cells to differentiate into chemosensitive CD138+ cells. These cells can be toggled back and forth depending on MIF-1 activity or levels, offering new insight into the epigenetic control of MM growth. Too little MIF-1 and cells dedifferentiate; too much MIF-1 and cells differentiate.
CD138+ cells secreted MIF-1 more rapidly, although secretion at a lower level is still detected in the pure CD138-populations. MIF-1 is known to associate with multiple receptors, including CD74, CXCR2/4/7 and CD44 [27][28][29], and one differential expression may start the interconversion process. Differential gene expression in the CD138-and CD138+ MM cells has been described further supporting the idea that they represent two different functional populations, and expression of genes associated with stemness, such as NANOG, Wnt, and Hedgehog, are preferentially detected in CD138-precursor cells [30]. Additionally, our study demonstrates that small changes in MIF-1 activity or levels in both cell lines and in primary MM tumor material can shift the set point in one direction or the other. Cytokines are also secreted from the BM microenvironment, and both chemical and physical cues, such as adhesion and oxygen tension, may influence interconversion [31][32][33]. MIF-1 has emerged as a candidate controlling plasticity and interconversion [33][34][35][36], and the logical extension of this work will be to validate this in more physiological in vivo settings. Perturbation or modulation of the MIF-1 levels could shift the balance in one direction or the other, and thus represents a viable way to increase the percentage of CD138+, chemosensitive cells. In our preliminary cohort of patients, the addition of 4-IPP to bone marrow derived MM cells increased or maintained the CD138-population in 5 out of 6 patients, suggesting that similar mechanisms are at play in vivo. We are intrigued by the observation that some patients had very high CD138pools at harvest, and this might explain intrinsically different patient responses to chemotherapy. While we examined clinical parameters and outcomes (Fig 7SA), it is difficult to make conclusions from this small sample size. In our tissue culture assay, cells are only viable for a short term, and in the future culturing cells in a 3D matrix will permit more detailed analysis.
Recently, others have reported that MIF-1 can regulate the adhesion of MM tumor cells to BM [37]. Higher levels of MIF-1 expression are seen in MM tumor cells than in normal plasma cells, and loss of MIF-1 reduced MM adhesion to BMSC in vitro. While MIF-1 expressing cells formed tumors in the bone when injected into SCID-mice, cells lacking MIF-1 only formed tumors in the abdomen or extramedullary space, suggesting that MIF-1 enhances BM colonization. While these authors did not directly examine interconversion, there was an increase in CD138cells in the tumors taken from extramedullary space in the MIF-1 KD animals. It is entirely possible that loss of MIF-1 and an increase in CD138-CSCs would affect BM homing and adhesion. They also found that the inhibition of MIF-1 either through KD or 4-IPP treatment caused apoptosis in MM cells after treating with Melphalan in the presence of accessory cells [37]. While the mechanism of action of Melphalan and Bortezomib are very different, our data is in agreement with theirs that altering MIF-1 levels affects chemotherapeutic response, highlighting MIF-1 as an important target.
Our model suggests that when presented with the appropriate environment, any myeloma tumor cell might have the ability to acquire "stemness", which increases the difficulty in treating a moving target. Our very use of chemotherapy and its change to the microenvironment may permit the reactivation of the chemoresistant clone. If interconversion provides a way to continually renew the chemoresistant CSC population, this would contribute to the eventual relapse seen in this disease, and call for the development of therapies to specifically target plasticity.
Flow cytometry and sorting
Cells were stained with CD138 PE-Cy5 (Beckman Coulter), CD38 PE, Isotype Control IgG1 PE, CXCR4 PE, or CD44 FITC (BD Pharmingen), CD74 FITC (Abcam), and Blimp1 Alexa-Fluor 488 (R&D) by standard procedures. All flow cytometry analysis used the BD FACScan, the BioRad S3 Cell Sorter and data was analyzed using CellQuest software. Cells were stained with Vybrant DyeCycle Green Stain (Invitrogen) and cell cycle analysis was performed using CellQuest software. Freshly sorted populations were also resuspended in methylcellulose (Stem Cell Technologies) at 10 4 cells/ml as per manufacturer's instructions. Colonies were counted manually and photographs were taken using a light microscope under 4X magnification. Colonies were measured using the annote tab to compare pixel number to the scale created by the Kodak film software.
Tracking assay
Cells were treated with 5μM PD 0332991 for 72 h., before washing in fresh media and staining with 5μM CFSE for 10 min. Cells were further stained with CD138 PE-Cy5 and CD38 PE and sorted into CFSE hi CD138 + CD38 + and CFSE hi CD138 -CD38 + and re-plated as above.
Cytokine array and analysis
Pure CD138 + CD38 + and CD138 -CD38 + populations were replated in complete media, and harvested at days 1,2, 4 for staining with CD38 PE and CD138 PE-Cy5, while media for each time point was stored at -20˚C for later assessment using the Human Cytokine Array Panel A (R&D). Capture antibodies against the cytokines listed in Fig 4A were spotted in duplicate on nitrocellulose membranes. Cell culture supernatant is mixed with a cocktail of biotinylated detection antibodies, and the sample/antibody mixture is then incubated with the array. Any cytokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Arrays were analyzed using IRDye 800CW Streptavidin (LICOR) and the LICOR Imaging System following vendor's instructions. Raw values for each cytokine detected in media alone were subtracted from values for each cytokine at each time point. Data presented is representive of N = 2 experiments.
Chemosensitivity assay
Sorted populations were treated with 0.01μM Bortezomib (SelleckChem) for 24 h. Chemosensitivity was measured as the change in viability before and after treatment as measured by trypan blue exclusion. MTT Assay (Promega) used to measure viability as described.
Primary MM patient samples
All research involving human participants was approved by the authors' Institutional Review Board (IRB) of SUNY Downstate Medical Center, and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Informed consent, written or oral, was obtained from all participants. Bone marrow aspirates from MM patients at Kings County Hospital or University, obtained with informed consent, were purified by passage through the MM enrichment kit (RosetteStep). RosetteStep MM removes cells with CD2, CD14, CD33, CD41, CD45RA, CD66b and glycophorin A on red blood cells (RBCs). When centrifuged over a buoyant density medium, the unwanted cells pellet along with the RBCs. The purified multiple myeloma cells are present as a highly enriched population at the interface between the plasma and the buoyant density medium. Purified plasma cells were either grown in tissue culture +/-4-IPP or immediately fixed and stained with CD38 PE and CD138 PE-Cy5. Viability of cells was checked by trypan blue staining. Cells were grown using the patient's own serum, obtained from peripheral blood.
Statistics
All data are shown as the mean ± SD. The statistical analyses were performed using the Exact Mann-Whitney 2-sided tests, a semi-parametric (SAS PROC MULTTEST with bootstrapping) approach was used to (1) apply a t-test of monotonic trend, allowing for possible non-normality; (2) correct p-values derived from pair-wise t-tests for possible non-normality and for multiple testing. The Satterthwaite adjustment for heteroscedasticity was also applied. Selected pair-wise comparisons were made using t-tests (with Satterthwaite corrections to allow for possible unequal variances). The distribution of the dependent variable was checked for symmetry and for the presence of outliers. All other statistics used Student t-test (2 tail). O) Dot Plot of Sorted CD138+ and CD138-NCI-H929 cells. The CD138null population (bottom in the dot plot) was non-viable and was gated out of all analysis. P, Q) Sorted populations of CD138-and CD138+ cells. R) Cell counts for experiment the plated, pure, sorted CD138and CD138+ population. Growth rates were calculated and are the mean of the growth seen over a 5 day period (1.1 for CD138-and 1.2 for CD138+). S) Cell counts plotted. T) CD138plated experiment. 250000 cells were plated at day 0. 0.73% of 250,000 is 1825 contaminating CD138+ cells. We predicted that this population would expand to 2190 cells at day 2, given the growth rate of 1.2 seen for these cells. However, we detected 76,480 CD138+ cells or 23.9% of the total population of 320,000 cells. U) CD138+ plated experiment. 250,000 cells were plated at day 0. 0.17% of 250,000 is 425 contaminating CD138-cells. We predicted that this population would expand to 466 cells at day 2, given the growth rate of 1.1 seen for these cells. However, we detected 13,200 CD138-cells or 3.3% of the total population of 400,000 cells. | v3-fos-license |
2019-04-30T13:06:03.699Z | 2017-11-22T00:00:00.000 | 139302574 | {
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} | pes2o/s2orc | CHARACTERIZATION OF ALGINATE PREPARED FROM BROWN SEAWEEDS IN THUA THIEN-HUE PROVINCE , VIETNAM
The characterization of alginate prepared from brown seaweeds in TT-Hue province of Vietnam was investigated. Composition and the sequential structure of alginate were determined by 1 HNMR, 13 C-NMR, TOCSY, 1 H1 H COSY, and 1 H13 C-HSQC spectrum. It is found that alginate with molecular weight around 101 KDa is composed of homopolymeric regions of M and G blocks, interspersed with very small amount of regions of alternating structure of MG block. This information is essential for application guideline of alginate in TT-Hue province.
INTRODUCTION
Alginate, a family of non-branched binary copolymers of 1-4 glycosidically linked -Dmannuronic acid (M) and α-L-guluronic acid residues can be considered as patterns of homopolymeric blocks of G and M, respectively, and blocks with an alternating sequence, all in coexistence.Alginate contains all four possible glycosidic linkages: diequatorial (MM), diaxial (GG), equatorial-axial (MG), and axial-equatorial (GM) [1].Chemical block structures of alginate are presented in Fig. 1.
It has been known that the physical properties of alginates depend on the relative proportion of four types of blocks.The industrial utilization of any particular alginate will depend on its properties and therefore on its uronic acid composition, however, the composition and sequential structures of alginate of different resource are different from one place to others.From a scientific point of view, the uronic acid composition of alginate from different sources had to be examined.ThuaThien-Hue province is the zone in which some brown seaweed species are mainly distributed with the yield estimated in wet state to be about 3,000 metric tons per year [2].Attempts to study on the brown seaweed have been made so far.Vo Thi Mai Huong et al [3,4] investigated the technology of alginate extraction from sargasssum of TT Hue and some of their bio-physical and bio-chemical specifications.However, no single paper studying on the composition and the sequential structure of alginate extracted from brown seaweeds in TT-Hue has been reported so far.In previous papers [5 -7], the authors demonstrated the use of the second order orthogonal design in the extract of sodium alginate from brown seaweeds in TT-Hue province.The desired quality of alginate can be obtained from appropriate technology regimes based on regression equations.In the present work, the characterizations of obtained alginate are discussed.
EXPERIMENTAL
Alginate was prepared from the brown-seaweed of TT-Hue in the manner described in the previous paper [5 -7].The viscosity average molecular weight of alginate was calculated using classical Mark-Houwink equation: is the intrinsic viscosity of alginate.The intrinsic viscosity of alginate in 0,1M NaCl solution were measured in triplicate for each of six different concentrations by recording the efflux time of the solution in Ubbelohde viscometers maintained in a constant-temperature water bath at 25 0.2 o C. K = 7.3.10 - and = 0.92 are constants for given solute-solvent system and temperature [8,9].The obtained alginate was characterized by 1 H-NMR, 13 C-NMR, 1 H-1 H TOCSY (TOal Corelated SpectrometrY), COSY (Correlated SpectrometrY), HSQC (Heteronuclear Single Quantum Correlation) using Bruker Advance 500 MHz, NaOD 5% solution in D 2 O were used as the solvent.The carbon atoms in mannuronate and guluronate residues are denoted and named as G1, G2, G3, G4, G5, G6, M1, M2, M3, M4, M5 and M6 as shown in Fig. 1.
RESULTS AND DISCUSSION
The resultant calculation of the viscosity average molecular weight showed that the viscosity average molecular weight of alginate is around 101 KDa as shown in Table 3. Fig 2 shows 1 H-NMR of sodium alginate. 1H-NMR spectrum of alginate consists of ten peaks in correspondence to 10 proton resonances of mannuronate and guluronate.Two peaks of 5.473 and 5.090 ppm located at the lowest magnetic field side should be assigned to G1 hydrogen and M1 hydrogen, respectively because of the strongest deshielding by two oxygen atoms adjacent to them and the deshielding by oxygen atom from axial linkage is stronger than that by oxygen atom from equatorial linkage.From TOCSY spectrum as shown in Fig 3, it could be seen that G1 hydrogen interacts with the protons of G5, G4, G3, and G2 at 4.883, 4,571, 4.446, 4.318 ppm.Among them, the interaction signal of G1 and the other proton at 4.883 ppm is the smallest so the peak at 4.883 ppm should be attributed to G5.In order to interpret the neighboring proton of G1, the 1 H-1 H COSY of alginate has been performed (Fig. 4).G1 hydrogen only interacts with the proton at 4.318 ppm.Then the peak at 4.318 ppm should be assigned to G2 hydrogen.In turn, G2 hydrogen interacts with the proton located at 4.446 ppm.The peak at 4.446 ppm is the resonance signal of G3 hydrogen.The rest of 4.571 ppm is G4 hydrogen.In the same manner, the TOCSY spectrum confirms that M1 hydrogen interacts with the protons having resonance signals of 4.446, 4.318, 4.152, 4.135 ppm.From COSY spectrum, the peak at 4.446 ppm resulted in interaction of M1 should be attributed to the proton of M2.Referring back to TOCSY spectrum, in addition with the interaction of proton of M2, M1 proton interacts with two protons at 4.152 and 4.318 ppm.Two these peaks should be assigned as the protons of M3 and M4.On the other hand, the interaction of M2 with the proton at 4.152 ppm is observed in COSY spectrum.As resulted, the resonance signal at 4.152 ppm is obviously of M3 hydrogen.The rest of 4.318 ppm is assigned to M4 hydrogen.In both COSY and TOCSY spectrum, it could be seen that M4 proton interacts with the other proton at the highest field and consequently, the signal at 4.135 ppm is certainly of M5.The resultant assignment of alginate peaks is listed on Table 1 and Fig. 2.However, 1 H-NMR could not provide information of MG and GM blocks so the 13 C-NMR was performed.Drawing horizontal lines from identified peaks of 1 H-NMR and vertical lines from peaks of 13 C-NMR, resonance signals of corresponding carbons are listed in Table 2. 13 C NMR spectrum of C-1 carbons is shown in Fig. 7. C-1 carbons of mannuronate and guluronate show peaks in the range of 100-102 ppm, and each peak is separated owing to the vicinal residue bonded with carbon.MG, GG, MM and GM in the order from lower magnetic field side should have been observed [10].In that case, only two peaks around 101 ppm was obtained.The question is whether these two peaks are assigned to (MG-GG), (GG, MM), or (MM-GM) or not.
CONCLUSION
This is the first study on the structure of alginate prepared from brown seaweeds in TT-Hue province.It was found that alginate consists of block copolymers composed of homopolymeric regions of M and G, interspersed with very small regions of alternating structure of MG block.We hope that this work will be useful for applications of the investigated alginate to agriculture and medicine.
Table 2 :
Chemical shift of protons of D-mannuronic acid (M) and L-guluronic acid (G).Similarly, guluronic acid may form homopolymeric structure.Peaks around 100.5 and 100.2 ppm of GM and MG are very small but visible which demonstrates that GM and MG blocks are very shorter in comparison with homogeneous blocks.The amount ratio of mannuronic acid and guluronic acid (M/G ratio) in the alginate and the fraction of MM block and GG block are calculated by integrated areas of H1 of M and G.These values are also determinated by the intensities of carbon M1 and G1 but it isn't certain result.The resultant calculations are shown in Table3.The unequivalent amount of M and G as seen in Table3can be contributed to negligible amounts of MG and GM block.It is concluded that the obtained alginate is composed of chains of homopolymeric block M and G in which chain of block M is longer, interspersed with very small amounts of regions of alternating structure of MG block.
Table 3 :
Chemical characteristics of alginate. | v3-fos-license |
2020-06-20T05:05:48.693Z | 2020-06-19T00:00:00.000 | 219889620 | {
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} | pes2o/s2orc | Comparison of hydrophilic ophthalmic media on silicone oil emulsification
The aim of this study was to investigate whether and how the biological media which are in contact with silicone oil play a role in the silicone emulsification process. Commercially available Oxane 1300 silicone oil and potential hydrophilic phases of the emulsions in the eye (porcine aqueous humor, porcine vitreous and balanced salt solution) were investigated separately and in a mixture or emulsions by means of surface tension, rheological, zeta potential measurements and microscopic investigation. The surface tension of biological media (vitreous and aqueous humor) was significantly lower than that of non-biological media, especially in the case of aqueous humor, which indicates a remarkable emulsification tendency with these phases. The biological media are able to form both oil-in-water and water-in-oil emulsions, which can be observed in the clinical practice as well. It was established that the vitreous has a more expressed emulsification ability compared with the aqueous humor because smaller and more stable droplets can form with silicon oil when the vitreous is still there. It can be concluded that the vitreous has a higher impact on emulsification than the aqueous medium, which can predict that the vitreous remaining after vitrectomy has a key role in emulsion formation in the eye with silicone oil endotamponade.
Introduction
Silicone oil endotamponade has frequently been applied in the last decades since its introduction by Armaly [1] and Cibis in 1962 [2]. After implantation, silicone oil forms a spherical bubble in the vitreous cavity, but some residual vitreous can remain at the vitreous base, especially in phakic patients. Intraocular silicone oil is in contact with the residual vitreous, and aqueous humor secreted by the ciliary body. Silicone oil, however, may undergo emulsification in 4-72% of the cases and lead to vision-threatening complications affecting nearly all ocular structures. Complications can include corneal decompensation, band keratopathy, acute and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 chronic changes in intraocular pressure, lens opacities, epiretinal membrane, retinopathy, optic neuropathy, and extraocular extension (such as migration into the optic nerve, chiasm and even into the cerebral ventricular system) [3][4][5][6][7][8]. The emulsification of silicone oil depends on the interfacial tension between the oil and the hydrophilic phases (aqueous, vitreous or balanced salt solution (BSS)), which means decreased interfacial tension results in increasing emulsification tendency. The time course and level of emulsification are rather variable [9], but it was established to occur within the first year and mainly after the 5 th postoperative month [10].
Besides the physicochemical characteristics of silicone oils, biological factors such as biological environment, emulsifiers and mechanical effects can influence the development of emulsification. On the other hand, based on literature information, to date there is no data on how the complex ocular biological media can influence emulsification.
The purpose of the current study was to develop an in vitro model for the complex investigation of the phenomenon of silicone oil emulsification in the presence of potential ophthalmic hydrophilic phases such as aqueous humor, vitreous and BSS. Our aim was to evaluate the emulsification ability of the biological media and to analyze the formed emulsions.
Materials
Silicone oil. Original silicone oil Oxane 1300 (Bausch&Lomb GmbH, Germany) (SiO) and potential hydrophilic and lipophilic phases of the emulsions were investigated separately and in a mixture or emulsions.
Hydrophilic phases. For the hydrophilic phase of the emulsions balanced salt solution (BSS, Alcon Laboratories, Inc., USA) and biological aqueous phases such as porcine aqueous humor (AH) and porcine vitreous (VB) were used. Porcine eyes were freshly obtained from a slaughterhouse (Pick Szeged Zrt., Szeged, Hungary) (animals were not sacrificed for the study). The aqueous was aspirated through corneal paracentesis and the vitreous was removed with scissors 2-5 hours postmortem from 100 porcine eyes. The humors were homogenized, chilled and stored at -20˚C. They were melted at room temperature just before the application. Our applied microscopical and zeta potential methods required high optical purity, but the biological fluids contain often pigments and other non-soluble components. Based on our preformulation studies, membrane filters with pore size 0.45 and 0.20 microns were not suitable for the separation of the non-soluble components. Therefore the clear biological aqueous phases were separated from the pigments by centrifugation in Vivaspin 15R 5,000 MWCO Hydrosart tubes (Sartorius, Stonehouse, UK) with Hermle Z323K, (HERMLE Labortechnik GmbH, Wehingen, Germany). Centrifugation was performed at 5000 rpm (9000 rcf), for 30 min at 4˚C. This separation method resulted optically clear biological fluids. Mixtures of the vitreous and BSS were made to model in vivo vitrectomized (and silicone oil filled) eyes with residual vitreous.
In vitro emulsions. Silicone oil and the hydrophilic phases were mixed with a Vortex stirrer at 50% of maximum speed for 5 min. The emulsions/mixtures were measured immediately after preparation. Each emulsion was prepared three times. Two ratios of the oil and aqueous phase were applied 2:8 and 8:2.
Methods
Surface tension. Original silicone oils and aqueous phases were analyzed by means of surface tension measurements, by OCA 20 instrument (Dataphysics Instruments GmbH, Filderstadt, Germany) using the pendant drop method. The instrument detects the contour of the pendant drop and calculates the surface tension. The mean of ten parallel measurements was used for calculations.
Microscopic investigation. For the microscopic observations, the hydrophilic phases of the emulsions were stained with 0.001% Fluorescein sodium (Sigma-Aldrich GmbH, Germany) in a 100:1 ratio. 50 μL of emulsions were put onto microscope slide and covered with cover glass. Microscopic images were made by a Leica DMB6 microscope (Biomarker GmBH, Hungary) in fluorescence mode (Leica DFC7000 T fluorescence camera, Biomarker GmBH, Hungary). The magnification was 200x. Droplet size and droplet size distribution were analyzed on the basis of the black and green contour of the oily and aqueous phase using the LAS X core software of the microscope.
Each composition was prepared three times, and each sample was scanned by microscope. The images, where droplets were observed, were analyzed by the software.
Emulsions with both ratios (2:8 and 8:2 oil and aqueous phase ratios) were also investigated.
Zeta potential. The zeta potential of the in vitro emulsion was measured by Zetasizer Nano ZS (Malvern Instrument, UK) with electrophoretic light scattering. Emulsion droplets are surrounded by an electric double layer. This double layer is formed when surface-chargecarrying emulsion droplets are surrounded by counter-ions of charge opposite to that of the droplet surface. A given amount of these counter-ions move together with the droplet, hence a slipping plane beyond the emulsion droplet. The electrical potential at this slipping plane is the zeta potential (z, mV). As a result of increased electrostatic repulsion, the coalescence of the oil droplets is hindered. An emulsion with a high absolute value of zeta potential is more stable in comparison to that with lower zeta potential absolute values [11].
With this method, emulsions with 2:8 oil and aqueous phase ratio were investigated. Rheology. The viscosity of silicone oil and in vitro formed emulsions/mixtures was measured by a Physica MCR 101 rheometer (Anton Paar, Austria). The measuring device was cone and plate type (diameter 25 mm, gap height in the middle of the cone 0.046 mm, cone angle 1˚). The flow and the viscosity curves of the samples were plotted. The shear rate was changed from 0.1 to 100 s -1 . The viscosity of the samples was evaluated at 100 s -1 . The experiments were carried out in triplicate.
With this method, emulsions with 2:8 oil and aqueous phase ratio were investigated.
Statistics
Unpaired t-test was performed using GraphPad Prism (GraphPad Software Inc., USA). P value < 0.05 was considered to be statistically significant. A level of p � 0.05 was taken as significant, p � 0.01 as very significant, and p � 0.001 as highly significant.
Surface tension
All hydrophilic substances had significantly lower surface tension than that of BSS. Aqueous humor had the lowest surface tension (54.99 ± 1.98 mN/m), which was followed by the value of the vitreous (61.16 ± 1.30 mN/m), while BSS had the highest value (72.62 ± 0.08 mN/m) (Fig 1). The lower surface tension of the hydrophilic phase can mean lower interfacial tension. The surface tension of fresh and freeze-thaw vitreous was also compared in order to investigate the effect of the freezing. Our results indicated the surface tension remained the same (S1
Microscopic investigation
For easy distinction, a fluorescent dye was applied during the microscopic measurements. Green fluorescein sodium can dye the aqueous phases of the emulsions, while silicone oil can be seen with black color in the pictures. A few large and many small black oil droplets can be observed in the green aqueous phase in the microscopic images (Fig 2a, 2b and 2c) when a high amount of aqueous phase (80%) was applied. At this oil:water ratio an oil-in-water type emulsion was formed.
The microscopic image of the emulsion containing VB showed a greater amount and smaller oil droplets in the aqueous phase compared with that of the emulsion with AH, while in the case of BSS the typical picture of the emulsion was a main giant continuous oil droplet and a few bigger ones (Fig 2a).
When a higher amount of silicone oil (80%) was applied, a water-in-oil type emulsion could be observed (small green aqueous droplets in the black oil phase) (Fig 3a, 3b and 3c). There was no difference between the two emulsions containing biological media, while the droplet frequency was lower when BSS was used as the hydrophilic phase of the emulsions.
The sharp contour of the droplets made it possible to analyze droplet size and droplet size distribution using the software of the microscope (Fig 4). The emulsions containing BSS proved to have greater droplet frequency at bigger droplet size (above 100 μm). The droplet size distributions of the emulsions containing VB or AH were almost similar, but in the case of VB a higher amount of smaller droplets could be observed (more remarkable frequency below 20 μm).
Droplet size and zeta potential
Zetasizer was used to measure droplet size from nanometer to several microns using dynamic light scattering, and zeta potential using electrophoretic light scattering. This type of droplet size measurement can avoid the errors from microscope resolution and from the selection of the photo imaging.
In our work the droplet size and the zeta potentials of emulsions containing 20% silicone oil were only measured (Fig 5 and S1 Table), since a larger percent of oil could not be measured by this method.
The average droplet size (Z ave ) of emulsions containing VB was significantly smaller (about 20 μm) (p = 0.018), while that of emulsions containing AH was bigger (over 60 μm). This observation correlates with the results of the droplet size distribution presented in the microscopic investigations (Fig 4).
The absolute zeta potential of the emulsion with VB was remarkably higher than that of AH (Fig 6). The zeta potential value can indicate the formation of an electric double layer surrounding the oil droplets stabilizing the emulsion droplets. The increase of the absolute values can predict an improvement in the stability of the disperse system.
Rheology
The viscosity of the original oil Oxane 1300 was measured to be 1313.3±5.8 mPa � s (mean ±SD). Emulsions were prepared on the basis of the "In vitro emulsion section", where the Oxane concentration was 80%, while the aqueous phase (AH or VB) concentration was 20%. Each emulsion was prepared in triplicate.
PLOS ONE
Comparison of hydrophilic ophthalmic media on silicone oil emulsification In the case of emulsions containing AH, the viscosity values were lower than those of Oxane, and the standard deviation (SD) was very high. Contrarily, the emulsions containing VB revealed elevated viscosity values with moderate standard deviation (Table 1.)
Discussion
The implanted silicone oil forms a giant continuous oil droplet in the vitreous cavity, which is surrounded by the thin layer of the hydrophilic phase remaining in the eye. This hydrophilic phase can be a leftover vitreous, aqueous humor secreted by the ciliary body, or buffer solution applied during vitrectomy. Silicone oils produce high interfacial tension opposite to the hydrophilic phases, and this behavior enables these materials to exert an intraocular endotamponade effect in the vitreous body. Despite the high surface tension, in practice silicone oils undergo emulsification, which can lead to several complications including cataract, keratopathy, glaucoma and silicone retinopathy [13]. Silicone oils have excellent chemical stability, but many studies proved that they are not inert biologically, some impurities such as retinol [14], lowmolecular-weight components (LMWC) [4], residual catalysts [15], OH-endgroups [16], and cholesterol [17] were detected in silicone oils, which can probably improve emulsification due to the modification of the surface tension of the oil. The other possible cause of emulsification is the mechanical effect, therefore, some literature analyzed the effect of different agitators and eye movement on emulsification using special eye chambers, which mimic the eye movement in vitro [18,19].
A new mechanism of SiO emulsification was also proposed, where break-ups of SiO lead to its adherence to ocular tissue and thus forming emulsified droplets. This mechanism can decrease with the application of high-molecular-weight SiOs [20,21] and preactivated SiOs, the latter is able to form a cohesive gel in the vitreous cavity [22].
Impurities and mechanical effects can be largely patient-dependent because these factors can be disease or surgical action related. In our work we focused on different biological and surgical hydrophilic phases which can be present during vitrectomy and can be responsible for the early development of emulsification. These hydrophilic phases can be in contact with silicone oil and are able to form an interface. When the surface tension at the interface is low and a mechanical effect occurs, it promotes the formation of droplets. In our work the surface tensions of the possible hydrophilic phase were investigated, and it was found that the biological media have significantly lower surface tension compared to the non-biological ones (BSS) (about 61 and 54 mN/m compared with 72 mN/m). On the basis of Antonov's rule and its later corrections and modifications [23], these lower surface tension values can mean lower interfacial tension between silicone oils and biological aqueous media, and the lower interfacial tension can result in increased emulsification.
In our work we formulated in vitro emulsions with silicone oil and the possible hydrophilic phases in order to investigate the structure and stability of the emulsions, and also to compare the emulsification ability of the different hydrophilic phases. If an inhomogeneous system is formed (not a stable emulsion system), it will result in varying viscosity data, as we can see for emulsions containing AH. When an emulsion is formed (aqueous droplet in the oily phase), a higher viscosity value can be measured due to the viscosity increasing effect of the dispersed droplets (this tendency can be observed in the case of emulsions with VB) [12]. https://doi.org/10.1371/journal.pone.0235067.t001 Two types of emulsion can develop after silicone oil implantation, both of them can be observed in clinical practice. In the case of the first emulsion type, the oil droplets get loose from the giant continuous oil droplet and form an oil-in-water emulsion (o/w emulsion). These emulsified oil droplets are able to move easily in the hydrophilic phase, thus they can be present in the aqueous humor, which can be seen in the anterior chamber in the human eye after undergoing vitrectomy with silicone oil endotamponade. On the other hand, the migration of small oil droplets can lead to secondary silicomacrophagocytic open-angle glaucoma [24]. In our study this type of emulsion could be observed when we applied a high amount of biological hydrophilic phase. It had already been established in earlier studies that the components of the biological medium have a strong effect on emulsification. The role of fibrinogen, fibrin, γ-globulin, VLDL, alfa-1-glycoprotein in emulsification had already been clarified in vitro [16]. In our work the complex hydrophilic phases were compared concerning the emulsification tendency. The aqueous phases were indicated by fluorescent dye, thus black oil droplets were perceptible in the green aqueous phase on the microscope slide of our in vitro emulsions (Fig 2). The number of the oil droplets was higher, and the droplet size was smaller in the case of VB compared with the BSS and AH containing systems. This observation was confirmed by the droplet size and zeta potential measurements. Zeta potential is a sensitive indicator of the stability of disperse systems such as emulsions. Higher absolute Zeta potential means an effective stabilizing layer around the droplets, which hinders the adhesion and the fusion of the droplets. In contrast with microscopic investigation, where the presented and analyzed slides are chosen by the investigator, these types of measurements are more investigator-independent, a bigger amount of the sample can be analyzed using dynamic and electrophoretic light scattering, which results in more representative data from the sample. In this measurement, similarly to the microscopic investigation, smaller oil droplets were present in the case of VB, furthermore the absolute Zeta potential of the emulsions containing VB was also higher compared with emulsions containing AH. These statements can mean that the residual vitreous body, which can form an interface with the silicone oil droplet, has more emulsification potential, and it can be the starting point of further emulsification. On the other hand, the formed oil droplets are more stable in the vitreous (indicated by the higher absolute Zeta potential value), predicting that the oil droplet will remain and will not fuse with the continuous oil droplet later.
Earlier studies established the role of the characteristics of the silicone oil and the surfaceactive agents in the biological environment in the emulsification. The presence of the very small amounts of emulsifier can facilitates the formation of an emulsion. Various biologic surface-active agent can accumulate in hemorrhagic and inflammatory situations in the aqueous biological media such as fibrin, fibrinogen, gamma globulins, acidic alpha 1-glycoprotein, very low-density lipoprotein [10]. In our present biological media were collected from healthy animals and were investigated as a complex fluid. We cannot denominate only one two or three components whose concentration or presence can be the key factor(s) in the emulsification, but we can clearly see, the normal biological fluids have remarkable emulsifying effect. In addition, it is also clear from the results that the residual vitreous can have a much greater effect on subsequent emulsification than aqueous.
As for the second emulsion type, droplets from the hydrophilic phase can form and enter the oil phase resulting in a water-in-oil type emulsion (w/o emulsion). This type of emulsion can be observed in the vitreous cavity when the opalescence of silicon oil is noticed in clinical practice. In our study this type of emulsion was obtained when lower hydrophilic phase was applied (20%). The microscopic investigation indicated hydrophilic droplets in the continuous oil phase in all cases, but the biological media showed remarkable emulsification tendency. In order to compare the emulsification ability of the two biological media as a water-in-oil emulsion, rheological investigation was performed and the viscosity of the systems was evaluated. This method is considered to be a more investigator-independent test than the zeta potential measurement. In our work the viscosities of the w/o emulsions containing biological media were compared with the original oil viscosity data. When a nearly stable w/o emulsion is formed, the emulsified droplets increase the viscosity of the continuous phase, in our case this phase is silicone oil. Considering the viscosity data of the formulated in vitro w/o emulsions, mixtures with AH exhibited varying viscosity data indicating an unstable system, while the viscosity of the emulsion containing VB showed an elevated value, which predicts a real, stable emulsion formation. The latter suggests the VB droplets are more stable in the continuous silicone phase.
Conclusions
As a practical conclusion, the residual vitreous body can increase the risk of the emulsification of silicone oil because it has remarkable emulsification potential concerning both emulsion types (w/o and o/w). The amount of the residual can be also critical because a higher amount can form a larger interface between the oil and the vitreous, and it can result in increasing the risk of starting the emulsification. Our multimodal complex in vitro model provided evidence on the clinically known feature, namely that as complete a removal of the vitreous should be performed as possible in order to minimize the emulsification ability of biological media. | v3-fos-license |
2020-12-17T09:06:04.936Z | 2020-12-16T00:00:00.000 | 230603068 | {
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} | pes2o/s2orc | The intermittent fasting-dependent gut microbial metabolite indole-3 propionate promotes nerve regeneration and recovery after injury
The regenerative potential of mammalian peripheral nervous system (PNS) neurons after injury is critically limited by their slow axonal regenerative rate1. Since a delayed target re-innervation leads to irreversible loss of function of target organs2, accelerated axonal regeneration is required to enhance functional outcomes following injury. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms3. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration4. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and release of neurotrophins, can be activated by intermittent fasting (IF)5,6. IF has in turn been shown to increase synaptic plasticity7,8 and neurogenesis9, partially sharing molecular mechanisms with axonal regeneration. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in the mouse via an unexpected mechanism that relies upon the gram + gut microbiome and an increase of the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype that was confirmed by the inhibition of neutrophil chemotaxis. Our results demonstrate for the first time the ability of a microbiome derived metabolite, such as IPA, in facilitating regeneration and functional recovery of sensory axons via an immune-mediated mechanism. Since IPA is a naturally occurring metabolite with a very favourable toxicity profile, it represents a realistic translational possibility for human axonal injuries.
Abstract
The regenerative potential of mammalian peripheral nervous system (PNS) neurons after injury is critically limited by their slow axonal regenerative rate 1 . Since a delayed target re-innervation leads to irreversible loss of function of target organs 2 , accelerated axonal regeneration is required to enhance functional outcomes following injury. Regenerative ability is in uenced by both injury-dependent and injury-independent mechanisms 3 . Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration 4 . Several of these pathways, including modi cations in gene transcription and protein synthesis, mitochondrial metabolism and release of neurotrophins, can be activated by intermittent fasting (IF) 5,6 . IF has in turn been shown to increase synaptic plasticity 7,8 and neurogenesis 9 , partially sharing molecular mechanisms with axonal regeneration. However, whether IF in uences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in the mouse via an unexpected mechanism that relies upon the gram + gut microbiome and an increase of the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for e cient axonal regeneration, and delivery of IPA after sciatic injury signi cantly enhances axonal regeneration, accelerating recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype that was con rmed by the inhibition of neutrophil chemotaxis. Our results demonstrate for the rst time the ability of a microbiome derived metabolite, such as IPA, in facilitating regeneration and functional recovery of sensory axons via an immune-mediated mechanism. Since IPA is a naturally occurring metabolite with a very favourable toxicity pro le, it represents a realistic translational possibility for human axonal injuries.
Main
Injuries to the peripheral nervous system (PNS) have a high prevalence with millions of affected individuals worldwide and are typically followed by long-lasting neurological disability without effective treatments beyond surgical reconstruction, which is only effective in a small percentage of cases [10][11][12] .
These injuries frequently result in partial or total loss of sensory, motor and autonomic function due to ine cient and slow axonal regeneration 13,14 . The study of cellular and molecular responses to injury 15,16 , of neurodevelopmental pathways 17,18 , of regenerative organisms 18,19 , as well as molecular screening approaches 20 have contributed to the identi cation of a number of mechanisms that in uence regenerative ability. However, our knowledge of the cellular and molecular mechanisms underpinning axonal regeneration and functional recovery remains incomplete, undermining the development of effective treatments for clinical injuries.
Recently, accumulating evidence implicates healthy lifestyle choices such as exercise and environmental enrichment in priming and enhancing the regenerative potential of sensory neurons 4 . Similarly, dietary regimens including intermittent fasting (IF) have been shown to promote metabolic and signalling pathways with the potential to favour wound repair and recovery in several disease states, including following nervous system injury [21][22][23][24] . Therefore, we investigated whether IF would prime sensory neurons for enhanced axonal regeneration and recovery in a model of sciatic nerve injury and whether it would enable the identi cation of speci c molecular mechanisms with translational potential for nerve repair.
Intermittent fasting promotes axonal regeneration following sciatic nerve crush via the gram + gut microbiome Axonal regeneration was initially assessed 24 hours after a sciatic nerve crush (SNC) following 10 or 30 days of IF versus an ad libitum (AL) diet. Both 10 and 30 days IF regimens signi cantly enhanced regeneration past the crush site to a similar extent (Extended data Fig. 1a-c), therefore all subsequent experiments were performed following 10 days of IF. Importantly, IF also promoted axonal regeneration when the recovery time was extended to 72 hours after SNC (Fig. 1a-c). In con rmation of these ndings, IF was followed by an increase in neurite outgrowth in cultured dorsal root ganglia (DRG) neurons (Extended data Fig. 1d-f). Importantly, IF did not alter critical modi ers of axonal regeneration such as Schwann cell and macrophage recruitment 25,26 to the nerve crush site (Extended data Fig. 2a- Since IF modi es cell metabolism 32,33 it was hypothesised that changes in metabolism may underpin the IF-dependent regenerative ability. To this end, semi-targeted gas chromatography coupled to mass spectrometry (GC-MS) of extracted serum following IF vs AL was performed, enabling identi cation of 79 metabolites, 14 of which were differentially enriched upon IF ( Fig. 1d-f, Extended data Fig. 3). These metabolites could be clustered into 2 groups: microbiome derived and host metabolites (Fig. 1e).
Interestingly, the 4 most enriched metabolites upon IF were the microbiome derived 3-indolelactic acid 2, 2,3-butanediol 2, xylose 2 and indole-propionic acid (IPA) (Fig. 1f). To assess whether the IF-dependent microbiome promotes sciatic nerve regeneration, microbiota collected from IF fed animals were transplanted via faecal transplantation (FT) into AL mice: indeed, FT from IF-mice led to a signi cant increase in regenerating bres past the crush site in AL mice after SNC (Extended data Fig. 4). Since indole metabolites are primarily synthesized from tryptophan by the gram-positive bacteria Bi dobacterium (indole-3-lactic acid) 34 , Lactobacillus and Clostridium sporogenes (indole-propionic acid) 35 , the requirement of gram + bacteria for IF-dependent axonal regeneration was investigated.
Vancomycin treatment, which depletes gram + bacteria, abolished IF-dependent axonal regeneration (Fig. 2a-c), indicating that gram + bacteria are required for IF-dependent regeneration.
To identify metabolites underpinning the microbiome-dependent regenerative phenotype, serum samples were pro led by GC-MS following IF vs AL with and without vancomycin ( Fig. 2d-k, Extended data Fig. 5). Remarkably, IPA was the only metabolite signi cantly affected by vancomycin treatment with the most signi cant interaction p-value, suggesting that its amount critically depended on IF and the presence of the gram + gut microbiome (Fig. 2e, Table 1). Indeed, following IF, serum IPA was found among the highest increased metabolites, which was completely depleted by vancomycin (Fig. 2f), as con rmed by targeted Liquid Chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 2g, h). As expected, vancomycin signi cantly reduced bacterial diversity as shown by 16S rDNA sequencing (Extended data Fig. 6a, b, Supp File 1 and 2) and decreased the number of Bacteroidetes and Firmicutes, which were increased after IF (Extended data Fig. 6c). Importantly, the vast majority of Firmicutes belongs to the order of Clostridiales (Extended data Fig. 6d), and the top 5 most increased bacteria following IF are also Clostridiales (Extended data. Figure 6e), con rming Vancomycin treatment impacts IF-dependent pathways.
Together, these data suggest that gram + bacteria producing IPA are responsible for IF dependent axonal regeneration.
IPA promotes axonal regeneration after sciatic nerve crush via neutrophil chemotaxis and IFNγ Next, it was investigated whether IPA synthesis by the gut microbiome is required for axonal regeneration after SNC. Following vancomycin-mediated depletion of gram + bacteria in mice, the gut was recolonised with either a mutant Clostridium sporogenes dC (C.s. dC) bacterial strain that cannot produce IPA 36 or with a C.s. WT strain and axonal regeneration was measured 72 hours after a SNC (Fig. 3a-d). Depletion of IPA from the serum of mice recolonised with the C.s. dC (Fig. 3b) resulted in signi cantly reduced axonal regeneration compared with the C.s. WT (Fig. 3c, d). Conversely, IPA gavage signi cantly and robustly increased axonal regeneration of the sciatic nerve ( Fig. 3e-h).
Together these studies demonstrate that bacterial production of IPA in the gut critically affects axonal regeneration. Since changes in IPA production in the gut are re ected by variations in the serum, IPA was administered by intraperitoneal (i.p.) injection and axonal regeneration was assessed 72 hours after SNC.
Remarkably, systemic IPA delivery mirrored the increase in axonal regeneration elicited by IPA gavage (Extended data Fig. 7a-c), indicating that serum IPA might affect DRG neuron growth either directly or indirectly via neuronal intrinsic or extrinsic mechanisms respectively.
When IPA was administered directly into the media of cultured DRG neurons no changes in neurite outgrowth were observed (Extended data Fig. 7d-f), suggesting that IPA affects growth via an extrinsic regenerative mechanism to the DRG neurons.
To investigate IPA-dependent molecular signatures potentially affecting the regenerative ability of DRG neurons, RNA sequencing was performed from DRG preceding (naïve) or 72 hours after SNC following IPA or PBS oral gavage. IPA treatment affected the DRG gene expression programme as shown by differential gene expression analysis (Extended data Fig. 8a-c, Supp File 3 and 4), increased the expression of the gene encoding for the IPA nuclear receptor Pregnane X Receptor (Nr1i2), supporting activation of IPA signalling, and it affected mainly genes related to immune-regulatory GO categories, with the most signi cant enrichment for "neutrophil chemotaxis" at 72 hours following SNC (Fig. 4a, b, Supp File 4 and 5). Notably, the neutrophil ligand Cd177 and endothelial neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 (Cxcl1) were selectively upregulated by IPA after SNC (Extended data Fig. 8c), suggesting that IPA might promote neutrophil chemotaxis towards the DRG via a CXCR2 mediated mechanism.
In support of this model, we found that IPA treatment increased the number of neutrophils within the DRG tissue, but not at the nerve crush site (Extended data Fig. 9a-h). The number of other immune cells including CD4 and CD8 T-cells, macrophages, NK and B-cells, did not differ between IPA and vehicle (Extended data Fig. 10). DRG immunolabelling showed that the majority of neutrophils were positive for CXCR2 (Extended data Fig. 9i, j), a marker of neutrophil activation and chemotaxis, suggesting that they might be activated and attracted by a CXCR2 mediated mechanism. To test this hypothesis, neutrophil chemotaxis was inhibited at the time of IPA treatment by using an anti-CXCR2 neutralising monoclonal antibody that signi cantly impaired IPA-dependent regeneration after SNC ( Fig. 4c-g, Extended data Fig. 11j-k). Accordingly, neutrophil depletion with anti-Ly6G monoclonal antibody signi cantly decreased axonal regeneration mediated by IPA compared with IgG control antibody (Extended Data Fig. 11a-i). Together these data indicate that IPA-dependent neutrophil chemotaxis is required for axonal regeneration after SNC.
RNA sequencing also identi ed an IPA-dependent increase in IFNγ signalling in the DRG, indicating a potential signalling mechanism downstream of neutrophil recruitment (Extended data Fig. 12a). Indeed, monoclonal antibody mediated neutralisation of IFNγ signi cantly reduced IPA-dependent sciatic nerve regeneration (Extended data Fig. 12b-c). In line with these data, IFNγ treatment in cultured DRG neurons and in vivo IFNγ delivery followed by ex vivo analysis of DRG neuronal outgrowth in culture revealed a signi cant increase in regenerative growth (Extended data Fig. 12d-g).
IPA delivery after sciatic nerve crush injury improves sensory neurological recovery and epidermal innervation Next, whether IPA administration at a clinically suitable time 24 hours after injury was able to accelerate the rate of sensory recovery following sciatic nerve injury was investigated. Both recovery of physiological sensory function by assessing responses to thermal stimulation and the presence of mechanical allodynia, which can be an undesired consequence of maladaptive epidermal re-innervation, were evaluated ( Fig. 4h-k). IPA treated mice showed a faster speed of recovery of thermal nociception as shown by the Hargreaves test, displaying a reduced withdrawal response time compared to PBS treated mice between day 7 and day 19 (Fig. 4j), however, no changes in mechanical allodynia, measured with Von Frey laments, between IPA-and PBS treated groups were observed (Fig. 4k). These data show that IPA leads to an accelerated recovery in thermal nociception while it does not induce mechanical allodynia following sciatic nerve injury. Importantly, anti-PGP9.5 immunostaining of the hind paw interdigital skin at 16 days post-SNC revealed a signi cantly increased epidermal innervation following IPA vs PBS, providing an anatomical substrate for functional recovery (Fig. 4l-n). Together, these experiments indicate that IPA induces sensory recovery including by improving epidermal innervation without causing neuropathic pain in a sciatic crush model of peripheral nerve injury.
Conclusions
The present study identi es a novel dietary-dependent regenerative mechanism that relies on the gut gram + Clostridiales bacterial production of the metabolite IPA. IPA accelerates axonal regeneration, epidermal innervation and sensory neurological recovery after SNC via the recruitment of neutrophils to the DRG. The gut microbial metabolite IPA, a product of bacterial metabolism of tryptophan in the lower gut, and its xenobiotic nuclear receptor PXR have been implicated in a variety of immune responses in several tissues and cell types [37][38][39][40][41] , including in the gut microenvironment, where an immune suppressive and anti-in ammatory function for IPA and PXR signalling have been described [37][38][39] . Conversely, PXR activation in vascular endothelial cells has been shown to initiate an innate in ammatory response 41 , which results in neutrophil recruitment 42 , supporting our model that suggests an IPA-dependent increase of trans-endothelial neutrophil chemotaxis.
While neutrophils are required for axonal regeneration in the PNS 43 and CNS [44][45][46] via changes in their recruitment at the lesion site, our study supports a regenerative role for neutrophils by communicating regenerative signals such as IFNγ at the level of the DRG where the neuronal cell bodies reside.
IFNγ-dependent axonal regeneration is in line with increased in ammatory signalling by neutrophils, however, a role for additional cytokines supporting IPA-dependent axonal regeneration cannot be ruled out.
Since the gut microbiome undergoes profound changes in human subjects upon nervous system injury 47 , identifying gut microbiome derived regenerative small molecules for oral or intravenous administration, could provide promising new therapeutic possibilities to heal nerve injury, promote axonal regeneration and enhance neurological recovery.
Animal Husbandry
All animal experiments were carried out according to UK Home O ce regulations in line with the Animals (Scienti c Procedures) Act of 1986 under personal and project licenses registered with the UK Home O ce. Mice were maintained in-house under standard housing conditions (12-h light/dark cycles), 24 hr access to water and standard chow diet. All mice were of a C57BL6 background (male, 20-30 g, 6-8 weeks of age). Experimenters were blinded to experimental groups during scoring and quanti cations.
Sample sizes were chosen in accordance with similar previously published experiments.
Intermittent Fasting
Body weight-matched 6-8 week old male C57BL/6 mice were randomly assigned to intermittent fasting (IF), or ad libitum (control) treatment groups. The IF group did not have access to food (fasting) during the rst 24 h and then every second day after that (e.g. fasting during 0-24 h, 48-72 h, 96-120 h) with ad libitum access to food on the alternating days (24-48 h, 72-96 h, 120-144 h). Pre-weighed food was provided in the food hopper of their home cage at 9:30 am (unless a fasting day for the IF groups), and leftover food was weighed 24 h later.
Sciatic nerve crush (SNC) surgery
Mice were anesthetized with iso urane (5% induction, 2% maintenance), shaved on the hind limbs and lower back, sterilised with iodine, and an ophthalmic solution was applied to the eyes to prevent drying. An incision was made on the skin and the biceps femoris and the gluteus super cialis were opened by blunt dissection and the sciatic nerve was exposed using a surgical hook. The sciatic nerve crush was performed orthogonally for 20 seconds (45 seconds for reinnervation experiment) using a 5 mm surgery forceps (91150-20 Inox-Electronic). The crush was performed at approximately 20 mm distally from the sciatic DRG.
Faecal transplantation
Fresh faeces were collected every day from the cage of 5 healthy C57BL/6 mice, approximately 200 mg/ mouse. A fresh cage was used each day.. 1g of faeces was then homogenized in 10 ml of PBS for 30 seconds at 3000 rpm at 4°C. Thereafter, the supernatant was transferred to a new tube and centrifuged for 5 min at 12000 rpm at 4°C. The pellet was resuspended in 2.5 ml PBS and 500 µl gavaged to each mouse.
Antibiotic administration
Vancomycin (Sigma, V2002) was administered through the drinking water (50mg/kg/day; 214.27 mg/l), which was replenished every second day.
Clostridium sporogenes recolonization
WT and dC (mutant for (R)-phenyllactyl-CoA dehydratase beta subunit of the phenyllactate dehydratase complex [ dC]) Clostridium sporogenes were cultured overnight in trypticase yeast extract medium anaerobically. Bacterial cultures were mixed with glycerol to reach the nal concentrations of: WT 6.01E+06 CFU/ml and C.s. dC mutant 5.46E+06 CFU/ml in hermetically sealed glass vials. Mice were pre-treated with vancomycin in drinking water for 3 days. C.s. WT or C.s. were transplanted to the cecum via oral gavage for 10 consecutive days at 1E+06 CFU/day.
Serum and cecum preparation
Blood was extracted via heart punctuation and collected into a covered test tube. The blood was allowed to clot for a minimum of 30 min at room temperature and centrifuged at 2000 g for 10 min at 4 °C. The supernatant was then collected into a fresh tube and stored at -20 °C until use.
Cecum content was removed from the cecum using sterile forceps into a tube and snap-frozen in liquid nitrogen immediately.
Gas chromatography -Mass Spectrometry (GC-MS) untargeted metabolomics
Serum samples (100 μl) were prepared as follows: I) samples were spiked with 10 μL internal standard solution (myristic acid-d27, 750mg/ml), II) 850 μL of ice cold methanol were added, followed by centrifugation for 20 min (4 o C, 16000 g), III) 750 μL of supernatants were transferred to silanized dark 2mL autosampler vials and were evaporated to dryness in a rotational vacuum concentrator (45 o C, 20 mbar, 2 h), IV) 50 μL of methoxyamine solution (2% in pyridine) were added and the samples were incubated overnight at room temperature and nally, V) 100 μL of N-methyl-trimethylsilyltri uoroacetamide (MSTFA) +1% trimethylchlorosilane (TMCS) solution were added, the samples were incubated at 60 ºC for 1 h and were transferred to dark autosampler vials with 250 μL silanized inserts. The samples were analysed in an Agilent 7890B-5977B Inert Plus GC-MS system. 2 μL of each sample were injected in a split inlet (1:10 ratio). The chromatographic column was an Agilent ZORBAX DB5-MS (30 m X 250 µm X 0.25 µm + 10m Duraguard). The temperature gradient was 37.5 min long and the mass analyser was operated in full scan mode between 50-600 m/z. The detailed instrumental conditions are described elsewhere (Agilent G1676AA Fiehn GC/MS Metabolomics RTL Library, User Guide, Agilent Technologies, https://www.agilent.com/cs/library/usermanuals/Public/G1676-90001_Fiehn.pdf). Quality Control (QC) samples were created by pooling equal amounts of every serum sample of the study and were analysed interspaced in the analytical run. Study samples were randomized before sample preparation. Peak deconvolution, alignment and annotation and were performed with the use of the Fiehn library via the software packages AMDIS (NIST), Mass Pro ler Pro and Unknowns (Agilent technologies) in the pooled QC samples. Peak picking was performed with the GAVIN package (A software complement to AMDIS for processing GC-MS metabolomics data. doi: 10.1016/j.ab.2011.04.009) 48 . Non-reproducible (CV>30% in QC samples) and contaminated (blank > 20% of the mean QC levels) metabolic features were removed from the dataset. This data set was then used to build orthogonal partial least-squares-discriminant analysis (OPLS-DA) (IF vs AL) or partial least-squares-discriminant analysis (PLS-DA) (IF, IF-V, AL, AL-V), focusing on the differences among the experimental groups. The OPLS algorithm derives from the partial least-squares (PLS) regression method 49 . The method explains the maximum separation between class samples Y (n dummy variables for n classes) by using the GC-MS data X. Here the ropls R package (http://bioconductor.org/packages/release/bioc/html/ropls.html) was used, which implements the PCA, PLS-DA and OPLS-DA approaches 50 . It includes R 2 and Q 2 goodness-of-t and goodness-of-prediction statistics, permutation tests, as well as scores, loadings, predictions, diagnostics, outliers graphics 50 . R2X describes the percentage of predictive and orthogonal variation in X that is explained by the full model. Isotopes) as internal standard (IS) were used. Acetonitrile, methanol and formic acid were of ULC-MS grade was supplied from Bio-Lab. Water with resistivity 18.2 MΩ was obtained using Direct 3-Q UV system (Millipore). Standard curve was built using concentration range of 3-indole propionic acid 0.01-12 µg/mL, with nal concentration of IS 100 ng/mL.
Extract preparation
Plasma (30 µL) and IS (10uL, 1 ug/mL) was incubated 10 min, then 500 µL of methanol was added. The mixture was shaken at 10°C for 30 min (ThermoMixer C, Eppendorf), and centrifuged at 21,000 g for 10 min. Collected supernatant was evaporated in speedvac and then in lyophilizer. Before LC-MC analysis, the obtained residue was re-suspended in 100 µL of 20%-aq methanol, centrifuged twice at 21,000 g for 5 min to remove insoluble material. The soluble part was placed to insert of LC-MS vial. Some samples were diluted 1/20 with 20%-aq methanol.
LC-MS analysis
The LC-MS/MS instrument consisted of an Acquity I-class UPLC system (Waters) and Xevo TQ-S triple quadrupole mass spectrometer (Waters) equipped with an electrospray ion source and operated in positive ion mode. MassLynx and TargetLynx software (version 4.1, Waters) were applied for the acquisition and analysis of data. Chromatographic separation was done on a 100 mm × 2.
16S rRNA amplicon sequencing data analysis
Only single-end R1 sequences were analysed. Data processing was carried out in R statistical environment version 3.5.2 according to the DADA2 pipeline 51 as follows; primers were trimmed from demultiplexed sequences, amplicon sequence variants (ASVs) were generated using DADA2 package version 1.10.1, and taxonomic assignment was performed using the SILVA rRNA database version 132 [https://www.arb-silva.de/]. ASVs with no phylum assigned were excluded from analyses to yield a nal number of n = 906 annotated ASVs. Alpha and beta diversity analyses were carried out using phyloseq package version 1.26.1 52 . Alpha diversity (Shannon Index) was calculated using raw counts 53 . Groups were compared using Mann Whitney U statistical tests. For beta diversity analyses, data was normalised by log-transformation with a pseudo count of +1. Differential abundance of groups were compared in the DESeq2 package version 1.22.2 54 using the Wald test with Benjamini and Hochberg adjustments for multiple testing (p value set as 0.05). Alpha diversity is a measure of diversity or "microbial variation" within a (single) sample while beta diversity is a measure of diversity across (several) samples.
RNA sequencing DRG were collected from animals that had undergone 10 days of IPA or PBS treatment and/or followed by sciatic nerve crush. Sciatic DRG were extracted 72h after sciatic nerve crush or from naïve animals (surgeries were performed as described above) and collected into RNAlater. DRG were crushed with RNase free micro pestle and RNA was then immediately extracted using RNAeasy kit (Qiagen), according to manufacturer's guidelines. Residual DNA contamination was removed by treating the spin column with 40 units of RNase-free DNase I (Qiagen) for 15 min at 23 °C prior to RNA elution. RNA concentrations and purity were veri ed for each sample following elution with the Agilent 2100 Bioanalyzer (Agilent). RNA with RIN factors above 8.5 were used for library preparation. cDNA libraries for each sample were generated by the Imperial BRC Genomics Facility using the TruSeq Sample Preparation Kit A (Illumina, San Diego CA) and sequenced using Illumina HiSeq 4000 (PE 2x75 bp) sequencing. Gene ontology (GO) was performed on differentially expressed genes with DAVID 6.8 (Database for Annotation, Visualization, and Integrated Discovery (http://david.abcc.ncifcrf.gov/)). Differentially expressed genes were selected using a threshold of P<0.05 and |1.5|<FC (fold change) or no FC cut-off. To identify IPA-dependent speci c differentially expressed genes following SNC, we compared IPA-SNCvsPBS and PBS-SNCvsPBS groups and selected the uniquely up-or downregulated genes in the IPA-SNCvsPBS group.
Sciatic nerve regeneration 24 or 72 hours following the surgery, sciatic nerves were dissected and post-xed in 4% PFA, incubated at 4°C for 1h and transferred into 30 % sucrose for at least 3 days. Subsequently, the tissue was embedded and frozen in Tissue-Tek OCT and maintained at -80°C until cut into 11 µm sagittal sections. Tissue sections were immunostained for SCG10 (1:1000, rabbit, Novus) a marker for regenerating axons. The crush site was identi ed by deformation of the nerve and disruption of axons coinciding with highest SCG-10 intensity. The SCG-10 intensity was measured in 500 µm intervals along the length of the nerve distal to sciatic nerve crush site. The intensity was normalised to the SCG-10 intensity before the crush site and plotted as fold-change. 4-6 sections per animal were analysed and imaged with a HWF1 -Zeiss Axio Observer with a Hamamatsu Flash 4.0 fast camera using 10x magni cation.
Glass coverslips were coated with 0.1 mg/ml PDL, washed and coated with mouse Laminin 2ug/ml (Millipore) for 1-2 hours each previous to the start of the experiment. Sciatic DRG from adult animals were dissected and collected in Hanks balanced salt solution (HBSS) on ice. The DRG were transferred into a digest solution (5mg/ml Dispase II (Sigma), 2.5 mg/ml Collagenase Type II (Worthington) and incubated in a 37 °C water bath for 45 min, with occasional shaking for 30 seconds . Thereafter, the DRG were washed and manually dissociated with a 1ml pipette in media containing 10 % heat inactivated FBS (Invitrogen) and 1x B27 (Invitrogen) in F12:DMEM (Invitrogen). Pipetting was continued until DRG were fully dissociated and no clumps could be observed. Next, the cell suspension was spun down at 1000 rpm for 4 min and resuspended in culture media containing 1x B27 and Penicillin/Streptomycin in F12 : DMEM. 3500 cells were plated on each coverslip (laminin and PDL coated) and maintained in a humidi ed culture chamber with 5% CO2 at 37 °C, for 12 hours before xed with 4% PFA and immunostained.
Microscopy
Photomicrographs were taken with a Nikon Eclipse TE2000 microscope with an optiMOS scMOS camera using 10x or 20x resolution Zeiss Axio Observer with a Hamamatsu Flash 4.0 fast camera using 10x or 20x magni cation. Confocal images were taken with a Leica TCS SP8 II confocal microscope at 40X or 60X magni cation and processed with the LAS-AM Leica software (Leica).
Image Analysis for IHC and ICC
Image analysis was conducted using ImageJ (Fiji) software. All analysis was performed by the same experimenter who was blinded to the experimental groups. DRG images were taken using a Nikon Eclipse TE2000 microscope with an optiMOS scMOS camera at either 10x or 20x magni cation. Images were analysed by counting the number of cells per area, measuring the intensity against the background or calculating percentage of cells with positive staining.
For neurite length analysis between 15 and 20 images were taken per coverslip and analysed using NeuronJ plugin for Image J software (Image J). All analyses were performed in blind. Approximately 45-60 cells were analysed per animal and condition.
Statistical analysis
Results are graphed as mean ± SEM. Statistical analysis was carried out using GraphPad Prism 7.
Normally distributed data was evaluated using a two-tailed unpaired Student's t-test or a one-way ANOVA when experiments contained more than two groups. Dunnett multiple comparisons test or multiple comparison testing corrected by FDR with Benjamini and Hochberg were applied when appropriate. The two-way ANOVA, Tukey's or Sidak's test, was applied when two independent variables on one dependent variable were assessed. A threshold level of signi cance was set at P<0.05. Signi cance levels were de ned as follows: * P<0.05; ** P<0.01; *** P<0.001; **** p<0.0001. All data analysis was performed blind to the experimental group.
Competing interests
The authors declare no competing interests.
Author contributions | v3-fos-license |
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} | pes2o/s2orc | Hypoglycemic Effects of Insoluble Fiber Rich Fraction of Different Cereals and Millets
The present global pandemic of Diabetes is accounted for by westernization of life style, population growth, ageing and urbanization, with consequent dietary change, sedentary life style and obesity. Diabetes Mellitus(DM) is also a common endocrine disease in middle aged to older cats and is often called as “sugar diabetes’’. In dogs with naturally occurring insulin dependent DM a high insoluble fiber diet may aid in glycemic control. Incidence of DM is lower in population with high fiber intake mostly in rural areas and tribal belt which might be due to feeding of different cereals and millets with high fiber content. The term dietary fiber was first adopted in 1953 by Hipsley to describe the plant cell wall components of food. Millets are small sized grains, containing large proportions of husk and bran; require dehusking and debranning prior to consumption [1]. The nutritive value of millets is comparable to other cereals, some of them are even better with regard to average protein and mineral contents [2].
Introduction
The present global pandemic of Diabetes is accounted for by westernization of life style, population growth, ageing and urbanization, with consequent dietary change, sedentary life style and obesity. Diabetes Mellitus(DM) is also a common endocrine disease in middle aged to older cats and is often called as "sugar diabetes''. In dogs with naturally occurring insulin dependent DM a high insoluble fiber diet may aid in glycemic control. Incidence of DM is lower in population with high fiber intake mostly in rural areas and tribal belt which might be due to feeding of different cereals and millets with high fiber content. The term dietary fiber was first adopted in 1953 by Hipsley to describe the plant cell wall components of food. Millets are small sized grains, containing large proportions of husk and bran; require dehusking and debranning prior to consumption [1]. The nutritive value of millets is comparable to other cereals, some of them are even better with regard to average protein and mineral contents [2].
The protein contents of the dehusked millets varied between 8.7% (kodo millet) and 13.8% (pearl millet), whereas in case of milled grains it varied from 5.8% (finger millet) to 12.7% (pearl millet). Milled grains contained nearly 70% of total fat of whole seeds. The calcium and phosphorus contents of milled millets varied from 2.3 mg% to 162.8 mg% and 105 mg% to 425 mg%, respectively. Milling removed nearly 50% calcium and about 65% of phosphorus from whole seeds. The bran fraction from small millets other than finger millet, contain 23.0-27.0% oil. Whereas pearl millet bran contain 15% oil. The total dietary fiber content of debranned millets is ranging from 9.0 to 16.0 %. This indicates the millets, even after removal of husk and major portion of bran, contained appreciable amounts of dietary fiber. The millet bran, besides containing considerably higher proportion of oil, appears to be a good source of dietary fiber, out of which 10-15% was soluble fraction [3].
Dietary fibers are not uniform chemically or in their nutritive and biological properties, the only common ground being their resistance to mammalian digestive enzymes. The AOAC [4] method for total fiber is subjected to interference from ash, protein, tannins, and resistant starches. These interferences can be reduced by urea enzymatic dialysis. The measurement of soluble and insoluble fiber is nutritionally relevant since physical properties greatly modify dietary effects of fiber. Insoluble fiber is conveniently measured as neutral detergent fiber. This procedure has been improved by reducing the starch interference and the time of analysis. Physical and biological properties of dietary fiber can be measured by using relevant procedures for hydration capacity and rate of fermentation. The lignin and tannin content modify the characteristics of dietary fiber [5]. Breads made from a combination of wheat potato and/or oat has relatively high in total dietary fiber. Wheat breads with different ash contents or breads made from a combination of wheat and rye had clearly higher total dietary fiber content [6]. Schieber et al. [7] has pointed out that agricultural byproducts could be exploited as a potential source of fibers and functional compounds for food application. Dietary fiber is unique among feed constituents because it is defined only on a nutritional basis (i.e. in terms of digestive and physiological effects that it elicits) but must be measured chemically. The usefulness of dietary fiber results vary from its value as an indicator of physiological health benefits to its value as a predictor of digestibility and energy value of feeds. Numerous methods have been proposed for measuring dietary fiber and some have become routine analyses for research and practical use. Fiber extraction methods are typically categorized in to three types (chemical-gravimetric, enzymatic-gravimetric or enzymatic-chemical) based on ways fibrous residues are isolated and measured. Isolation of dietary fiber residue is done by extraction in chemical solution, enzymatic hydrolysis of nonfibrous constituents or a combination of two. After fibrous residue is isolated it is measured either gravimetrically (weighing the residue) or chemically (hydrolyzing the residue and measuring individual components such as sugars and lignin) [8]. There are several AOAC official methods for measuring total dietary fiber (TDF), Insoluble dietary fiber (IDF), soluble dietary fiber (SDF). The first AOAC method for TDF is 985.29-Total dietary fiber in food, enzymatic gravimetric method which did not allow separation of dietary fiber in to soluble and insoluble fraction. Insoluble fraction can be determined using AOAC official method 991.42-Insoluble dietary fiber in foods and food products, enzymatic gravimetric method [8]. Certain physiological responses have been associated with the consumption of dietary fiber and physical and chemical properties of individual dietary fiber compounds appear to be important in determining the physiological response to sources of dietary fiber in the diet [9]. The post prandial glycemic response is most effectively reduced with sources of viscous polysaccharides. The importance of viscosity in this response has been demonstrated in several studies suggesting that the ability to form a gel matrix may be important in mediating the physiological response to these fiber sources. Also the physical properties of digestibility are key determinants in the metabolism of fiber rich diets. Fiber modulates and slows the rate of digestion and absorption by at least three mechanisms. 1) The rate of gastric filling and emptying can be slowed by certain fibers; 2) The activity of digestive enzymes in the small intestine could be diminished in presence of fibers; 3) The diffusion and absorption of nutrients enzymes and substrates in the intestinal tract may be altered by certain fibers via these mechanisms and its fermentation in the large intestine. Fibers affect the metabolic process [10]. The fermentability of polysaccharides as well as their bulking ability in the large bowel is important for determining the physiological effects of soluble versus insoluble fibers. In this part of gut soluble fibers can be readily degraded by bacteria because the water holding capacity allows the bacteria penetrate the fiber matrix. The increase in bacterial mass due to fermentability leads to increase in faecal bulk. In contrast insoluble fiber cannot be penetrated as well by bacteria and cannot be broken down as extensively hence a residual fiber is present and a matrix is maintained in large bowel content and bacterial mass is increased [11]. The rate at which food is emptied from the stomach determines the rate of nutrient absorption from the intestine. Hence a delay in gastric emptying determines the rate of nutrient absorption from the intestine [9]. The findings that several dietary fibers can decrease the activity of human pancreatic amylase, lipase, trypsin, and chymotrypsin may be attributable at least in part to enzyme inhibitors [12]. Alternatively this decreased activity after incubation with several dietary fibers could be due to nonspecific adsorption of enzyme molecules [13]. With high fiber intake glucose absorption is slowed down and spread out along a greater length of the intestine. This allows uptake of glucose by the intestine keeping in pace with the gastrointestinal absorption after initial stimulation of insulin release there by regulating plasma glucose level [14][15][16]. Dietary fibers coming from various sources do not seem to be equally effective in delaying glucose absorption in intestine. Tanchoco et al. [17] It is estimated that more than 50% post prandial insulin secretion is triggered by intestinal peptide hormone. In presence of elevated blood glucose, glucagons like peptide 1 (GLP-1) stimulates the release of insulin by interacting with specific receptors on pancreatic beta cells. In addition to potentiating glucose induced insulin secretion GLP-1 stimulates proinsulin gene expression and proinsulin biosynthesis [18]. By stimulating insulin release and increasing insulin dependent glucose disposal, GLP-1 enhances glucose tolerance [19]. The potential action of this hormone on carbohydrate metabolism makes it potentially applicable in the treatment of non-insulin dependent diabetes mellitus. It is found that long term ingestion of dietary fiber by rats ingesting similar amounts of energy, proteins, lipid, glucose, vitamins and minerals stimulates small chain fatty acids (SCFA) production and proglucagon m-RNA abundance and increases post prandial glucagons like peptide 1, insulin and c-peptide concentration. High carbohydrate/high fiber diet significantly improves blood glucose control and reduces plasma cholesterol levels in diabetic patients compared with a low carbohydrate low fiber diet. In addition a high carbohydrate/high fiber diet does not increase plasma and triglyceride concentration despite the higher consumption of carbohydrate. Ability of dietary fiber to retard food digestion and nutrient absorption certainly has an important influence on lipid and carbohydrate metabolism. The fiber content and physical form of the food can influence the accessibility of nutrients by digestive enzymes thus delaying digestion and absorption. The fiber content and physical form of the food can influence the accessibility of nutrients by digestive enzymes thus delaying digestion and absorption. The identification of these foods with a low glycemic response would help to enlarge the list of foods particularly suitable for diabetic patients [20]. The polysaccharides composing the major part of dietary fiber in fruits and vegetables are beneficial to diabetes and heart patients since the fibers lower blood sugar and serum cholesterol levels [21,22]. In fact the most likely explanation for the reduction of post prandial hyperglycemia by viscous fibers is decreased amylase activity [23] and a direct delaying effect on glucose absorption in the gastro-intestinal tract due to alternation in the diffusion of digestion end product with the lumen [24,25]. Starch degradation and glucose diffusion are delayed in the presence of mango fiber. Viscous solutions which reduce starch digestibility in vitro can decrease glycemic post prandial response. Hence mango fiber could be of potential benefit in controlling plasma glucose [26].
Collection of sample
Grain samples of different millets and cereals (Table1) were collected from Boudh district, Odisha. These samples were then cleaned properly, shed dried and grinded manually and sieved to collect the bran. Whole grain was grinded using electrically operated grinder to desired size. Then these bran samples and whole grain powder samples were stored in sealed containers till their use in different experimental procedures ( Figure 1,2).
Proximate analysis of bran and whole grain powder
Estimation of moisture content: Dry weight of moisture cup (W 1 ) was first taken. Then weight of moisture cup with bran/whole grain sample (W 2 ) was taken and it was kept in hot air oven over night (12 hrs) at 100°C. Weight of moisture cup with sample was again taken after drying (W 3 ). Moisture content is calculated as
Estimation of ether extract:
The instrument used for estimation of ether extract was Socs plus (Pelican Equipments, Chennai, India). Moisture free samples (2 gm each) from the above experiment were taken in different thimbles. Dry weight of flasks was taken (W 1 ). The thimbles were placed in the flasks. 150 ml of petroleum ether (60-80°C) was taken in each flask. The attachment of the instrument were made properly and was run for 1 hr at 90°C and further for 30 minutes at 180°C.
After collection of ether extractives the flask were removed from the apparatus and kept in the hot air oven (100°C) for 12hrs to make the flasks free from petroleum ether. The weight of flasks was taken again (W 2 ) and ether extract percentage was calculated by using the formula 2 1 W -W Ether extract% or Crude fat% = 100 Weight of dried sample ×
Estimation of crude fiber (by Von Soest method):
Fat free samples (W 1, 2 gm each) were taken in the sintered crucible of Fibra Plus Fes 6 Instrument (Pelican equipment's, Chennai, India). Then it was treated with 1.25% H 2 SO 4 (v/v) at 400°C for 45 min for acid digestion followed by alkali digestion with 1.25% NaOH (W/V) at 400°C for 45 minutes. Then the crucible containing fiber sample was washed with distilled water and dried in oven at 100°C for 24 hrs. Then weighed (W 2 ) and placed in muffle furnace at 550°C for 6 hr and weighed (W 3 ) again. Percentage of crude fiber was determined by formula 2 3 Estimation of crude protein: Protein content of sample was estimated using Nitrogen Autoanalyser manufactured by Pelican Equipment's, Chennai, India. 0.2 gms of dried sample, 3 gms kelpac, 10 ml H 2 SO 4 were mixed and digested at 400°C for 3hrs using KES 06L Digestion Chamber of Pelican instrument. Distillation was done by Kel Plus (Classic DX) apparatus manufactured by Pelican. Solutions used in equipment were 40% NaOH and 4% Boric acid with distillation time 9 minutes. Then the distillate was titrated against 0.1 N HCl. The nitrogen content was titrated/estimated using Metrohm Autotitrator (Switzerland). Protein percentage was calculated by multiplying nitrogen content with 6.25.
Estimation of total ash: The dry weight of porcelain crucible was taken (W 1 ). Then weight of crucible with moisture free sample was taken (W 2 ) and kept in muffle furnace at 550°C for 6 hrs. After cooling weight of crucible with ash content was taken (W 3 ). Total ash% calculated by formula 3 1 All the above procedures were followed both for bran and whole grain powder of different millets and cereals to estimate their proximate composition.
Preparation of insoluble dietary fiber (IDF) from bran sample
The moisture free bran samples were first made fat free by using Socs Plus (Pelican Equipment, Chennai). The fat free samples were used for extraction of IDF by Von Soest method with acid and alkali treatment using Fibra plus Fes-6 (Pelican Equipment, Chennai). Acid digestion of samples was performed at 400°C for 45 min using 1.25% H 2 SO 4 followed by Alkali digestion using 1.25% NaOH at 400°C for 45 min. Then the contents were filtered and washed thoroughly with distilled water to make the sample free from alkali and kept in hot air oven at 100°C for drying. Then the fiber samples were weighed and stored till further use.
Preparation of alcohol insoluble solids (AIS) from bran samples
Alcohol insoluble solids (AIS) were prepared from the bran sample by using the method described by [27]. Briefly, 3 gms of bran sample was homogenized with boiling alcohol (ethanol) 850 ml/lit at high speed followed by further boiling for 40 min. The bran to alcohol ratio was 1:30 (w/v). AIS was collected by filtration and washed with ethanol (700 ml/lit), air dried and stored properly for further use.
Preparation of water insoluble solid (WIS) from bran sample
The water insoluble solids (WIS) were prepared according to the method of [28]. WIS was separated from bran samples by homogenizing the bran in cold distilled water (1:10 w/v) at high speed for 1 min. After filtration WIS was washed with ethanol (700 ml/lit), air dried and stored for further use.
Study of hypoglycemic activity in vitro
Determination of glucose adsorption capacity (GAC): The glucose adsorption capacity (milimoles per gram) was determined according to method described by [28] with slight modification. 0.1 gm of fiber sample was mixed with 10 ml of glucose solution of different concentration (i.e. 5 mmol/lit, 10 mmol/lit, 25 mmol/lit, and 50 mmol/ lit ) and then incubated for 5 hrs at 37°C using constant shaker cum incubator (Rotek-LIS, Pelican Equipments, Chennai ). The final glucose content in the supernatant was measured after centrifuging at 3500 rpm for 15 min by glucose assay kit (Corals glucose assay kit, GOD-POD method) to estimate the amount of glucose adsorbed on fiber sample. A control test was done without addition of fiber.
Determination of glucose diffusion and glucose dialysis retardation index (GDRI):
Glucose dialysis retardation index was determined on the basis of [28] with slight modifications. A mixture solution was prepared by mixing 0.125 gm of fiber sample in 6.25 ml of glucose solution (10 mmol/lit) and was dialyzed against 40 ml of distilled water at 37°C using a dialysis membrane with a molecular weight cut off value of 12,000 D. After incubation of 10, 30, 60, and 120 min the glucose content in the dialysate was measured by glucose assay kit (Corals glucose assay kit, GOD-POD Method) for estimation of GDRI. A control test was also prepared without addition of fiber. GDRI was calculated using formula GDRI = 100 -Glucose content in dialysate with fiber 100 Glucose content in dialysate of control ×
Determination of starch digestibility:
The effect of different fibers on starch digestibility was determined as per [28] with slight modifications. A mixture was prepared by mixing 0.1 gm of fiber and 0.02 gm of diastase in 5 ml of potato starch solution (4 gm/100 ml) and was dialyzed against 100 ml of distilled water at 37°C using a dialysis membrane with molecular weight cut off value of 12,000 D. After incubation of 10, 30, 60 and 120 min the glucose content in the dialysate was determined using glucose assay kit (Coral glucose assay kit, GOD-POD Method). A control experiment was also performed without addition of fiber.
Determination of residual amylase activity:
The effect of fiber on glucose production rate and residual amylase activity was determined as per [28], with slight modifications. An incubation mixture containing 0.25 gm of fiber sample and 1 mg of diastase in 10 ml of potato starch solution (4 gm/100 ml) was incubated at 37°C for 60 min. Starch digestion then stopped by addition of 20 ml of 0.1 N NaOH. Then it was centrifuged at 3500 rpm for 15 min and glucose content of supernatant was measured by Glucose Assay kit (Corals glucose assay kit, GOD-POD method). A control experiment was also done without addition of fiber. The residual amylase activity was defined as the percentage of glucose production rate with fiber addition over the control.
Proximate composition
The proximate composition of whole grains and bran samples were shown in percentage ( Table 2 and 3) for whole grain and bran samples respectively. Moisture content in whole grains varied slightly ranging from 7.63 ± 0.581% to 8.89 ± 0.12%, barnyard millet being the highest in moisture content and kodo millet being the lowest. Highest crude fiber content (17.42 ± 1.066%) was found in finger millet and was lowest in sorghum (10.48 ± 0.494%). The dry matter content was highest in kodo millet (92.37 ± 0.581%) and lowest in barnyard millet (91.11 ± 0.068%). The ether extractives were highest in barnyard millet (4.35 ± 0.164%) and lowest in Proso millet (1.003 ± 0.057%) whereas, the crude protein content was highest in barnyard millet (10.39 ± 0.248%) and was lowest in kodo millet (5.48 ± 0.449%). The total ash content was found to be highest in both kodo millet (3.59 ± 0.246%) and Proso millet (3.59 ± 0.234%) and lowest in sorghum (1.29 ± 0.085%). The NFE content was highest in Sorghum (78.47 ± 0.968%) but lowest in barnyard millet (70.47 ± 0.747%).
From the proximate composition of bran samples (Table 3) it was observed that the moisture content was highest in finger millet (9.22 ± 0.56%) and lowest in kodo millet (5.53 ± 0.67%) and the dry matter content was highest in kodo millet (94.47 ± 0.67%) and lowest in Ragi (90.78 ± 0.56%). The ether extract content was highest in sorghum (4.69 ± 0.60%) but lowest in Proso millet (2.8 ± 0.53%). The crude fiber content in bran samples varied from 38.4 ± 0.66% to 11.32 ± 0.73, barnyard millet bran being the highest and wheat bran being the lowest whereas, the crude protein content was highest in wheat bran (12.19 ± 0.62%) and was lowest in Proso millet bran (4.13 ± 0.56%). The total ash content was found to be highest in finger millet bran (10.49 ± 0.85%) and lowest in kodo millet bran (7.74 ± 0.89%). The bran sample of Sorghum was highest in NFE (63.51 ± 0.48%) but the barnyard millet contained lowest NFE (43.29 ± 1.48%). From proximate composition analysis of both whole grains and bran samples it was clear that the crude fiber content was more in bran samples than the corresponding whole grains so the bran samples were used for the extraction of fibers for further studies.
Hypoglycemic effect of insoluble fibers in vitro
In vitro Hypoglycemic effect of insoluble fibers of different cereal and millet samples was studied by assessing the effect various fibers on glucose adsorption capacity (GAC), glucose diffusion, GDRI (Glucose dialysis retardation index). Starch digestibility and alpha-amylase activity.
Effect of insoluble fibers on glucose adsorption capacity in vitro:
A Series of different concentration of glucose (5 Millimole/l, 10 Millimole/l, 25 Millimole/l and 50 Millimole/l) were used to investigate the in vitro glucose adsorption capacity (Table 4, Figure 3). The results showed that all the dietary fibers could bind glucose and the adsorption capacity also increases in almost all the samples studied with increase in concentration of glucose. Glucose adsorption capacity (GAC) at 5 Millimole/l concentration of glucose was almost similar in IDF of all the millets and wheat ranging from 0.04 ± 0.01 in case of Barnyard millet IDF to 0.06 ± 0.01 in Sorghum, Ragi and Kodo. In case of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC) at 5 Millimole/l concentration of glucose ranged from 0.04 ± 0.01 in wheat to 0.10 ± 0.01 in Ragi and sorghum whereas, that in case of WIS ranged from 0.07 ± 0.02 in Barnyard millet and wheat to 0.18 ± 0.01 in Ragi. Glucose absorption capacity at 5 Millimole/l concentration of glucose was highest in Ragi fibers. Glucose adsorption capacity (GAC) at 10 Millimole/l concentration of glucose was ranging from 0.12 ± 0.01 in case of Ragi IDF to 0.25 ± 0.02 in wheat which is interestingly reverse as compared to the 5 Millimole/l concentration of glucose. In case of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC) at 10 Millimole/l concentration of glucose ranged from 0.12 ± 0.01 in Proso millet to 0.31 ± 0.02 in Ragi whereas, that in case of WIS ranged from 0.08 ± 0.02 in Proso millet to 0.25 ± 0.02 in Ragi. Glucose absorption capacity at 10 Millimole/l concentration of glucose was interestingly highest in wheat fibers. Glucose adsorption capacity (GAC) at 25 Millimole/l concentration of glucose was ranging from 0.19 ± 0.01 in case of kodo millet IDF to 0.71 ± 0.02 in wheat. In case of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC) at 25 Millimole/l concentration of glucose ranged from 0.15 ± 0.02 in Barnyard millet and Ragi to 0.62 ± 0.02 in wheat whereas, that in case of WIS ranged from 0.08 ± 0.02 in Barnyard millet to 0.51 ± 0.02 in wheat. Glucose absorption capacity at 25 Millimole/l concentration of glucose was also highest in wheat fibers.
Glucose adsorption capacity (GAC) at 50 Millimole/l concentration of glucose was ranging from 0.51 ± 0.01 in case of Barnyard millet IDF to 1.65 ± 0.02 in wheat. In case of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC) at 50 Millimole/l concentration of glucose ranged from 0.3 ± 0.02 in Barnyard millet to 1.31 ± 0.01 in Sorghum (Great millet) whereas, that in case of WIS ranged from 0.26 ± 0.01 in Barnyard millet to 1.02 ± 0.05 in wheat. Glucose absorption capacity at 50 Millimole/l concentration of glucose was also highest in wheat fibers amongst all the fibres studied. From the results it was clear that at 5 mM/lit, the GAC was more for WIS in all sample except in Kodo millet, but in 50 mM/lit the GAC was highest for IDF in all six samples studied. Figure 4) showed the variation in glucose diffusion with addition of insoluble fibers compared to that of control as a function of time. With increases in time from 10 to 120 min the glucose content in the dialysate with addition of various fiber samples were increased in all six samples. When compared with control, test of all the fibers from six different samples could significantly (p<0.05) decreased the amounts of diffused glucose in dialysate within 120 min. After 10 minutes of incubation the glucose content of the dialysate was reduced invariably by all the fibers
Effect of insoluble fibers on GDRI (Glucose dialysis retardation index):
The retardation in glucose diffusion by fibers was expressed by values of GDRI in% (Table 5, Figure 5). GDRI is a useful in vitro index to predict the effect of fiber in the delay in glucose absorption inside the gastrointestinal tract. Glucose diffusion retardation index (GDRI) of different fibers after 10 minutes interval varied from 3.92 ± 7.26 in sorghum IDF to 71.61 ± 3.12 in sorghum WIS and that after 30 minutes of incubation ranged from 8.34 ± 4.58 in wheat AIS to 75.69 ± 1.17 in kodo millet AIS. GDRI of different fibers after 60 minutes of incubation varied from 8.40 ± 0.49 in ragi AIS to 78.12 ± 1.17 in kodo millet AIS whereas, that after 120 minutes of incubation ranged from 6.72 ± 0.57 in ragi AIS to 69.44 ± 0.71 in kodo millet WIS.
Effect of insoluble fibers on starch digestibility:
The effects of various insoluble fibers on starch digestibility were demonstrated by changes in the glucose content in dialysate as a function of time when starch, fiber and diastase were dialysed against distilled water (Table 6, Figure 6). After 10 min of incubation the comparable glucose content in dialysate with fiber addition indicated that there was significant difference in starch digestibility in first 10 min in all fibers from six samples. Kodo millet IDF retarded the starch digestibility to the greatest extent after 10 min of incubation (glucose in dialysate being 1.84 ± 0.28 micromole) and the wheat IDF retarded the least (glucose in dialysate being 25 Values (mean ± SE, n=6) in same column with different letter superscripts are significantly different, (p<0.05) micromoles of glucose in dialysate. As the incubation time increases the results showed that glucose content in the dialysate increased with subsequent increases of time. The three types of insoluble fibers in all six samples were found to retard starch digestibility effectively along the enzymatic digestion process. When compared with control, the glucose content in the dialysate with all insoluble fibers was less than that of control excepting the sorghum WIS after 60 min of incubation. In case of kodo millet, proso millet, and barnyard millet glucose content in dialysate was higher in WIS than in IDF and AIS but in wheat it was highest in IDF at 10 and 30 min highest in WIS at 60 and 120 min whereas, in case of finger millet glucose content in dialysate was highest in AIS at 10, 30 and 60 min but highest in WIS at 120min.
Effects of various insoluble fibers on alpha-amylase activity:
In (Table 7,8, Figure 7,8) the effects of various insoluble fibers on alphaamylase activity are presented in terms of glucose production rate and residual amylase activity (%). The reduction in glucose production rate was lower than that of control in all the samples studied. Glucose production rate was higher in AIS than IDF and WIS of all the samples. Among the six samples finger millet have highest glucose production rate in IDF, AIS and WIS (8.61 ± 1.00, 19.67 ± 1.27 and 18.69 ± 1.56 respectively) and Sorghum IDF showed the lowest value (4.55 ± 0.86). The reduction in glucose production rate could also be presented in another way by means of decrease in residual amylase activity (%) which is the percentage of glucose production rate by addition of fiber over control. In this case also the AIS of all six samples have higher residual amylase activity as compared to their corresponding IDF and WIS, finger millet being the highest (97.67 ± 8.76) and sorghum being the lowest (53.89 ± 5.99). Sorghum IDF reduced the α-amylase activity to the lowest extent (21.7 ± 3.32).
Discussion
Degenerative diseases such as diabetes mellitus become prevalent in the population due to more sedentary lifestyle in name of modernization and westernization. Dietary fiber plays an integral role in the management of diabetes mellitus. High dietary fiber content of millets and less prevalence of metabolic disorders in tribal people regularly consuming it prompted us to undertake a comparative study on hypoglycemic and anti-oxidative efficacy of insoluble fiber rich fractions of different cereals and millets commonly grown in tribal regions of Odessa.
Proximate analysis
Proximate composition analysis was performed for both whole grains (Table 1) and bran samples ( Table 2). As dried and stored grains were procured so there was slight variation in moisture and dry matter content in all the grain samples studied ( Table 1). The ether extractives varied from 4.35 ± 0.164% in barnyard millet to 1.003 ± 0.057% in proso millet and high crude fiber content was observed in all the grains studied ( Table 1). The crude protein content of grains ranged from 10.39 ± 0.248% in barnyard millet and 10.25 ± 0.530% in wheat to 5.48 ± 0.449% in kodo millet. Ash content also varied from 3.59 ± 0.246% in kodo millet and 3.59 ± 0.234% in proso millet to 1.29 ± 0.085% in great millet whereas; the NFE% was almost similar in all the cases. These types of variations in the composition might be due to the difference in genetic makeup and environment in different grains and our results were in accordance to [2,29]. It was clear from the results that the crude fiber% and the ash content were higher in bran sample than the whole grain in all six samples studied ( Table 2). Hadimani and Malleshi [3] reported similar findings in different millets. Thus the bran samples were used for extraction of fiber and in vitro evaluation of hypoglycemic effect of insoluble fibers whereas, for evaluation of antioxidant, antibacterial antifungal property and the polyphenol content whole grain powder was used with methanol as solvent.
Hypoglycemic effect of insoluble fibers from millets in vitro
In vitro hypoglycemic effect of insoluble fibers from different cereal and millet samples was studied by assessing the effect of various fibers on glucose adsorption capacity (GAC), glucose diffusion, GDRI (Glucose dialysis retardation index), starch digestibility and alphaamylase activity to have a simulation of digestion and absorption of carbohydrates in the intestine. These experiments were designed to form the basis of simple methods to measure the potential biological effects of various insoluble dietary fibers. Discussion of each of these tests was given separately to have better understanding. Effect of insoluble fibers on glucose adsorption capacity: (Table 3 Figure 1) revealed that all fiber samples at different glucose concentration (5, 10, 25 and 50 milimol/lit) could bind glucose effectively and amount of glucose bound to these fibers were concentration dependant and increased with higher concentration of glucose. There is significant difference (P<0.05) in the glucose adsorption capacity (GAC) of different insoluble dietary fibers in all the concentrations of glucose (Table 3). Ou et al [30] have reported that insoluble fiber derived from wheat bran could adsorb glucose at different concentrations to decrease the concentration of glucose available in small intestine. The finger millet WIS showed highest GAC (0.18 ± 0.01 mM/g) at normal (5 mM/l) concentration whereas, at a higher glucose concentration of 50 mM/l wheat IDF exhibited highest GAC (1.65 ± 0.02 mM/g). At low concentration of glucose WIS in all cases showed higher adsorption of glucose but interestingly it was reversed at higher concentration of glucose and IDF in all grain samples showed high GAC. Adsorption of glucose by insoluble fibers might be attributed to the increased water holding capacity of the fibers. When the glucose concentration reduced to 5 mmol/litre, the GAC found to be very low which indicated that the insoluble fiber might help to retain glucose to small extent in the intestinal lumen even at a low glucose concentration. Our findings were in binding with the findings of [31]. Further validation of these effects in vivo is required to confirm the hypoglycemic ability of these fibers.
Effect of insoluble fibers on diffusion of glucose: Diffusion of glucose from the dialysis membrane was affected by dietary fibers (Table 4, Figure 2). Diffused glucose for different insoluble fibers was less affected at 10 and 30 minutes but at 60 and 120 minutes it was much affected and showed higher retardation in glucose diffusion in all the cases as compared to control. By hypothesis the effect of dietary fiber on diffusion was mainly due to their viscosity. The diffusion rate would decrease as time increased because of the gradual increase in viscosity of the medium. The diffusion rate of glucose was decreased by insoluble fibers even if they contribute less to viscosity [30]. This phenomenon can be explained by adsorption of dietary fiber for glucose. Some authors indicated that the retardation in glucose diffusion and absorption due to fibers was affected by viscosity of intestinal content [32]. The retardation effect of insoluble fibers which contribute little to viscosity of solution might be probably attributed to their adsorption capacity. At beginning of dialysis glucose diffusion might be affected by adsorption of glucose on fiber and viscosity so diffusion rate was slow (though glucose concentration is higher inside the dialysis bag) with progress of time diffusion of glucose affected by only viscosity of fiber. The retardation of glucose diffusion might be due to the physical in the jejunum. Ability of these fibres to retard the absorption of glucose in the gastrointestinal tract is a function of their viscosity [36]. The modest effects on glucose dialysis retardation index seen with insoluble fibres are in keeping with their effect on glucose absorption in man. Based on these results, it was conceived that these insoluble fibers could effectively adsorb glucose, delay the glucose diffusion and subsequently postpone the glucose absorption in the gastro-intestinal tract.
Effect of insoluble fibers on starch digestibility:
The effects of various insoluble fibers on starch digestibility were presented in (Table 6, Figure 4) IDF of all the grains excepting wheat retarded the starch digestibility significantly (P<0.05), even at 10 minutes of incubation there was 70 to >90% retardation of starch digestibility. Similar was the case with increasing time to 30, 60 and 120 minutes ( Table 6). According to view of [37] dietary fibers can be adsorbed to starch and thus hinder hydrolysis of starch by alpha-amylase.
The various millet fiber fractions modified enzymatic starch digestion similarly. Compared to control initial rate of starch digestion was not modified by the presence of AIS and WIS fibers but a decrease in final rate and total starch degradation was noted. This decrease may be attributed to a direct effect of fiber on amylase activity due to adsorption of enzyme on the fibers or a decrease in activity due to viscosity or pH modification of medium. Presence of fiber might have also influenced the accessibility of the enzyme to its substrate. In the present study, the apparent reduction in the glucose contents in the dialysate by the presence of insoluble fibers indicated that both the starch degradation and glucose diffusion could be delayed by the insoluble fibers of the millets, even though glucose is actually absorbed into the small intestine through an active process in the human body.
In most of the millets taken, the IDF has more effect on decreasing starch digestibility. The apparent reduction in glucose content in the dialysate by presence of insoluble fibers (Table 6) indicated that both the starch degradation and glucose diffusion could be delayed by the insoluble fibers even through glucose is actually absorbed into the small intestine through an active process in the human body.
Effect of insoluble fibers on alpha-amylase activity: Effect of various insoluble fibers from different millet species showed that the IDF insoluble fibers could exhibit significant (P<0.05) effect in decreasing the alpha-amylase activity (Table 7, Figure 5 and 6) and starch digestibility (Table 6, Figure 4) This effect of insoluble fibers of millets might be due to several possible factors such as fiber concentration, presence of inhibitor on fibers, capsulation of starch and enzyme by fibers, reduced accessibility of the starch and direct adsorption of enzyme on fibers leading to the decrease in amylase activity [31]. The variation in the inhibitory activity to α-amylase among the different insoluble fibers suggested that the inhibition depended on the kind of fiber.
In this in vitro study the abilities of insoluble fibers from millet to adsorb glucose, slowed down glucose diffusion and starch digestibility and decrease the activity of alpha-amylase suggested that they might have hypoglycemic effects in delaying release of glucose from starch, reducing rate of glucose adsorption and hence controlling concentration of post prandial serum glucose [33].
The potential hypoglycemic effects of these fibers suggested that they could be incorporated as low-calories bulk ingredient in high fiber foods to lower post prandial serum glucose level and reduce calories levels. Further detailed studies are needed to investigate whether the insoluble fibers from the millets and cereals studied are competent inhibitors of α-amylase or simply act as a barrier between the amylase and starch.
Conclusion
• In-vitro studies on hypoglycemic effects of insoluble fibers of different cereals and millets (kodo millet, proso millet, barnyard millet, finger millet, wheat and sorghum) indicated that, • All these grains are having nutritional composition either comparable or better than most of the commonly used grains so could be used as staple food.
• All the three types of insoluble fibers from the six millet and cereal samples taken have potent hypoglycemic effect. Thus they can be included in diet having low carbohydrate and high fiber content, which will be beneficial in management of diabetes both in human being and pet animals. | v3-fos-license |
2020-04-02T09:27:16.212Z | 2020-03-01T00:00:00.000 | 214749085 | {
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} | pes2o/s2orc | Biomass Smoke Exposure Is Associated With Gastric Cancer and Probably Mediated Via Oxidative Stress and DNA Damage: A Case-Control Study
PURPOSE We investigated the association between gastric cancer and environmental and dietary exposures. In addition, we explored probable mechanistic pathways for the influence of biomass smoke on gastric carcinogenesis. PATIENTS AND METHODS The study was conducted in Lusaka, Zambia. Questionnaires were used to collect data on risk factors, whereas enzyme-linked immunosorbent assays and high-performance liquid chromatography were used to measure biologic exposures. Study data were analyzed using contingency tables and logistic regression. RESULTS We enrolled 72 patients with gastric adenocarcinoma and 244 controls. Gastric cancer was positively associated with rural residence (odds ratio [OR], 2.9; 95% CI, 1.5 to 5.3), poverty (OR, 4.2; 95% CI, 1.9 to 9.1), and daily consumption of processed meat (OR, 6.4; 95% CI, 1.3 to 32) and negatively associated with consumption of green vegetables (OR, 0.2; 95% CI, 0.1 to 0.5). Gastric cancer was also associated with biomass smoke exposure (OR, 3.5; 95% CI, 1.9 to 6.2; P < .0001), an association that was stronger for intestinal-type cancers (OR, 3.6; 95% CI, 1.5 to 9.1; P = .003). Exposure to biomass smoke in controls was associated with higher urinary levels of 8-isoprostane (P < .0001), 8-hydroxydeoxyguanosine (P = .029), and 1-hydroxypyrene (P = .041). Gastric cancer was not associated with biochemical measures of current exposure to aflatoxins or ochratoxins. CONCLUSION In Zambia, exposure to biomass smoke, daily consumption of processed meat, and poverty are risk factors for gastric cancer, whereas daily consumption of green vegetables is protective against gastric cancer. Exposure to biomass smoke was associated with evidence of oxidative stress and DNA damage, suggesting mechanistic plausibility for the observed association, and the association was restricted to intestinal-type gastric cancer.
INTRODUCTION
Gastric cancer is one of the leading causes of cancerrelated deaths worldwide, 1 but in Africa, data on its occurrence are scanty. 2 Scarce resources limit the ability to diagnose, treat, and stage gastric cancer, compromising the ability to clearly understand the epidemiology of the disease in Africa. 3 Furthermore, there are limited population-based registries and poor record keeping in many African centers. 4 Our preliminary work on gastric cancer in Zambia provided the basis to further explore risk factors associated with gastric carcinogenesis. We observed that the diagnosis of early-onset gastric cancer was unexpectedly high and that patient outcomes were poor. [5][6][7] These preliminary data also showed no association between gastric cancer and the Helicobacter pylori virulence factor CagA. 6,8 Therefore, we endeavored to evaluate environmental and dietary influences on gastric cancer in Zambia.
The role of nutrition in the development of gastric cancer has been widely investigated with inconsistent results, 9 and much of the data have been obtained from observational studies. 10 Mycotoxins are one of the most common dietary contaminants in Africa. [12][13][14] They are small-molecular-weight compounds produced by filamentous fungi or molds under suitable temperatures and humidity. Aflatoxins are such toxins, produced by fungi of the Aspergillus species and Author affiliations and support information (if applicable) appear at the end of this article.
Accepted on February 24, 2020 and published at ascopubs.org/journal/ go on March 31, 2020: DOI https://doi. org/10.1200/GO.20. 00002 known to contaminate peanuts and maize, whereas ochratoxins mainly contaminate cereals, coffee, and grape berries. These mycotoxins have the potential to be carcinogenic. Biomass smoke is produced by the combustion of any organic matter, such as firewood, charcoal, grass, or dung. These are common sources of fuel in Africa, with . 70% of the population depending on biomass fuels. 15 However, the health consequences of long-term, or even lifetime, exposure to biomass smoke have been poorly investigated. Several researchers have linked squamous cell carcinoma of the esophagus to biomass smoke exposure, [16][17][18] but the evidence for gastric cancer is scanty. For example, a study involving fire fighters in Sweden showed that they had an increased risk of developing gastric cancer, 19 but this was indirect evidence.
We explored a possible link of gastric carcinogenesis to dietary factors including exposure to mycotoxins and biomass smoke. In addition, we explored measurable biomarkers including urinary 1-hydroxypyrene (1-OHP), a metabolite of one of the major constituent groups of biomass smoke, polycyclic aromatic hydrocarbons. Evidence of oxidative stress measured by urinary 8-isoprostanes was also evaluated. Oxidative stress to DNA double bonds was estimated using urinary 8-hydroxydeoxyguanosine (8-OHdG) and serum γ-H2AX. H2AX is one of the histone octamers that DNA is wrapped around and could be used to estimate double-stranded breaks. Although some of these biomarkers reflect only short-term exposure, we looked for early evidence that they could illuminate the connection between exposure and cancer development. The University of Zambia Biomedical Research Ethics Committee (reference No. 000-03-16) approved this study.
Patient Recruitment
This was a case-control study conducted at the University Teaching Hospital (UTH) in Lusaka, Zambia, between July 2016 and April 2018. UTH is the largest referral hospital in Zambia, serving patients from all ten provinces of the country. Patients referred for a diagnostic esophagogastroduodenoscopy (EGD) were targeted for enrollment. Patient cases were patients diagnosed with gastric cancer, whereas controls had no endoscopic or histologic evidence of malignancy. All patients presenting to the UTH endoscopy unit for EGD were considered for study enrollment. Patients who gave written informed consent were included in the study. From these, we excluded individuals with prior history of gastric or esophageal cancer diagnosis or therapy. For assessment of lifestyle risk factors, we used interviewer-administered questionnaires, which included questions on diet, biomass smoke exposure, and socioeconomic factors. Biologic samples were collected to search for biochemical corroboration of reported exposures.
EGD
After obtaining consent, an EGD was performed in fasted patients. During the EGD, biopsies were taken from gastric lesions suspected of being malignant. In all cases, at least six biopsies were taken from various sites of the lesions. For those without suspicious lesions, six biopsies were taken, two each from the antrum, incisura, and body. All biopsies were immediately placed in formalin and sent for histopathology. An experienced technician using standard methods processed biopsies sent for histopathology. The study histopathologist (A.S.) evaluated all the slides to determine the histologic diagnosis. Gastric adenocarcinomas were divided using the Lauren classification into intestinal type, diffuse type, and mixed type (composed of both the intestinal and diffuse types). Peripheral blood (serum later extracted) and urine samples were collected and stored at −80°C until analysis using the techniques outlined in the following sections.
Key Objective
There is a high proportion of early-onset gastric cancer among Zambian adults. This cannot be explained by the high incidence of Helicobacter pylori infection alone. Therefore, we set out to investigate other factors that could be associated with gastric cancer.
Knowledge Generated
We found that exposure to biomass smoke, poverty, and rural residence were risk factors for gastric cancer. Exposure to biomass smoke on its own was associated with oxidative stress. We also found that daily consumption of green vegetables was negatively associated with gastric cancer. We found no association with either aflatoxin or ochratoxin exposure.
Relevance
Our study indicates that improvement of socioeconomic status, use of cleaner fuels, and healthier diets could affect the occurrence of gastric cancer in Zambia.
Optical density was read at 450 nm. Results were corrected for creatinine excretion and reported as nanograms of 8-OHdG per milligram of creatinine.
Urinary 8-Isoprostanes
Following the manufacturer's instructions, urinary levels of 8-isoprostane were measured using ELISA (Detroit R&D, Detroit, MI). Optical density was read at 450 nm. These readings were also corrected for creatinine, and the final results were reported as nanograms of 8-isoprostanes per milligram of creatinine.
Serum Human H2AFX
Sera were analyzed for human H2AFX (histone H2AX) by ELISA (MyBioSource) at 1:10 dilution, as instructed by the manufacturer. Optical density was again read at 450 nm, and the results are reported in picogram per milliliter.
Urinary 1-OHP
High-performance liquid chromatography (HPLC) was used to measure urinary concentration of 1-OHP after enzymatic hydrolysis of the conjugates. The phenolic compound stock standard (Clincal; reference No. 9925; Recipe Chemicals, Munich, Germany) was used as the calibrator to make up standards from concentrations of 0.55 to 3.00 mg/L in deionized water. To all samples, calibrators, and controls (600 mL volume), β-glucuronidase enzyme mix (300 mL) was added. The enzyme mix was prepared by adding β-glucuronidase (50 mL) to 0.1 M of sodium acetate buffer pH 5 (5 mL). All samples, controls, and calibrators were incubated at 37°C for 4 hours followed by analysis on the HPLC system. Two certified reference controls (ClinChek; reference Nos. 8923 and 8924; Recipe Chemicals), levels 1 and 2, were used and run after the calibration and after every 10 samples. The percent recovery of the 2 certified reference controls, ClinCheck levels 1 and 2, are 97% and 95%, respectively. The limit of quantitation was 0.052 mg/L. A Waters (Milford, MA) system HPLC was used, with a 1525 binary pump, 717 autosampler, and 2475 fluorescence detector. The mobile phase consists of a methanol-to-water ratio of 3:1. The volume injected was 100 mL, and separation was performed on a Phenomenex SphereClone 3 mm ODS (2) 80Å 100 × 4.6 mm column (Phenomenex, Torrance, CA). The excitation wavelength was set at 242 nm and the emission wavelength at 388 nm. The flow rate was 1 mL/min for 5 minutes. The levels of 1-OHP were reported in microgram per gram of creatinine.
Testing for HIV
Serum was tested for HIV antibodies using Uni-Gold rapid diagnostic kits (Trinity Biotech, Wicklow, Ireland).
Statistical Analysis and Data Availability
Medians and interquartile ranges were used to summarize continuous variables. Two-way analyses were used to look for associations between gastric cancer and the exposures of interest using either the Fisher's exact or χ 2 test. In addition, stepwise unconditional logistic regression was used to assess the relative contributions of different
RESULTS
We recruited and studied 388 participants, 92 of whom (24%) had gastric cancer seen during endoscopy and 296 of whom were initially recruited as controls. For analysis of risk factors, only those with confirmed adenocarcinoma were included as patient cases (n = 72). The remaining 20 patients with gastric cancer seen during endoscopy had either no confirmatory histology report (n = 8) or other types of gastric cancer, including squamous cell or unclassified carcinomas (n = 8), gastric stromal cancer (n = 2), non-Hodgkin lymphoma (n = 1), or a hematolymphoid tumor (n = 1). Patients without any detectable gastric premalignant lesions seen on histology were used as controls (n = 244). We excluded patients with either gastric atrophy or intestinal metaplasia from the final risk factor analysis (Fig 1).
Characteristics of Enrolled Patients and Their Association With Gastric Cancer
Patients with gastric cancer were significantly older than those without cancer (Table 1). Therefore, age was adjusted for in subsequent analyses. Gastric cancer was significantly associated with poor housing, poor water, and lack of a kitchen (all P , .05; Fig 2). Patients with gastric cancer had higher odds of having a low socioeconomic status assessed using the previously mentioned parameters (OR, 4.2; 95% CI, 1.9 to 9.1; P = .0002). Applying an unconditional logistic regression analysis adjusted for age, sex, and residence, patients with gastric cancer had significantly higher odds of not having good housing (OR, 5.3; 95% CI, 2.1 to 13.5; P , .0001) or a kitchen (OR, 3.4; 95% CI, 1.5 to 7.4; P = .003).
Association Between Gastric Cancer and Biomass Smoke Exposure
Overall, 110 (35%) of 316 participants reported that they were completely reliant on biomass fuel for cooking, whereas another 107 (34%) of 316 used it occasionally because they had access to electric stoves. Thirty-nine percent of participants (99 of 316 participants) did not use biomass fuels in their homes at all. There was an association between gastric cancer and reliance on biomass fuel (OR, 3.5; 95% CI, 1.9 to 6.2; P , .0001). Two of the 3 unconditional logistic regression models factoring in parameters used to assess socioeconomic status and basic characteristics showed that biomass smoke exposure was an independent risk factor gastric cancer (Table 2). Of the patients with results for Lauren tumor classification, 28 (62%) of 45 had intestinal, 15 (34%) of 45 had diffuse, and 2 (4%) had mixed type of gastric cancer. Intestinal-type gastric cancer was associated with biomass smoke (OR, 3.6; 95% CI, 1.5 to 9.1; P = .003), whereas the diffuse type was not (OR, 0.9; 95% CI, 0.2 to 3.1; P = 1.00).
Associations Between Biomass Smoke Exposure and Measurable Biomarkers in Patients Without Gastric Cancer
We compared levels of 1-OHP, 8-OHdG, γ-H2AX, and 8-isoprostanes between patients frequently, occasionally, and rarely exposed to biomass smoke. Patients with history of cigarette smoking (n = 19) were excluded from this analysis. The median 1-OHP level among the patient cases was 0. creatinine) than controls (median, 4.0 ng/mg creatinine; IQR, 2.0-10 ng/mg creatinine; P = .012).
We then analyzed the influence of biomass smoke exposure on these biomarkers among controls only. Using the nonparametric test for trend, measured levels of 1-OHP, 8-OHdG, and 8-isoprostanes were significantly higher with increased use of biomass fuels (Fig 3). Table 3 compares daily and regular (at least once a week) consumption of various food types and groups in patients with gastric cancer and controls. There was a significant negative association between gastric cancer and regular consumption of green vegetables, eggplants, or fruit (Table 3). Despite the small numbers, daily consumption of processed sausages was associated with gastric cancer. Using unconditional logistic regression adjusted for age, sex, and residence, gastric cancer was negatively associated with daily consumption of green vegetables (OR, 0.2; 95% CI, 0.1 to 0.5; P = .0001) and positively associated with daily consumption of processed meat (OR, 6.4; 95% CI, 1.3 to 31.8; P = .022).
Gastric Cancer and Dietary Exposure to Mycotoxins
Of 254 patients with aflatoxin M1 results, 149 (63%) had detectable toxin in their urine. The median urinary aflatoxin M1 level was 33 ng/mL (IQR, 22-53 ng/mL); after correcting for urine creatinine, the median level was 35 ng/mg creatinine (IQR, 11-397 ng/mg creatinine). This was not dependent on age (P = .38) or sex (P = .37) but was significantly higher in patients living in urban areas (Fig 4).
Having aflatoxin in urine was not affected by lack of basic household goods (P = .40) or good housing (P = .34). Of the patients with ochratoxin results, 278 (96%) of 289 had evidence of ochratoxin A in their blood. The median level was 0.1 ng/mL (IQR, 0.06-0.16 ng/mL). There was no significant difference in ochratoxin levels for patients living in rural or urban areas (Fig 4). Similarly, ochratoxin levels were not different between patient cases and controls (Fig 4). Age, sex, and socioeconomic class had no influence on ochratoxin levels (P = .65, .56, and .82, respectively).
DISCUSSION
In this study, gastric cancer was associated with low socioeconomic status, frequent exposure to biomass smoke (an effect restricted to intestinal-type cancers), and regular consumption of processed meat. Regular consumption of fruits and vegetables was protective against gastric cancer, and exposure to biomass smoke was associated with evidence of oxidative DNA damage in controls.
As previously published, the median age for patients with gastric cancer was at least a decade lower than that reported from developed countries, with 20% of the patients being under the age of 45 years (early-onset cancers). 6 The prevalence was not significantly different between males and females, which is another point of distinction from industrialized countries, where male patients predominate. 1 Biomass smoke was associated with gastric cancer, a finding that requires further corroboration. Controls exposed to biomass smoke had significantly higher levels of urinary 8-OHdG and 8-isoprostanes than those not frequently exposed, suggesting a link between biomass smoke and oxidative stress. This is consistent with a Norwegian study that showed that oxidative damage to DNA and repair was induced by wood smoke particles in human A549 and THP-1 cell lines. 23 Cigarette smoking is a well-described risk factor for gastric cancer. 24,25 Some of the carcinogenic compounds that are found in cigarette smoke (eg, benz[a]anthracene and benzo[a]pyrene) are also found in biomass smoke. 26 There may be molecular parallels between swallowed biomass smoke and inhaled tobacco smoke, both of which increase cancer risk by inducing oxidative stress in epithelial cells.
Measured urinary levels of 1-OHP, a metabolite of polycyclic aromatic hydrocarbons (PAHs), were not associated with gastric cancer but were increased with exposure to biomass smoke in a stepwise manner. 1-OHP is not the best biomarker for assessment of long-term biomass smoke exposure because metabolism of PAH is rapid, and therefore, checking for metabolites in urine might not necessarily be an accurate reflection of such exposure in patients whose cancer has already developed and who would already have adapted to severe ill health. Changes in behavior when gastric cancer developed could have led to reduced exposure to biomass smoke, resulting in lower 1-OHP levels, perhaps because ill health reduces activities of daily living. In addition, measured PAH metabolites could have also come from other sources such as fumes from diesel engines, resulting in higher readings in patients not reliant on biomass fuels. This might be especially true in urban areas or near main roads. A study cohort including 256,357 men in Sweden showed that there was an increased risk of gastric cancer in workers exposed to diesel fumes. 27 In addition, a 15-year United Kingdom (UK) cohort study involving more than 34 000 employees in eight UK oil refineries found that gastric cancer risk was increased in laborers with long service compared with the UK general population. 28 All these data illustrate the need for further research on environmental pollution and gastric cancer.
Studies from developed countries have linked gastric cancer to low socioeconomic status. 29 In this study, patients with gastric cancer had less basic household items and were living in poorer-quality houses without ready access to piped or treated water. This effect remained significant after adjusting for rural residence, which is where many of the patients lived. We believe that the effect of poverty may operate through some of the risk factors we have identified. Many Zambians do not eat fruit on a daily basis, and the widely available or affordable fruits are predominantly seasonal. These data add more evidence in support of the protective properties of regular fruit intake. Some investigators have reported a benefit of Allium vegetables, such as onions, for protection against gastric cancer. 30, 31 Our data did not show any significant protection of Allium vegetables against gastric cancer probably Gastric Cancer and Biomass Smoke Exposure in Zambia because of the low number of patients not eating onions on a daily basis. Regular consumption of eggplants (aubergines) was found to reduce the odds of having gastric cancer. Eggplants are not part of the traditional Zambian diet, but their consumption is increasing, particularly in urban areas. They are solanaceous plants containing glycoalkaloids, which are believed to have some anticarcinogenic properties. 32 This could explain their negative association with gastric cancer.
The proportion of patients with detectable aflatoxin M1 in their urine was high, particularly for those living in urban areas. It could be an indication of poor grain storage, especially maize, which is the staple food in Zambia. Socioeconomic status did not show any influence on exposure to aflatoxins, suggesting that the source of the toxin could also be from well-packaged commercially available maize and groundnut products consumed by urban dwellers, rich or poor alike. Patients with gastric cancer had significantly lower aflatoxin M1 levels than the controls. This could be a result of changes in food intake as a result of the illness itself. In addition, the metabolism of aflatoxins is quite rapid, and the assay we used was only validated to determine exposure of aflatoxin ingestion in the prior 2 to 3 days. 33 Exposure to ochratoxins was high in this patient group. The proportions found were much higher than those reported among Koreans (42%). 34 However, unlike the aflatoxins, there was no significant difference between urban and rural residents or between patient cases and controls.
Despite the known limitations of a hospital-based study, mapping the town of permanent residence showed a fairly good representation of patients across the country, correlating well with the population distribution in Zambia. We do acknowledge that these data represent a convenience sample based on referral for EGD and not a populationbased sample; therefore, they may not reflect epidemiology across the whole nation.
In conclusion, biomass smoke exposure is a risk factor for gastric cancer, possibly mediated through oxidative stress. Regular consumption of green vegetables and eggplants reduces the odds of developing gastric cancer, whereas regular consumption of processed meat increases the odds of developing gastric cancer. | v3-fos-license |
2020-04-21T13:03:50.182Z | 2020-04-21T00:00:00.000 | 216028087 | {
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} | pes2o/s2orc | Length-Dependent Structural Transformations of Huntingtin PolyQ Domain Upon Binding to 2D-Nanomaterials
There is a strong negative correlation between the polyglutamine (polyQ) domain length (Q-length) in the intrinsically disordered Huntingtin protein (Htt) exon-1 and the age of onset of Huntington's disease (HD). PolyQ of Q-length longer than 40 has the propensity of forming very compact aggregate structures, leading to HD at full penetrance. Recent advances in nanobiotechnology provided a new platform for the development of novel diagnosis and therapeutics. Here, we explore the possibility of utilizing 2D-nanomaterials to inhibit the formation of supercompact polyQ structures through the so-called “folding-upon-binding” where the protein structure is dependent on the binding substrate. Using molecular dynamics simulations, we characterize two polyQ peptides with Q-length of 22 (Q22, normal length) and 46 (Q46, typical length causing HD) binding to both graphene and molybdenum disulfide (MoS2) nanosheets, which have been applied as antibacterial or anticancer agents. Upon binding, Q22 unfolds and elongates on both grapheme and MoS2 surfaces, regardless of its initial conformation, with graphene showing slightly stronger effect. In contrast, initially collapsed Q46 remains mostly collapsed within our simulation time on both nanosheets even though they do provide some “stretching” to Q46 as well. Further analyses indicate that the hydrophobic nature of graphene/MoS2 promotes the stretching of polyQ on nanosheets. However, there is strong competition with the intra-polyQ interactions (mainly internal hydrogen bonds) leading to the disparate folding/binding behaviors of Q22 and Q46. Our results present distinct Q-length specific behavior of the polyQ domain upon binding to two types of 2D-nanomaterials which holds clinical relevance for Huntington's disease.
INTRODUCTION
Neurodegenerative Huntington's disease (HD) is caused by expansion of the trinucleotide CAG repeats in exon-1 of the HD gene, the mutation that encodes an extended polyglutamine (polyQ) tract within the N-terminal exon-1 of the Huntingtin protein (Htt) (Macdonald et al., 1993). There is a strong negative correlation between polyQ length (Q-length) and the age of onset of HD (Saudou et al., 1998;Wexler and Res, 2004;Gray et al., 2008). Expanded polyQ regions form oligomers that aggregate into large, insoluble protein complexes, which may subsequently mature to observable fibrils (Perutz et al., 1994). PolyQ length and structure are critical for posited neurotoxicity mechanisms caused by oligomers and larger aggregates (Miller et al., 2011). Recent work from our group has found that at longer Q-lengths, increased β-sheet content seems to promote the formation of supercompact structures (Kang et al., 2017). The increased compactness at long Q-lengths indicates that the polyQ domain may result in increased neural toxicity by inducing distinct morphological changes throughout the entire Htt exon-1 protein (Kang et al., 2017).
It is nontrivial to structurally characterize the Httexon-1 protein due to its inherently disordered nature. It is also known that when the other two domains of Htt exon-1 protein, N17 (the first 17 residues of the Htt N-terminal region) and C38 (38 residues near the Httexon-1 C-terminus, polyproline-rich segment), are absent and the Q-length is 20 or greater, polyQ collapses into a disordered but compact globule (Perutz et al., 1994;Chen et al., 2002;Crick et al., 2006;Miettinen et al., 2012;Heck et al., 2014;Hoop et al., 2016). The supercompact structures of polyQ (not found in other globular proteins) are mainly owing to the "glue-like" propensity of glutamine sidechains to form many hydrogen bonds (Kang et al., 2017). The sidechains tend to be "buried" inside the protein so that the polyQ domains themselves are insoluble.
Meanwhile, there is a growing interest in applying nanobiotechnology for biomedical applications. For example, biocompatible two-dimensional (2D) nanomaterials with unique compositional, structural and physicochemical features, such as graphene and MoS 2 , have gained increased attention in the biomedical field as potential antibacterial and antitumor agents. In this study, we explore the possibility of interrupting the formation of a supercompact polyQ using novel engineered nanomaterials like graphene or MoS 2 . Some intrinsically disordered proteins (Arai et al., 2015;Shammas et al., 2016;Bonetti et al., 2018) and switch sequences (Chen and Elber, 2014;Chen et al., 2016;Porter and Looger, 2018) are thought to utilize the folding-upon-binding mechanism where the protein structure is dependent on the binding substrate. Both graphene and MoS 2 nanosheets have a highly hydrophobic surface with large water contact angles (despite that MoS 2 has partial charges on Mo and S atoms), offering a biomimetic environment for protein binding when interfaced with water (Mathesh et al., 2016;Gu et al., 2017;Zhang et al., 2017). Previous studies have observed a variety of effects of graphene on proteins, including disruption of protein structures (Zuo et al., 2011;Chong et al., 2015;Wang et al., 2015), enhancement of enzymatic activity (Mathesh et al., 2016), and potential interference of protein-protein interactions (Luan et al., 2015(Luan et al., , 2016aFeng et al., 2016). Comparatively, there are less studies on MoS 2 's effect on proteins partly due to the lack of appropriate potential function parameters for MoS 2 simulation. The recently developed MoS 2 force field parameters that are compatible with the TIP3P water model ) might help facilitate a wider application of computer simulations on the interaction of MoS 2 with biomolecules.
Herein, we use all-atom molecular dynamics (MD) simulations to study Htt-polyQ adsorption onto graphene and MoS 2 nanosheet. We investigate two poly glutaminelengths, 22 (Q22, healthy Q-length) and 46 (Q46, typical Q-length associated with HD at full penetrance) to determine if the binding mechanism of the polyQ on these nanosheets is Q-length dependent. We find that Q22 exhibits a similar binding mode on both graphene and MoS 2 surfaces (with graphene showing a slightly stronger effect) regardless of its initial configuration -the final conformation of Q22 is fully extended. On the other hand, the initially collapsed Q46 remains mostly collapsed when adsorbing onto graphene or MoS 2 nanosheet, indicating that neither graphene nor MoS 2 is sufficient to fully stretch the supercompact Q46 structure (again with graphene showing slightly stronger effect). Further energetic and hydrogen bonding analyses indicate that the difference in Q22 vs. Q46 binding conformations is determined by the competition between polyQ internal interactions (mainly intra-hydrogenbonds) and polyQ-nanosheet interactions. Insights derived from our simulations of polyQ interaction with these novel 2D-nanomaterials may lead to future potential applications in diagnoses and therapeutics for Huntington's disease (HD).
SIMULATION METHODS
In this work, we study two poly glutamine peptides of length 22 (Q22) and 46 (Q46) adsorbing onto graphene and MoS 2 nanosheets. Figure 1 illustrates the initial configurations of polyQ on the graphene surface simulation systems: fully extendedQ22 at the graphene interface ( Figure 1A), collapsed Q22 at the graphene interface ( Figure 1B), fully extended Q46 at the graphene interface ( Figure 1C), and collapsed Q46 at the graphene interface ( Figure 1D). The equivalent simulation systems of polyQ on the MoS 2 surface are shown in Figure S1. In each initial configuration, the polyQ was separated from the graphene/MoS 2 nanosheet by 0.75 nm to prevent large initial interactions. The OPLS-AA force field (MacKerell et al., 1998) was used for the polyQ. The force fields for graphene and MoS 2 nanosheets are the same as in our previous MD studies (Tu et al., 2013;Luan and Zhou, 2016;Luan et al., 2016b). Sodium and chlorine ions were added in each system to yield an electrolyte concentration of 100 mM. The TIP3P model (Jorgensen et al., 1983;Neria et al., 1996) was used for water and the standard force field was used for ions (Beglov and Roux, 1994).
All MD simulations were performed with GROMACS (Hess et al., 2008). To investigate both adsorption of Q22 and Q46 on the nanosheets as well as to capture protein conformational changes, each simulation was run for 600 ns for Q22 and 1000 ns for Q46 under the NVT (T = 300 K) ensemble. Following similar protocols used in our previous studies (Zhou et al., 2001Zhou, 2003Zhou, , 2004Li et al., 2005;Das et al., 2009;Xia et al., 2012), we first performed energy minimization and 100 ps MD equilibration under the NPT ensemble (pressure = 1 bar) prior to production runs with the positions of protein and nanosheets FIGURE 1 | MD simulation systems of a fully extended (A) and collapsed (B) Q22 peptide near the water-graphene interface, and a fully extended (C) and collapsed (D) Q46 near the water-graphene interface. Atoms in graphene and PolyQ are shown as spheres and cartoon, respectively. Water molecules and ions are not shown. Graphene is colored in cyan; the Q22 is colored in blue; and the Q46 is colored in red. The equivalent simulation systems of polyQ on the MoS 2 surface are shown in Figure S1. restrained to relax the solvent. A smooth cutoff (cutoff distance of 12 Å) was used to calculate the van der Waals (vdW) interactions. The particle-mesh Ewald method (with the grid size ∼1 Å) was applied for the electrostatic interactions. The motion equation was integrated using the Verlet algorithm, with a time step of 2 fs. The v-rescale temperature coupling method was employed in our simulations (Bussi et al., 2007).
RESULTS
To investigate the influence of graphene and MoS 2 nanosheets on the polyQ structure, we calculated the radius of gyration (Rg) and root-mean-square deviation (RMSD) of Q22 and Q46 as a function of time (Figure 2). For comparison, we also analyzed the trajectories of the control systems containing either Q22 or Q46 alone in aqueous solution. For the control systems, the polyQ peptide starts from an initially fully extended configuration with the Rg value of 2.76 nm for Q22 and 5.91 nm for Q46. As the polyQ peptides collapse in solution, the Rg decreases to stabilize at 0.76 nm for Q22 and 1.05 nm for Q46 (black in Figures 2E,F). In the polyQ + graphene and polyQ + MoS 2 systems, the initially fully extended Q22 remains extended with the Rg fluctuates around 1.52 nm (red and purple in Figure 2E). However, the initially collapsed Q22 unfolds and extends across the nanosheet surfaces, as also evidenced by the jump in Rg from ∼0.90 to 1.25 nm, with graphene showing slightly larger Rg than MoS 2 (green and blue in Figure 2E). In contrast, the collapsed Q46 peptide does not unfold when it binds to the nanosheets. Rather, the Rg of the initially collapsed Q46 increases somewhat and fluctuates around 1.36 nm when bound to graphene and around 1.22 nm when bound to MoS 2 (the Rg of the equivalent control system is 1.05 nm), again indicating graphene has slightly stronger denaturing capability toward Q46 than MoS 2 . The Rg of the initially fully extended Q46 is 1.94 nm on graphene and 2.67 nm on MoS 2 . The smaller Rg of the initially fully extended Q46 on graphene is a result of the extended peptide "winding back" toward itself, which should not be over-read in terms of significance, but rather as an indication of non-convergence in our simulations for the fully extended case. The collapsed case, on the other hand, should be more indicative. Collectively, these results indicate that the 2D-nanosheets can induce unfolding of the Q22 structure, but not Q46, with graphene showing slightly stronger capacity, which is consistent with previous studies where graphene displays stronger interaction with biomolecules than MoS 2 (Tu et al., 2013;Luan and Zhou, 2018;Wu et al., 2018).
The RMSD "trajectories" largely mimic the Rg results, namely there are RMSD jumps for the Q22 collapsed systems indicating unfolding (again with graphene showing somewhat larger jumps), while the rest of the systems fluctuates mostly randomly. The exception is when the initially fully extended Q22 and Q46 systems bind to graphene, their RMSDs fluctuate more widely when compared to the control systems, indicating large configurational diversity. Figures 2A-D show the representative final configurations of Q22 and Q46 on the graphene and MoS 2 surfaces. Both the initially fully stretched and collapsed Q22 adsorbed onto the hydrophobic graphene and MoS 2 nanosheets in a similar stretched manner, while the initially collapsed Q46 did not stretch out on either of the nanosheets like the collapsed Q22 did. For the graphene system, the collapsed Q22 starts to unfold and spread out on the surface of the graphene after 236 ns (Figure 2I). This transition process corresponds to the increase of Rg shown in Figure 2E at ∼236 ns. At 244 ns, the Rg reaches 1.27 nm, which is correlated with Q22 achieving a stretched configuration on graphene. Note that this configuration is not fully stretched but maintains some kinks and turns and remains stable for over 50 ns. The collapsed Q22 on MoS 2 nanosheet exhibits similar behavior to that of graphene except the unfolding is not as extended. However, the collapsed Q46 does not unfold on either the graphene or MoS 2 nanosheet (Figures 2C,D, bottom panel). In order to further characterize the structure of polyQ on the surface of nanosheets, we analyzed the contacts between the polyQ peptides and the nanosheets. This involved: (1) calculating the number of heavy atoms in contact with graphene and MoS 2 nanosheets (Figures S2A,B), (2) computing the average center of mass (COM) distances between polyQ sidechains and graphene, and between polyQ sidechains and the upper sulfur atoms in the MoS 2 sheet (Figures S2C,D), as a function of simulation time. Here, a contact between polyQ and nanosheets was defined if the distance between any polyQ heavy atoms is within a distance of 4.5 Å of any graphene/MoS 2 atoms. In both the graphene and MoS 2 systems, the atom contact number (ACN) and the COM distance of the initially fully extended Q22 show that Q22 quickly binds the nanosheets (<20 ns). The ACN and COM distance of the initially collapsed Q22 reveal more gradual, detailed binding and unfolding behavior. The initially collapsed Q22 systems exhibit two distinct binding stages: (i) In the early binding stage (0-236 ns in graphene, and 0-40 ns in MoS 2 ), a portion of the Q22 atoms are in direct contact with the graphene and MoS 2 nanosheets. The binding is not strong enough to restrain the Q22 motion, so the Q22 still diffuses along the graphene/MoS 2 surface, while the number of contacting atoms slowly increases. (ii) This is the stable binding stage (after 244 ns in graphene, and after 136 ns in MoS 2 ) where the ACN reaches the maximum, which is very close to the stable binding stage of the fully extended Q22. For the Q46 system, the final ACN of the initially collapsed Q46 are far less than the initially fully extended Q46, and the COM distances with the graphene (0.67 nm) and MoS 2 (0.92 nm) surface are far away from the initially fully extended Q46 (0.42 nm on graphene and 0.33 nm on MoS 2 ), indicating different nanosheet binding structures of Q46 based on different initial conformations or non-convergence in simulations (unfortunately, a fully convergent simulation is beyond our current reach with limited computational resources. Nevertheless, we believe the Q-length dependent behavior is clear even with the current simulation lengths).
The binding behavior of polyQ on 2D-nanosheetsis further characterized by monitoring the residue contacts and contact ratio of polyQ with nanosheets (Figure 3). The contact ratio is defined as the number of residues in contact with the nanosheet to the total number of residues. A residue is in contact with the nanosheet if any heavy atom is within 4.5 Å of the nanosheet. In the graphene system, the average residue contact number (RCN) of the initially collapsed Q22 in contact with the nanosheetis ∼16 with a contact ratio of 73%, close to the initially fully extended Q22 system with ∼19 contacting residues and 86% contact ratio (average of the last 300 ns of trajectory). The RCN of the initially collapsed Q46 on graphene is ∼25 with a contact Frontiers in Chemistry | www.frontiersin.org ratio of 54%, far less than the initially fully extended Q46 system with ∼34 contacting residues and 74% contact ratio. In the MoS 2 systems, the RCN of the initially collapsed Q22 in contact with the nanosheet is ∼18 and the contact ratio is 82%, similar to the initial fully extended Q22 system. The RCN and contact ratio of the initially collapsed Q46 on MoS 2 is ∼21 and 46%, far less than the initially fully extended Q46 values of ∼38 and 83%. The residue contact and contact ratio analyses indicate that when polyQ binds to the graphene/MoS 2 surface, most of the Q22 residues are in contact with the 2D-nanomaterials, typical of an unfolded, extended structure. In contrast, only part of the collapsed Q46 is in contact with the 2D-nanomaterials, so although the nanosheets induce an unfolding of Q22, they do not cause a full unfolding of Q46 within our simulation time. Next, we performed energetic analyses to gain a deeper understanding on the interactions between polyQ and nanosheets as well as among polyQ atoms (self-interaction). Although glutamine is normally a hydrophilic residue, isolated polyQ in water shows strong hydrophobic protein properties, including the propensity of forming a supercompact structure and self-aggregation. Therefore, polyQ adsorption onto the graphene/MoS 2 nanosheet should be favorable due to hydrophobic forces. This favorable binding is also clearly indicated in the time-dependent van der Waals (vdW) interaction energies between polyQ and graphene/MoS 2 nanosheets (Figure 4; here we used vdW energy for comparison as graphene has no partial charges on C atoms and the electrostatic energy is actually very small compared to vdW for MoS 2 due to its symmetry even though both Mo and S have partial charges; The electrostatic interaction energy between Qs is shown in Figure S3). For example, as shown in Figure 4A, when the collapsed Q22 unfolds and extends on the graphene nanosheet, there is a significant loss of the self vdW energy and a larger gain in the Q22-graphene interaction energy. Same for the Q22 on MoS 2 nanosheet ( Figure 4B). On the other hand, for the collapsed Q46, the self vdW energy remains strong without any significant loss, indicating no meaningful unfolding on either graphene or MoS 2 . Again, these results indicate that the hydrophobic nature of graphene and MoS 2 is energetically favorable for the unfolding of Q22 on the nanosheet surface, but probably not strong enough to unfold Q46.
PolyQ peptides are known to form hydrogen bonds using both the glutamine backbone and sidechain which is the main cause of the formation of polyQ supercompact structures (Perutz et al., 1994;Chen et al., 2002;Crick et al., 2006;Miettinen et al., 2012;Heck et al., 2014;Hoop et al., 2016;Kang et al., 2017). Here, we analyze the time evolution of polyQ hydrogen bonds and find interesting competition behavior between the intra-polyQ interactions (mainly hydrogen bonds) and polyQ sidechain-nanosheet interactions. Figures S4, S5 show the number of all hydrogen bonds (A and B) and sidechain-sidechain hydrogen bonds (C and D) of Q22 and Q46 for the graphene and MoS 2 simulations. The polyQ sidechain-nanosheet interaction is sufficiently strong in the Q22 systems to not only prevent the collapse of Q22 but also to unfold collapsed Q22, reducing the number of intrachain hydrogen bonds. However, the polyQ sidechain-nanosheet interaction is not strong enough to unfold the collapsed Q46 though it can prevent the collapse of initially extended Q46 on graphene and MoS 2 . Therefore, from our work, we find that the two-dimensional nanomaterial graphene and MoS 2 have the potential to inhibit the collapse of polyQ, but not for Q-lengths up to Q46.
CONCLUSION
To summarize, we have investigated polyQ peptides of two lengths (22 and 46) binding to both graphene and MoS 2 nanosheets using atomistic molecular dynamics simulations. Two different initial polyQ configurations, fully extended and collapsed, were employed to identify structure dependent folding/binding behavior. Our simulations reveal that both Q22 and Q46 bind to graphene and MoS 2 nanosheets effectively, but in a Q-length dependent manner. Upon binding, Q22 unfolds and elongates onto both 2D-nanomaterial surfaces, regardless of the initial conformation, with graphene displaying slightly stronger effect. In contrast, Q46 does not spontaneously stretch out onto the graphene/MoS 2 surfaces within our simulation time if starting from an initially collapsed structure. Detailed analyses indicate that the differential binding behavior of Q22 and Q46 is due to competition between the hydrophobic polyQ-nanosheet interactions and internal polyQ-polyQ self-interactions (mostly intra-hydrogen-bonds). We believe our current work reveals important polyQ lengthdependent folding/binding behavior upon binding to novel 2D-nanomaterials, which might have implications for future potential clinical applications to Huntington's disease.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.
AUTHOR CONTRIBUTIONS
MF: job design, MD simulation, data collection, draft paper, and approve final paper for publication. DB and WZ: make important changes to the paper and approve final paper for publication. ZW: data collection and approve final paper for publication. | v3-fos-license |
2018-08-28T09:51:17.020Z | 2018-05-16T00:00:00.000 | 52078572 | {
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"year": 2018
} | pes2o/s2orc | Dicationic ionic liquids as new feeding deterrents
Abstract In this study, new quaternary bis(ammonium) salts with alkyl-1,X-bis(dimethyldecylammonium) cation and saccharinate, acesulfamate, lactate and pyroglutamate anions were synthesized and characterized by 1H and 13C NMR spectroscopy. Thermal gravimetric and differential scanning calorimetry analyses confirmed that all salts were thermally stable and the majority of them exhibited melting points below 100 °C. The physicochemical properties (viscosity, density, refractive index values, and solubility) of the obtained salts were determined for three compounds with lactate anions. All the tested salts have suitable properties which, in practical application, will reduce the losses caused by the most important storage insects. Most of the synthesized ionic liquids had comparable or better deterrent activity than azadirachtin—an alkaloid known as the most active antifeedant. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s11696-018-0495-6) contains supplementary material, which is available to authorized users.
Introduction
The use of synthetic insecticides for stored-product protection from pests has led to problems such as disturbances of the environment, insect resistance to pesticides and lethal effects on non-target organisms (Geng et al. 2011). It is necessary to develop safe and cheap methods for fighting harmful insects and searching for new active compounds, especially of a natural origin. Substances that deter or inhibit insect feeding are called food deterrents or antifeedants (Isman 2002). Due to their specific properties, insect antifeedants are environmentally friendly crop protection agents. Antifeedants are allomone substances that mainly inhibit feeding on stored products, limit the potential of pest development and do not kill them directly (Lozowicka and Kaczyński 2013). A key characteristic of these compounds is selective action. Antifeedants exhibit activity against parasites and do not cause any negative effects for useful insects such as pest predators or pollinators (Pavela 2010). In addition, the use of deterrents may limit the application of toxic 1 3 insecticides and reduces the risk of contamination of food with dangerous compounds.
Dicationic ionic liquids (DILs) are a group of compounds consisting only of ions, they have a melting point of less than 100 °C. DILs are composed of two cations linked with a rigid or flexible linker and two anions. In the case of dicationic ionic liquids, the modification potential is much greater than in the case of monocationic ionic liquids. It is not only possible to select cations and anions, but also the type and length of the spacer (Claros et al., 2010(Claros et al., , 2012. Additionally, DILs may contain appropriate functional groups, such as thiol, ether, hydroxyl and amino groups in the structure of cations (Lohar et al. 2016).
In contrast to monocationic ionic liquids with a single cation, DILs are characterized by higher density, viscosity and glass transition temperature. They also exhibit very high thermal stability. (Shirota et al. 2011;Claros et al. 2010). Low volatility and excellent thermal properties allow for their use in processes carried out in high temperatures. (Fan et al. 2013).
The use of natural food deterrents would be the best solution due to ecological reasons. However, the cost of obtaining them is high and uneconomical. This cost limits the commercial application of natural compounds such as azadirachtin. Currently, research for new compounds with similar or better efficacy is being conducted.
Ionic liquids are used as cheap and safe feeding deterrents. These are mainly compounds containing ammonium cations such as didecyldimethylammonium. The presence of long alkyl chains determines the high activity of ionic liquids in relation to different types of beetles and pest larvae (Pernak et al. 2013).
The most favorable direction for the development of the third generation of ionic liquids, which are compounds exhibiting biological activity, applies to the synthesis of salts from natural and low-toxicity materials. Antifeedants in the form of ionic liquids may contain natural anions, e.g., lactate (Cybulski et al. 2008), theophyllinate (Markiewicz et al. 2014), fatty acids (Pernak et al. 2015) or abietate (Klejdysz et al. 2016). In addition, compounds based on artificial sweeteners also show good deterrent activity (Pernak et al. 2012).
The aim of this study was the synthesis of a new compound with antifeedant activity and containing gemini surfactant as a cation. As per the literature data, compounds having one long alkyl chain exhibited better biological properties than compounds with a short alkyl chain (Pernak et al. 2013). Compounds with a low toxicity (artificial sweeteners and natural acids) were used as a counterion. This combination was created to obtain a salt with the desired physicochemical properties and an increased biological activity. The reason for this research was the need to find new, cheaper and more active antifeedants.
Synthesis
Alkane-1,X-bis(decyldimethylammonium) dibromides were obtained by quaternization reaction between appropriate dibromo-alkane (0.1 mol) and decyldimethylamine (0.2 mol). The reactions were conducted in acetonitrile at 60 °C for 24 h. Next, the solvents were removed by vacuum evaporator and the product of reaction was mixed with ethyl acetate. After adding the solvent, dibromide bis(ammonium) precipitated as white solid and was isolated by filtration. The end product was dried under a reduced pressure at 70 °C for 24 h. The next step was the synthesis of dicationic ionic liquids. The first method was based on the bromide anion exchange reaction (metathesis reaction) in methanol according to the method described earlier (Aher and Bhagat 2016). The reaction was carried out between bis(ammonium) dibromide (0.01 mol) and two molar equivalents of potassium salts of appropriate acids (l-lactic acid, l-pyroglutamic acid) or commercially available saccharin sodium and acesulfame potassium salts. The precipitated inorganic salt was removed from the mixture by filtration and the solvent was evaporated under a reduced pressure. To remove all by-product, bis(ammonium) salts were dissolved in acetone. The white solid bromide sodium or potassium precipitated and was removed by vacuum filtration. Finally, the product was dried under a reduced pressure at 55 °C for 24 h.
The second method was based on acid-base neutralization reaction according to the method described previously (Niemczak et al. 2015). Quaternary bis(ammonium) dihydroxide was obtained by the anion exchange reaction of dibromide bis(ammonium) with potassium hydroxide in methanol at room temperature. After neutralization of the obtained quaternary bis(ammonium) dihydroxide (0.01 mol) with appropriate acids (0.02 mol), solvent was removed by vacuum evaporator and the product was dissolved in acetone to precipitate other pollutants. The solid was recovered by filtration and the solvent in the mixture was evaporated by vacuum evaporator. The last step was drying under a reduced pressure at 55 °C for 24 h.
Characterization methods
The melting point was examined on METTLER TOLEDO MP 90 melting point system. The solubility had been studied accordance to Vogel's method. The solubility had been determined in ten selected solvents, such as water, methanol, DMSO, acetonitrile, acetone, 2-isopropanol, ethyl acetate, chloroform, toluene and hexane.
Refraction index was measured for obtained salts which were liquid at room temperature. This test was made on Abbe Rudolph Research Analytical J357 Automatic Refractometer and it was set in a range from 20 to 80 °C. Viscosity has been examined by rotational viscometer Rheotec RC30-CPS. The measurement consisted of examining the change in viscosity with temperature (20-80 °C).
Density was determined using an automatic density meter DDM2911 with a mechanical oscillator method. The density of the samples (approx. 2.0 cm 3 ) was measured with respect to temperature-controlled conditions with Peltier, from 20 to 80 °C. The apparatus used was calibrated using deionized water as the reference substance.
The solubility of the prepared salts was determined according to Vogel's Textbook of Practical Organic Chemistry (A. I. Vogel and B. S. Furniss, Vogel's Textbook of Practical Organic Chemistry, Longman, 4th edn, 1984). Different solvents were selected for the solubility test, such as water, methanol, DMSO, acetonitrile, acetone, ethyl acetate, chloroform, toluene, and hexane. The solubility test was developed to classify the ionic liquid into one of three groups for each solvent. The first group included ILs, which dissolved (0.1 g of IL) in 1 ml of solvent; these compounds were described as "high solubility". The term "moderate solubility" refers to 0.1 g compounds that have dissolved in 2 or 3 ml of solvent. The last group included 0.1 g ILs that did not dissolve in 3 ml and identified the compounds as "low solubility". Tests were conducted at 25 °C under ambient pressure. 1 H and 13 C NMR spectroscopic analyses were carried out on a Varian Mercury 300 spectrometer operating at 300 and 75 MHz. Elemental analyses were performed at the Adam Mickiewicz University, Poznan (Poland). The internal standard was used in the analysis of tetramethylsilane and the solvent deuterated chloroform (CDCl 3 ). CHN elemental analyses were performed at A. Mickiewicz University, Poznan (Poland).
Thermal gravimetric analysis (TGA) was performed using a Mettler Toledo Star e TGA/DSC1 unit (Leicester, UK) under nitrogen. Samples (2-10 mg) were placed in aluminum pans and heated from 30 to 450 °C at a heating rate of 10 °C min −1 .
Thermal transition temperature was determined by differential scanning calorimetry (DSC), with a Mettler Toledo Stare DSC1 (Leicester, UK) unit under nitrogen. Samples (5-15 mg) were placed in aluminum pans and heated from 25 to 100 °C at a heating rate of 10 °C min −1 and cooled with an intracooler at a cooling rate of 10 °C min −1 to -100 °C and then heated again to 100 °C.
Methods of the deterrent activity experiments
Tests on feeding activity of ionic liquids were done on three species of most important pests of storage grain: granary weevil (Sitophilus granarius (Linnaeus, 1758)), confused flour beetle (Tribolium confusum Jacquelin du Val, 1868) and khapra beetle (Trogoderma granarium Everts, 1898). The above-mentioned insects were reared in the laboratory in an incubator at 26 (±) 10 °C and 60 (± 5) % relative humidity on uncrushed wheat grain (granary weevil) and shredded products from wheat grain: flour, bran and others (confused flour beetle and khapra beetle).
The experiment was conducted under identical conditions in which insects were reared (temperature and relative humidity). The choice and no-choice tests were done. The wafer discs were done from wheat flour. Discs were 1 cm diameter, 1 mm thick and its average weight was about 15 mg. Prepared wafers were saturated by dipping in either methanol only (control) or 1% solution of compounds in methanol and then left to air dry for 30 min. After this, wafers were weighed and offered to insects on Petri dish: -two discs previously only dipped in a solvent (control for experiment in the variant no-choice test); -two discs, one previously dipped in a solvent and second in 1% solution of tested compound (choice test); 1 3 -two discs previously dipped in a 1% solution of tested compound (no-choice test).
Both variants of experience and control were repeated five times. Experiments were performed on three species of insect: 3 adults of S. granarius, 20 adults and 10 larvae of T. confusum and 10 larvae of T. granarium. The number of insects used to experiment depended on the intensity of their food consumption. Adults used for experiments were unsexed, 7-10 days old, and the larvae were 5-30 days old. Insects were left in Petri dishes for 5 days, after this time, wafers were reweighed. Weight loss of the wafers was the basis to calculate three deterrency coefficients: R relative, A absolute and T total: C, CC is the amount of food from the control discs consumed.
E, EE is the amount of food treated with tested compound consumed.
Total coefficient of deterrence (T) was the sum of relative and absolute coefficient.
The total coefficient of deterrence, which ranged from -200 to 200, served as the index activity. The compounds with T values ranging from 151 to 200, are very good deterrents, those with coefficients values 101-150 are good deterrents and for the medium active T ranged from 51 to 100. Compounds with T values lower than 50 are weak deterrents. The coefficients of deterrence of group of compounds were analyzed by means of one-way ANOVA followed by the post hoc Tukey test with homogenous subset. Calculations were performed using Statistica 6.0.
Results and discussion
In our work, five new bis(ammonium) dibromides were synthesized (Scheme 1, Step I) with a yield of about 90%. The purity of compounds was measured and confirmed by NMR. The melting points of salts are presented in Table 1.
The next step was the synthesis of a compound with deterrent activity. This compound was obtained in an exchange reaction (Scheme 1, Step II). As a result of this reaction, 20 new salts were created ( Table 2).
The synthesis of ILs was conducted with two methods with a yield between 70 and 99%. All salts with cation hexamethylene-1,6-bis(dimethyldecylammonium) (2a, 2b, 2c, 2d) had the lowest efficiency. Three salts were liquid at room temperature and eleven were greasy with high viscosity and density. Fifteen of the obtained salts can be included as ionic liquids.
The impact of the anion on the chemical shifts of the alkyl chain was not significant in the analysis of the proton nuclear magnetic resonance. The shifts for hydrogen atoms at the last and penultimate carbon atom in the alkyl chain (-C 10 H 21 ) are, respectively, 0.88 and 1.26 ppm, The CH 2 groups had the lowest chemical shift for saccharinate anion, but the highest one for pyroglutamate anion. Chemical shifts of the atoms next to the quaternary nitrogen atom strongly depend on the anions. Compounds with lactate and pyroglutamate anions had the lowest values of chemical shifts, and the saccharinate anions and acesulfamate anions caused a shift towards a higher value. The viscosity, density and refractive index were examined for DILs which were liquid at 20 °C (RTILs). Figure 1 shows refractive index of obtained RTILs.
When analyzing the impact of temperature on the refraction index for three ionic liquids with a lactate anion (1c, 4c and 5c), a linear relationship was observed. The highest refraction index was observed for 4c in the entire temperature range. In addition, a linear relationship between density and temperature was also noted (Fig. 2).
Compound 1c had the highest density. The density of liquids 4c and 5c was almost the same as water. In the literature, data for single ionic liquids, the correlation between refractive index and the density were described. From these data, it follows that ionic liquids with the highest refractive index also had the highest values of density. In the case of the examined ionic liquids, an inverse relationship was observed. The lowest measured density values were recorded for the highest refractive index values. Figure 3 shows the temperature dependence of the viscosity for dilactate alkyl-1,X-bis(decyldimethylammonium). Viscosity was measured for compounds 1c, 4c and 5c. ILs 1c had the highest viscosity; on the other hand, liquid 5c had the lowest viscosity. Two intervals were observed. In the first interval, for a temperature of 20-40 °C, there is a sharp decrease in viscosity with a rise of temperature. In contrast, in the second interval (40-80 °C), a decrease in the measured viscosity value is much lower with an increase in temperature. Such a dependence is also observed for single ionic liquids with long alkyl chains. Table 3 presents solubility of synthesized salts. All of them were insoluble in nonpolar solvents such as ethyl acetate, toluene and hexane, but soluble in chloroform.
Salts with saccharin anions (1a-5a) were soluble in polar solvents, except for water. Synthesized salts with acesulfame anions (1b-5b) were characterized by weaker solubility in DMSO and isopropanol in comparison to 1a-5a salts. Compounds 1b-5b were insoluble in DMSO and dissolved poorly in isopropanol.
The salts with lactate and pyroglutamate anions were insoluble in solvents such as isopropanol and acetonitrile, but exhibited solubility in methanol and water. However, compounds 2d-5d dissolve better than salts 1c-5c and 1d. DILs 1c-5c and 1d-5d dissolved poorly in acetone and acetonitrile, except for 3d-5d. Thermal stability and differential scanning calorimetry tests were conducted for all synthesized salts. The results of the studies are presented in Table 4.
It was observed that salts with acesulfamate and saccharinate anions have lower values of glass transition temperature (T g ) than pyroglutamate or lactate anions. The lowest glass transition temperature for 1c-5c was recorded for an eight-carbon alkyl linker and the highest for ILs with four carbon atoms in the linker. In the case of the series with a pyroglutamate anion, compounds with the shortest linker had a very low T g , while the 3d-5d DILs had a similar T g and ranged from − 11.1 °C (5d) to − 15.3 °C (3d). Salts with saccharin anions had the highest glass transition temperature in the range from − 5.5 °C (5a) to 5.6 °C (1a). Only two compounds (3a and 2b) exhibited a temperature of crystallization (T c ) and a melting point (T m ) in the tested temperature range. The thermal stability of the tested salts was also determined. It can be seen that compounds with saccharinate anions (1a-5a) and acesulfamate (1b-5b) have a slightly higher stability than the other synthesized salts. DILs 1d-5d were characterized by a decrease in the T onset5% value for ILs with eight carbon atoms in the alkyl linker (3d). T onset values ranged from 237 to 320 °C. The lowest values were recorded for the 1c-5c series, where the minimum value was observed in the case of 3c DIL. On the other hand, the highest values were observed for compounds with the acesulfamate anion, except for 5b, where a decrease in the T onset50% parameter was noted. The other two series (1a-5a and 1d-5d) were characterized by an almost linear increase in stability with growth in the alkyl linker, except for DIL 5a.
Scheme 1 Synthesis of dicationic ionic liquids
Compounds with a lactate anion and the didecyldimethylammonium (DDA) cation described in the literature showed higher thermal stability, but the ones with benzyldimethylalkyl ammonium (BA) cation exhibited a lower value than in the case of all the obtained salts. In addition, compounds with DDA and BA cations had a glass transition temperature, which was not observed in bis(ammonium) compounds.
ILs with cationic species such as chlormequat (CC), diallyldimethylammonium (DADMA), didecyldimethylammonium (DDA), hexadecylpyridinium (HEXA) or benzylalkyldimethylammonium (BA), acesulfame and saccharinate anions have already been reported. Compounds with CC and DADMA cations exhibited higher thermal stability than the synthesized compounds, but in the case of DDA, HEXA and BA, the stability was lower or comparable with the tested substances. Antifeedants containing anion deriving from natural acids have also been reported in the literature. These compounds exhibited thermal stability at a similar level as bis(ammonium) compounds or slightly higher (Pernak et al. 2013;Cybulski et al. 2008;Hough-Troutman et al. 2009). DILs with lactate, pyroglutamate, saccharinate and acesulfamate anions were stated by very good or good deterrent properties (Fig. 4).
In the case of granary weevil (Sitophilus granaries), the synthesized series of quaternary ammonium compounds with acesulfamate, saccharinate and pyroglutamate anions had minimum activity for a compound with six carbon atoms in the linker alkyl.
Statistical analysis of the deterrent's activity confirmed very good antifeedant properties for all synthesized compounds with eight, ten and twelve carbon atoms in the linker alkyl, beyond the series with lactate anions (1c-5c). The same activity was presented by salts with an acesulfamate and pyroglutamate anion and four carbon atoms in the linker (Fig. 5).
In the case of an adult confused flour beetle (Tribolium confusum), all salts with acesulfamate anions (1b-5b) showed activity at a very good level. This series noted a linear increase with an extension linker between quaternary nitrogen. A similar relationship was observed for salts with saccharinate anions (1a-5a). The rest of the compounds exhibited the lowest activity for the shortest linker. Synthesized ILs can be included in three groups with medium, good and very good activity. Activity towards an adult confused flour beetle increased with the number of carbon atoms with the exception of salts with a pyroglutamate anions (1d-5d). The graph of larvae confused flour beetle (Tribolium confusum) showed similar activity for all salts obtained, at a very good activity level (Fig. 6).
Obtained ILs showed very good activity towards larvae khapra beetle (Trogoderma granarium). From each series of compounds, the lowest activity was noted for the salts with a linker with six carbon atoms (2c, 3d). Taking into account the data describing the biological activity for all the obtained compounds towards tested insects, the slight advantage of the salt with the cation long alkyl linkers (in particular for the linker with 12 carbon atoms) was noticeable, but there was no such dependence for all tested insects. ILs with 8, 10 or 12 carbon atoms in the bis(ammonium) cation were characterized by the highest activity than the reference compound (Azadirachtin) to insects such as the confused flour beetle or larvae of both tested species.
On the basis of previous data regarding ILs with acesulfamate, saccharinate and lactate anions, it can be concluded that synthesized DILs have high feeding deterrence activity.
Efficacy of salts with acesulfamate and saccharinate anion depends most likely on the amphiphilicity of the cation. Hence, acesulfamates and saccharinates comprising [2-(acryloyloxy)ethyl]trimethylammonium, [2-(methacryloyloxy)ethyl]trimethylammonium, 2-chloroethyltrimethylammonium, trimethylvinylammonium or (2-acetoxyethyl)alkoxymethyl-dimethylammonium cations were characterized by medium or weak biological activity (T coefficient did not exceed 114). However, antifeedant activity for ILs with more hydrophobic cations was significantly higher, and in several cases (i.e., alkoxymethyl(2-hydroxyethyl)dimethylammonium, didecyldimethylammonium salts) exhibited very good efficacy similar to the reference substance (azadirachtin-the compound of natural origin with the highest feeding deterrence activity). The feeding deterrence activity was determined also for precursor of ILs with didecyldimethylammonium cation-didecyldimethylammonium chloride. The obtained results allowed us to evaluate the impact of anion exchange on antifeedant activity-saccharinates or acesulfamates were characterized by increased biological activity in comparison to precursor with chloride anion. Therefore, we can conclude that used bis(ammonium) dibromides also have a deterrent activity. Furthermore, in terms of general efficacy against all of the tested insects, the obtained DILs exhibit the best efficiency (Czerniak et al. 2014;Pernak et al. 2013;Hough-Troutman et al. 2009;Pernak et al. 2007).
According to the previous papers, the highest deterrent activity (T parameter equal to 190) was reached for the IL with lactate anion. The obtained DILs with the same anion exhibited the highest possible efficacy according to the methodology (T equal to 200)-a noticeably better result in comparison to azadirachtin (Cybulski et al. 2008;Czerniak et al. 2014). So far, no studies on the feeding deterrence of the ILs with pyroglutamate anion have been reported.
Conclusions
Two methods of synthesis of bis(ammonium) salt and ionic liquids with sweet anions (acesulfamate, saccharinate), as well as anions occurring naturally (lactate and pyroglutamate) and gemini surfactant cations were described. The selection of cation and anion allowed to design the desired properties of the obtained compounds.
The effect of the anion and length of the alkyl linker on solubility was determined. The compounds with sweet anions had medium or weak solubility in water, in comparison to compounds with natural anions. An inverse correlation in solubility for solvents such as acetonitrile and isopropanol was observed. In addition, the influence of an alkyl linker on physicochemical properties such as density, viscosity and refractive index was determined for bis(ammonium) lactates. An important advantage of the synthesized compounds was high thermal stability (values ranging from 237 to 320 °C), which also allowed them to be exposed to high-temperature conditions. Furthermore, it was noted that most of the salts obtained had a comparable or better antifeedant activity than azadirachtin. The highest efficacy towards all examined storage insects was observed for compounds with twelve carbon atoms in the alkyl linker. | v3-fos-license |
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} | pes2o/s2orc | Efficient syntheses of 1,3-unsubstituted 1 H -pyrazolo[3,4-b ]quinolines
The synthesis of pyrazolo[3,4-b ]quinolines from 2-chloro-3-formylquinolines is described. Direct reaction of the chloro-aldehydes with hydrazine was unsuccessful, but a very efficient sequence was developed in which protection of the aldehyde group was followed by displacement of the halogen by hydrazine, release of the aldehyde then leading to spontaneous ring closure giving the tricycles. The products were obtained in excellent yields
Introduction
1H-Pyrazolo [3,4-b]quinolines are of interest in a number of diverse contexts: for example as antiviral agents 1 in particular as inhibitors of Herpex simplex virus type 1 replication, 2 as activators of caspases and inducers of apoptosis, 3 as antimicrobial agents, [4][5][6][7] as parasiticidic agents, 8 as antimalarials, 9 in photoluminescence 10 and electroluminescence 11 studies, and in electroluminescent devices. 12The use of 1H-pyrazolo [3,4-b]quinoline derivatives as optical brighteners has been long known 13 and interest in them as dyestuffs 14,15 and as colourants within polymers, 16 continues to the present.
Two main strategies have been employed for the synthesis of 1H-pyrazolo [3,4-b]quinolines: (1) some routes begin with a preformed pyrazole (or pyrazolone) the pyridine ring being constructed during the sequence; (2) other routes start from a preformed quinoline with the pyrazole ring being constructed during the sequence.The new work in this paper falls into the latter category.8][19] An alternative pyridine ring construction results from the condensation of a 5-halo-4-acylpyrazole with an aniline (Scheme 1 route b). 20,21ess frequently employed routes which also start from preformed pyrazoles include the condensation of 5-aminopyrazoles with ortho-halo aromatic aldehydes, 22 the Friedel-Crafts type closure of an ortho-pyrazol-5-ylamino benzoic acid 9 and the condensation of 4arylidenepyrazolin-5-ones with anilines. 23 Scheme 1 (2) The reaction of 2-chloro-3-cyanoquinolines with hydrazine producing 3-amino-1Hpyrazolo [3,4-b]quinolines (Scheme 2) has been used widely.
Scheme 2
There has been a similar wide interest in the combination of 2-chloro-3-acylquinolines with hydrazine or arylhydrazines to form 1H-pyrazolo [3,4-b]quinolines.In particular, and partly because of the ready availability of 2-chloro-3-formylquinolines from Meth-Cohn's convenient and efficient synthesis of such molecules from acetanilides and the Vilsmeier reagent, 29 most work has involved these chloro-aldehydes, though these are easily converted into corresponding ketones by Grignard addition then oxidation of the resulting alcohol. 24,30The reaction of such ketones with hydrazine or arylhydrazines produces 3-substituted 1H-pyrazolo [3,4-b]quinolines simply by refluxing in methanol (Scheme 3); 24,30 it was suggested 30 that the size of the ketone substituent favours formation of the Z-stereoisomer of a first-formed hydrazone, thus having the geometry required for a subsequent ring-closing displacement of the chloride.
Scheme 3
At the commencement of our work, there were conflicting reports as to the facility with which hydrazine and arylhydrazines would react, to give comparable products, with 2-chloro-3formylquinolines i.e. to produce 3-unsubstituted 1H-pyrazolo [3,4-b]quinolines.Thus, although some hetarylhydrazines were described as reacting with 6-bromo-31 or 7-methoxy-32 -2-chloro-3formylquinolines in methanol at reflux or with microwave heating, other reports of attempts to bring about such cyclocondensations implied difficulties.It was shown 28 for example, that an Ehydrazone is formed from such chloro-aldehydes which would not ring-close in refluxing ethanol, having the inappropriate imine geometry, however other workers apparently did not encounter such difficulties using hydrazine hydrochlorides in refluxing ethanol for extended reaction times, 33 or reflux in acetonitrile. 34Much more vigorous conditions can be applied to the interaction between 2-chloro-3-formylquinolines and hydrazine and arylhydrazines which overcome these difficulties: microwave heating with p-toluenesulfonic acid is recommended. 35n a similar study, a 2-methylthio-3-benzoylquinoline formed the undesirable hydrazone isomer which would not ring close; here again, the difficulty was overcome by reacting the ketone with hydrazine using microwave heating in the presence of p-toluenesulfonic acid. 36 device which was utilised first by Meth-Cohn 39,37 and later by Singh 28 to avoid the difficulties associated with formation of the undesired geometrical isomer of the hydrazone, was to initially protect the aldehyde as an acetal thus forcing a first step to be nucleophilic displacement of the chloride by the hydrazine, hydrolytic release of the aldehyde then leading to the desired pyrazolo-quinoline.
Results and Discussion
Our aim in this work was develop a simple and mild procedure for the synthesis of 1,3unsubstituted 1H-pyrazolo [3,4-b]quinolines (Scheme 4).We view such compounds as ideal starting materials for the flexible synthesis of a large range of substituted 1H-pyrazolo [3,4b]quinolines through manipulations at N-1 and C-3.Benzene ring substituents will be provided by the 2-chloro-3-formylquinoline ring syntheses, which, it will be recalled, are absolutely straightforward using Meth-Cohn's procedure and start from simple and readily available benzenamines.Alkylations at N-1 will involve pyrazole ring anions which can be easily formed using a strong base and which react with alkylating agents. 38N-Arylations will rely on highly efficient and well established copper- 39 or palladium-40 -catalysed processes.
The introduction of substituents at C-3 will involve the intriguing prospect of developing further the relatively little explored electrophilic substitution of indazoles, in this case 7azaindazoles.There is considerable interest in indazole analogues of the many biologically significant indoles, natural and synthetic, in the medicinal and biological fields.Halogenation of the target 1H-pyrazolo [3,4-b]quinolines, would be predicted to produce 3-halo-derivatives, with all the potential therein for formation of nucleophilic organometallic reagents or for use directly in cross-coupling processes.
2-Chloro-3-formylquinolines 1a-f were produced in good yields from precursor acetanilides (acetanilide itself, 2-methyl-, 3-methyl-, 4-methyl-, 3-methoxy-and 4-methoxyacetanilide). 29eaction of 1a with hydrazine produced a hydrazone which could not be cyclised by refluxing in ethanol.We concluded that the hydrazone had E geometry and thus was sterically prevented from attacking the quinoline C-2.In order to avoid this difficulty, each of the aldehydes was converted into an ethylene acetal 2, and aldehyde 1a was also converted into the corresponding thioacetal 5a.
With the aldehyde masked, each of the chloro-quinolines 2a-f and 5a was reacted with hydrazine hydrate in refluxing ethanol, giving the quinolin-2-ylhydrazines 4a-f and 5b in good yields.Finally, mild aqueous acidic removal of the acetal protection from 3a-f led directly, in one pot, to the cyclised 1H-pyrazolo [3,4-b]quinolines 4a-f.This protocol is far superior to the bismuth trichloride method employed 28 for the comparable ring closure of two examples, 3a and 3e, and requires nothing more than simple basification at the end of the process to liberate the
Experimental Section
General Experimental Procedures.All the 2-chloro-3-formylquinolines (1a-f) were prepared by the Meth-Cohn method. 29Melting points were recorded on a Philip Harris C4954718 apparatus and are uncorrected. 1H and 13 C NMR spectra were recorded on a Bruker Avance AQS 300 MHz spectrometer, at 300 MHz and 75 MHz respectively.Chemical shifts d are in parts per million (ppm) measured in CDCl 3 or DMSO-d 6 as solvent and relative to TMS as the internal standard.Infrared spectra were recorded on a Thermonicolet-Nexus 670 FT-IR instrument and elemental analyses were carried out on an Exeter analytical model CE440 (CHN) and a Leco elemental analyzer Truspec (CHNSO), and Cl was determined by Chemical Analyses Labs Ltd.
General method for the formation of ethylene acetals (2)
A solution of 2-chloro-3-formylquinoline (20 mmol) in benzene (100 mL) containing ethylene glycol (3.57g, 3.2 mL, 57 mmol) and a crystal of toluene-p-sulfonic acid was heated under reflux using a Dean-Stark water separator until no more water collected in the side arm (5-12 h).The cooled solution was treated with saturated aqueous sodium carbonate (50 mL), dried and evaporated giving 2 which was recrystallised from petroleum (b.p. 100-120 ºC)-toluene.
General method for the hydrazinolysis reactions giving 3
A solution of 2a-f (20 mmol) and hydrazine hydrate 80% (12.5 g, 12.1 ml, 200 mmol) was heated under reflux for 24 h.Then cooled solution was evaporated and residue recrystallised from ethanol to give 3a-f in high yields.In 50-mL flask fitted with a condenser and a dropping funnel was added cupric oxide (0.48 g, 6 mmol), anhydrous cupric chloride (1.61 g, 12 mmol) and acetone (40 mL).The resulting suspension was brought to reflux with vigorous stirring and a solution of 5b (1.31 g, 5 mmol) in acetone (9 ml) and DMF (1 mLl) was added over 5 min.Reflux was maintained for 30 min then the mixture was cooled and filtered.The insoluble material was washed with dichloromethane (3×20 mL) and the combined organic layers were washed with aqueous sodium carbonate (50 ml), dried over sodium sulfate and filtered.Evaporation of the solvent gave 4a as yellow powder in 85% yield. 7,[24][25][26][27][28] | v3-fos-license |
2018-04-03T04:39:34.765Z | 2007-07-13T00:00:00.000 | 39766313 | {
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} | pes2o/s2orc | Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes*
Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 212 = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.
Since cartilage has poor regenerative capacity by itself, its regeneration using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders that cannot be treated by conventional methods. Although tissue-engi-neered cartilage from autologous chondrocytes is already available for clinical use (1)(2)(3), the size is less than 1 ml and is limited to just a filler of a small defect or an injectable plaque of the affected cartilage. To broaden its clinical indication to major disorders such as osteoarthritic joints, microtia, and cleft lip/ palate, cartilaginous matrix should be abundantly produced by a sufficient number of autologous chondrocytes. Since the number of chondrocytes that can be isolated primarily from the native cartilage is limited, it should be markedly increased in culture. However, during their multiplication in culture through repeated passaging, chondrocytes inevitably lose their ability to produce cartilaginous matrix such as glycosaminoglycan (GAG) 2 and type II collagen (COL2) and begin producing type I collagen (COL1), which is called dedifferentiation (4). Although biochemical factors such as hormones and growth factors are known to stimulate redifferentiation of the dedifferentiated chondrocytes to restore the chondrocytic properties (5,6), there remains another inevitable limitation, that is, induction of hypertrophic differentiation of chondrocytes (7), which leads to endochondral ossification as seen in the growth plates.
To develop a clinically practical tissue engineering of permanent cartilage that overcomes the limitations above, the present study focused on 12 representative soluble factors (8) that are known to be potent regulators of proliferation or differentiation of chondrocytes and have already been approved for safe clinical use. We initially determined a combination of two factors among them that most efficiently induce redifferentiation of the dedifferentiated human chondrocytes under a serum-free condition using a statistical method termed "analysis of variance by fractional factorial design" (9). We then selected a third factor that prevents the redifferentiated chondrocytes from hypertrophic differentiation and investigated the underlying molecular mechanism. Finally, we evaluated the properties of the tissue-engineered cartilage by the selected combination to examine whether it is applicable to clinical use for the treatment of major skeletal disorders.
EXPERIMENTAL PROCEDURES
Cell Isolation and Monolayer Culture-All procedures of the present experiments were approved by the ethics committee of the University of Tokyo Hospital (ethics permission number 622). Both remnant auricular cartilage and costal from one of six microtia patients (10 -15 years old) were obtained during surgery in adherence to the Helsinki Principles. Chondrocytes were isolated with digestion using 0.15% collagenase (Wako Pure Chemical Industries, Osaka, Japan). Primary articular chondrocytes were purchased from Sanko Junyaku (Tokyo, Japan). The chondrocytes were seeded in a 100-mm plastic tissue culture dish at a density of 6,400 cells/cm 2 and cultured in Dulbecco's modified Eagle's medium/F12 containing 5% human serum supplemented with fibroblast growth factor-2 (FGF-2, 100 ng/ml) and insulin (5 g/ml), as we reported previously (8). Passaging was performed by trypsin-EDTA solution (Sigma) and repeated until 1000-fold increase of the total cell number.
Generation of Cartilage Pellet and Implant-For three-dimensional atelocollagen culture, the dedifferentiated chondrocytes were suspended in 0.8% atelocollagen solution (Kawaken Fine Chemicals, Tokyo, Japan) at a density of 10 7 cells/ml. The atelocollagen pellets were cultured in Dulbecco's modified Eagle's medium/F-12 (Sigma) medium with or without soluble factors for 1-3 weeks. To make the cartilage implant, a 100 l-atelocollagen aliquot containing 10 6 chondrocytes was added to a poly(L-latic acid) (PLLA) mesh (kindly provided by Unitika, Tokyo, Japan) (10). Animal experiments were performed according to the protocol approved by the Animal Care and Use Committee of the University of Tokyo.
The software JMP-5.1.1J (SAS Institute, Cary, NC) was used to choose 256 randomized combinations in which the 12 factors independently appeared in an incidence of 50%, from 2 12 ϭ 4,096 combinations (see the supplemental table). Auricular chondrocytes obtained from three different patients (5 ϫ 10 4 cells in 5 l of 0.8% atelocollagen gel) were cultured in Dulbecco's modified Eagle's medium/F-12 containing the combinations for 2 weeks, and the GAG accumulation was measured as described below. Parameter estimates of the GAG accumulation by one factor or two were calculated from the F values that represent the effects of the individual factors and the interaction terms of two factors by the analysis of variance using the software above.
GAG Measurement-The sulfated GAG content was measured using Alcian blue binding assay (Wieslab AB, Lund, Sweden). After digestion of the atelocollagen pellet containing chondrocytes in 0.3% collagenase for 1 h at 37°C, cell debris and insoluble material were removed by centrifugation at 6,000 ϫ g for 30 min. GAG in the supernatant was precipitated with Alcian blue solution, and the sediments by centrifugation at 6,000 ϫ g for 15 min were dissolved again in 4 M GuHCl-33% propanol solution. The spectrophotometrical absorbance of the mixture was measured at a wavelength of 600 nm.
Enzyme-linked Immunosorbent Assay for Type I and Type II Collagen-The collagen proteins of the pellets, the implants, and the native human auricular cartilage were quantified by enzyme-linked immunosorbent assay using a human Type 1 and Type 2 collagen detection kit (Chondrex, Redmond, WA). The pellets, the implants, or the native human auricular carti-lage were dissolved in 10 mg/ml pepsin, 0.05 M acetic acid at 4°C for 48 h and then in 1 mg/ml pancreatic elastase, 0.1 mM Tris, 0.02 M NaCl, 5 mM CaCl 2 , (pH 7.8 -8.0) at 4°C overnight. The samples were centrifuged at 9,100 ϫ g for 5 min to remove the residue. The collagen proteins were captured by polyclonal anti-human COL1 or COL2 antibodies and detected by biotinylated counterparts and streptavidin peroxidase. O-phenylenediamine and H 2 O 2 were added to the mixture, and the spectrophotometrical absorbance of the mixture was measured at a wavelength of 490 nm.
Histology, Histochemistry, and Fine Structure-The tissueengineered cartilage was fixed with 4% paraformaldehyde, embedded in OCT compound (Sakura, Tokyo, Japan), and cryosectioned into 10-m slices. The sections were stained with toluidine blue-O and TUNEL (ApopTag, Intergen, Purchase, NY) according to the manufacturer's instructions. For fluorescent immunohistochemistry, the sections were incubated with a mouse monoclonal anti-COL10 antibody (Sigma) and then with biotin-conjugated anti-mouse IgG ϩ IgA ϩ IgM antibodies (Nichirei, Tokyo, Japan) and fluorescein isothiocyanate-streptavidin. The localizations were observed by confocal laser scanning microscopy (Leica TCS-SL, Wetzler, Germany). For the ultrastructural observation, the specimens were immersed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde. After postfixation with 1% OsO 4 , the specimens were dehydrated in a graded ethanol series, replaced with propylene oxide, embedded in Poly/Bed 812 resin (Polysciences, Warrington, PA), and sliced into 60 -80-nm sections. The sections were observed under a transmission electron microscope (Hitachi H-7100, Tokyo, Japan).
Mechanical Properties-Mechanical properties of the pellet and implant were measured using a Venustron tactile sensor (Axiom, Fukushima, Japan). With computer control, the motordriven sensor unit automatically presses down the objects up to 0.5 mm in depth from the surface and provides the compression strength and the decrease in the resonant frequency. Young's modulus was calculated by the compression strength and the frequency decrease using the software Venus 42 (Axiom), based on a previous report (29).
Statistical Analysis-All data are expressed as means Ϯ S.E. Means of groups were compared by analysis of variance, and significance of differences was determined by post hoc testing with Bonferroni's method.
Determination of the Optimal Combination of Soluble
Factors-We attempted to determine the optimal combination of soluble factors for regeneration of permanent cartilage from cultured human chondrocytes that were isolated from remnant auricular cartilage of microtia patients during surgery. To gain a substantial number of cells, the chondrocytes were initially cultured in monolayer with repeated passaging until 1,000-fold increase of the total cell number as we previously reported (8), which took ϳ4 weeks. The dedifferentiated chondrocytes during the monolayer culture were further cultured in an atelocollagen gel in a three-dimensional condition under stimulation by combinations of 12 candidate factors, i.e. BMP-2, insulin, IGF-I, testosterone, PTH, IL-1RA, growth hormone, 17-estradiol, T3, 1␣-25-dihydroxy vitamin D3, FGF-2, and dexamethasone. The redifferentiation was determined by the GAG accumulation. Since exhaustive examination of 2 12 ϭ 4,096 combinations was impossible, we adopted the fractional factorial design (9) for the faultless reduction of the experimental units to a practical number of 256 combinations (see the supplemental table). As a single factor, the statistical analysis revealed that BMP-2, insulin, and IGF-I were the most potent factors of the GAG accumulation (Fig. 1A). Regarding two factors, the combination of BMP-2 and insulin (BI) was shown to provide the most abundant GAG accumulation (Fig. 1A).
We then examined expressions of COL1A1, COL2A1, and COL10A1 by real-time RT-PCR in the cultured human auricular chondrocytes just after isolation (Fig. 1B, primary), after the monolayer culture (pre-rediff), and after the threedimensional atelocollagen culture (Fig. 1B). The long term monolayer culture with repeated passaging led to an increase in COL1A1 and a decrease in COL2A1, confirming the dedifferentiation. The three-dimensional atelocollagen culture thereafter with no stimulation somewhat restored both COL1A1 and COL2A1 expressions, indicating redifferentiation, which was much more enhanced by the BI stimulation. However, BI also increased COL10A1, a marker of chondrocyte hypertrophic differentiation, which is a critical defect for the regeneration of permanent cartilage. Thus, we next searched among the other 10 factors for a third factor that not only enhances the COL1 inhibition and the COL2 stimulation by BI but also suppresses the COL10 induction and found that only T3 exhibited significant effects on all parameters. This indicates that T3 is the optimal third factor that enhances the advantage and cancels the disadvantage of BI for regeneration of permanent cartilage from human auricular chondrocytes.
When we used cultured chondrocytes from human articular cartilage (Fig. 1C) and human rib cartilage (Fig. 1D), the combination of BI and T3 (BIT) potently suppressed COL1A1 and COL10A1 and enhanced COL2A1 expressions, Optimal Factors for Chondrocyte Redifferentiation JULY 13, 2007 • VOLUME 282 • NUMBER 28 just as in the case of the human auricular cartilage. This indicates that BIT is universally efficacious for tissue engineering of permanent cartilage from human chondrocytes of various origins.
Mechanism Underlying the Preventive Effect of T3 on BI-induced Hypertrophic Differentiation of Redifferentiated Chondrocytes-To clarify the molecular mechanism whereby T3 prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes, we examined the involvement of putative signalings that regulate hypertrophic differentiation in the threedimensional atelocollagen culture of human auricular chondrocytes. Runx2, a member of the runt family of transcription factors, is known to be required for chondrocyte hypertrophy (30). Sox9, an essential factor for chondrogenic differentiation (31), functions as a potent inhibitor of chondrocyte hypertrophy (32). PTH/PTHrP via the cAMP-dependent protein kinase is also known to be a major factor that inhibits chondrocyte hypertrophy (33). Among the three factors above, T3 dose-dependently suppressed the BI stimulation of expression of Runx2 similarly to that of COL10A1, but not that of Sox9 or PTH/PTHrP receptor, implying that Runx2 is involved in the prevention of the BI-induced hypertrophic differentiation by T3 (Fig. 2). The concentration of T3 that exhibited the maximum suppression of COL10A1 and Runx2, as well as the stimulation of COL2A1, was 100 nM. To further investigate the intracellular signaling involved, activation of Smads, p38 MAPK, and phosphatidylinositol-3 kinase/Akt, which are known to lie downstream of BMP-2 or insulin (34,35) and also upstream of Runx2 (36,37), was examined by Western blot analyses (Fig. 3A). The BMP receptor-specific Smad1/5/8, p38 MAPK, and Akt were phosphorylated 10, 10, and 5 min, respectively, after the treatment of BI. Although T3 (100 nM) hardly or faintly affected Smad or p38 MAPK activation by BI, it suppressed the Akt activation just like an Akt inhibitor. When we examined COL10A1 and Runx2 mRNA levels in the three-dimensional atelocollagen culture, both levels stimulated by BI were dosedependently suppressed by the Akt inhibitor, similarly to those by T3 (Fig. 3D), suggesting that T3 prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes at least partially via the suppression of the Akt signaling. The inhibitory effects of T3 or the Akt inhibitor on Akt activation by BI continued for 24 h and then disappeared (Fig. 3B). Because the medium was changed twice/week throughout the experiment (1 week), T3 or the Akt inhibitor seemed to exert an inhibitory effect on the Akt activation in approximately one-third of the entire culture period and finally demonstrated the suppression of the Runx2 and Col10A1 expressions at the end of the culture (Fig. 3D). The phosphorylation of p38 MAPK seemed to be slightly increased by T3 (Fig. 3A), but the use of p38 MAPK inhibitor SB203580 instead of T3 rather down-regulated the expression of COL10A1 and RUNX2 (Fig. 3, C and D). The p38 inhibitor also suppressed that of COL2A1, but enhanced that of COL1A1, unlike T3 or the Akt inhibitor (Fig. 3C), suggesting that the slight up-regulation of the p38 MAPK activation by T3 may be associated not with chondrocyte hypertrophy but with enhancement of the early chondrocyte differentiation markers such as the collage type II or proteoglycan.
Properties of the Tissue-engineered Cartilage from Human Chondrocytes by BIT-We next examined the properties of the tissue-engineered cartilage pellet generated by the three-dimensional atelocollagen culture (2 ϫ 10 5 cells/20 l) with and without stimulation by BI or BIT from human auricular chondrocytes that were dedifferentiated after long term monolayer culture, as described above. The size of pellets with BI or BIT stimulation was much larger than that without stimulation (Fig. 4A, top). In addition, histological analyses of toluidine blue staining revealed more proteoglycan production shown by metachromasia with cartilage lacunae under the BI stimulation as compared with the control, which was further enhanced by BIT (Fig. 4A, bottom). Measurement of cartilaginous matrix protein levels also revealed that the redifferentiation markers GAG and COL2, but not the dedifferentiation marker COL1, were stimulated moderately by BI and strongly by BIT (Fig. 4B). However, the GAG level tended to be lower in all these groups as compared with that in native cartilage (Fig. 4B). The ratio of GAG/COL1 and that of COL2/COL1 were 0.77 Ϯ 0. 25 JULY 13, 2007 • VOLUME 282 • NUMBER 28 BIT, suggesting that these stimulations did not cause cell cycle acceleration or carcinogenesis (38) (Fig. 4C). Contrarily, the cell apoptosis shown by TUNEL staining was decreased by BI and BIT (Fig. 4D). We further examined the mechanical properties of the pellets using a tactile sensor and compared them with those of the native auricular cartilage of microtia patients taken as surgical specimens (Fig. 4E). Although the mechanical properties of the BIT-treated cartilage were significantly greater than those of the control and BI-treated ones, they were much less than those of the native human auricular cartilage.
Optimal Factors for Chondrocyte Redifferentiation
Aiming at regenerating permanent cartilage that is applicable for clinical use, we created implants by adding the human auricular chondrocytes cultured in the three-dimensional atelocollagen gel to the PLLA mesh scaffold (10 6 cells/columnar implant 10 mm in diameter and 1 mm in thickness). The implants were cultured for 3 weeks with and without stimulation by BI or BIT (Fig. 5B, top) and thereafter transplanted subcutaneously into nude mice for 2 months (Fig. 5A). Although the size of the excised implants of the three groups seemed similar (Fig. 5B, bottom), depending on the original PLLA scaffold size, the metachromatic proteoglycan production was much more enhanced in the BIT-treated implant than in the other two groups (Fig. 5C). The implants of the control (Fig. 5D, Control) and BI (data not shown) groups contained not only hyaline cartilage with COL2-like fibrils but also fibrous cartilage with COL1-like fibrils even in the center of the implants, showing the mingling with the hyaline cartilage and fibrous one. However, the BIT-treated implants were filled with hyaline cartilage (Fig. 5D, BIT). Biochemical analyses confirmed the advantages of BIT: that GAG and COL2, but not COL1, were significantly stimulated by the treatment (Fig. 5E). The GAG level in the BIT reached that of the native cartilage, whereas COL2 in BIT tended to be higher than in the native one (Fig. 5E). The ratio of GAG/COL1 and that of COL2/COL1 were 0.44 Ϯ 0.18 and 0.25 Ϯ 0.18, respectively, for the control, 0.78 Ϯ 0.43/0.71 Ϯ 0.23 for BI, and 1.61 Ϯ 0.61/1.68 Ϯ 0.61 for BIT. The decrease in these ratios as compared with those of the tissue-engineered cartilage pellets cultured in vitro or the native cartilage may be due to the difficulty in the removal of the COL1-based fibrous tissues around the in vivo regenerated cartilage. Here again, COL10-positive matrices were visible only in the BI-treated implant but not in the control or BIT-treated one (Fig. 5F). Furthermore, mechanical parameters of the control, BI-, and BIT-treated implants were higher than those of the PLLA scaffold without cultured cells (Fig. 5G). Among them, both parameters of the BIT-treated cartilage were not only greater than the control and BI-treated cartilage but also comparable with that of the native cartilage.
DISCUSSION
The present study succeeded in overcoming the two major limitations of tissue engineering of permanent cartilage from autologous human chondrocytes: dedifferentiation during the long term culture for multiplication and hypertrophic differentiation by stimulation for redifferentiation. For the efficient and exhaustive search for the optimal combination, we utilized the analysis of variance by the fractional factorial design (9). This method was originally developed in the agricultural science field; one could decide the optimal conditions to yield good crops out of many naturally fluctuating conditions including temperature, soil condition, rainfall, etc (39). Thereafter, this method was successfully adapted to industrial applications; e.g. it drastically reduced production errors of aircraft engine manufacturing to one-fifth through the grading of numerous parameters including cutting oil, cutting speed, groove ratio, tip-end angle, etc (40). Since this method is now widely approved as being useful for the maintenance of goods and services of high quality, the Food and Drug Administration Good Manufacturing Practices regulation and the International Organization for Standardization 9000 series now require its confirmation for process validation (9). This method, however, has not been applied for quality control in medical fields that contain tremendous parameter variations. The present study for the first time adopted the method to determine the optimal combination of putative factors for a regenerative medicine and demonstrated its usefulness.
BMP-2 was shown to have the strongest potency to produce GAG as a single factor, according to the result of analysis of variance by the fractional factorial design (Fig. 1A). BMP-2 is known to enhance not only chondrogenic differentiation but also further hypertrophic differentiation of chondrocytes in several cell cultures (7,20), which is a critical weakness for the regeneration of permanent cartilage. Insulin is also reported to induce both chondrogenic and hypertrophic differentiation in cultures (11). The third factor, T3, as well as its prohormone L-thyroxin (T4), are also known to play important roles in physiological skeletal growth by regulating terminal differentiation of chondrocytes. A childhood hypothyroidism, congenital hypothyroidism, is characterized by growth arrest and short stature, whereas childhood thyrotoxicosis causes accelerated growth and premature closure of the growth plate (41). Mice devoid of all isoforms produced from thyroid hormone receptor-␣ (TR␣ 0/0 ) exhibited dwarfism due to impaired hypertrophic differentiation of chondrocytes in the growth plate (42). In addition, several in vitro studies have shown that T3 around physiological concentrations (0.1-1 nM) stimulates the terminal differentiation markers COL10, alkaline phosphatase, and osteopontin in several chondrocyte culture systems (43,44). These seem to be inconsistent with the present finding that T3 prevented BI from inducing hypertrophic differentiation through suppression of the phosphatidylinositol-3 kinase/Akt signaling, a major pathway for chondrocyte differentiation (34). The thyroid hormones are generally believed to exert their functions through the genomic action by activating the nuclear receptors (TR␣1 and -2 and TR␣1, -2, and -3), causing a conformational change that leads to dissociation from a repressor complex and interaction with an activation complex containing
Optimal Factors for Chondrocyte Redifferentiation
histone acetylase (41). In contrast, T3 at higher concentrations has been suggested to exert a nongenomic action; e.g. T3 (0.1-100 M) caused a rapid activation of the Na ϩ /H ϩ exchanger activity in isolated rat alveolar type II cells or rat AT II cell line RLE-6TN (45). The present study showed that T3 at more than 100 nM caused inhibition of COL10 and Runx2 expressions with a rapid suppression of Akt phosphorylation induced by BI within 5 min. We speculate that the T3 action might be a nongenomic one that is somehow associated with the BI and Runx2 signalings. In Fig. 2, Sox9 that is a potent inhibitor of the chondrocyte hypertrophy (32) was down-regulated in BI, implying the induction of chondrocyte hypertrophy in BI. However, the addition of T3 to BI did not up-regulate the Sox9 expression, which may not follow the effect of BIT on the inhibition of the chondrocyte hypertrophy. It was reported that the thyroid hormone around the physiological concentration (T4, 30 ng/ml) inhibited the Sox9 expression and promoted chondrocyte hypertrophy in the rat chondrocyte pellet culture (46). These functions of the thyroid hormone around the physiological concentration seemed to work through a genomic action (44). The decrease in the Sox9 expression by the addition of 10 Ϫ7 M T3 in the present experiment may suggest that T3 down-regulated the expression of Sox9 through the genomic action, even at the pharmacological concentration. However, we think that T3 at the pharmacological concentration prevents hypertrophic differentiation mainly through a nongenomic machinery. The nongenomic effect may dominate the genomic action in the pharmacological condition, leading to the inhibition of chondrocyte hypertrophy.
Considering that some tens to 100 ml of regenerated cartilage is necessary for its clinical application to major skeletal disorders with severe cartilage defects, we should initially obtain a sufficient number of cells from a limited amount of specimen that is no more than 0.1 ml (10 6 cells), meaning that a 1,000-fold increase in cell number is needed. For efficient cell proliferation, many researchers have performed cell cultures with mitogenic growth factors in the presence of fetal bovine serum that is unfavorable for clinical use due to the risk of pathogen transmission and immune reaction (47). Previous trials of cultures using autologous human serum, however, could provide no more than 10 7 cells, which corresponds to at most 1 ml of regenerative cartilage (2,3). To overcome this problem, our preparatory study established a fetal bovine serum-free culture system of primary human chondrocytes that realize a 1,000-fold increase in cell number within 4 weeks under the stimulation of FGF-2 with insulin or IGF-I (8), although the cell dedifferentiation was inevitable during the repeated passaging. Based on the previous study, the present study further established a serum-free culture system that causes redifferentiation of the dedifferentiated cells and maintains the properties of permanent cartilage. Since we succeeded in regenerating a high quality cartilage column 10 mm in diameter and 1 mm in thickness that is ϳ80 l in volume, starting from 10 3 primary chondrocytes in a surgical specimen, it is estimated that 80 ml of cartilage can be regenerated from 0.1 ml of a specimen containing 10 6 cells by using our original two systems. We conclude that the BIT stimulation realized the tissue engineering of permanent cartilage whose quantity and quality satisfy the revolu-tionary treatment of major skeletal disorders. A clinical trial is now underway. | v3-fos-license |
2015-03-21T17:44:09.000Z | 2004-09-30T00:00:00.000 | 16227373 | {
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} | pes2o/s2orc | Molecular Sciences Density Functional Studies of the Dipole Polarizabilities of Substituted Stilbene, Azoarene and Related Push-pull Molecules
We report high quality B3LYP Ab Initio studies of the electric dipole polarizability of three related series of molecules: para-XC 6 H 4 Y, XC 6 H 4 CH=CHC 6 H 4 Y and XC 6 H 4 N=NC 6 H 4 Y, where X and Y represent H together with the six various activating through deactivating groups NH 2 , OH, OCH 3, CHO, CN and NO 2. Molecules for which X is activating and Y deactivating all show an enhancement to the mean polarizability compared to the unsubstituted molecule, in accord with the order given above. A number of representative Ab Initio calculations at different levels of theory are discussed for azoarene; all subsequent Ab Initio polarizability calculations were done at the B3LYP/6-311G(2d,1p)//B3LYP/6-311++G(2d,1p) level of theory. We also consider semi-empirical polarizability and molecular volume calculations at the AM1 level of theory together with QSAR-quality empirical polarizability calculations using Miller's scheme. Least-squares correlations between the various sets of results show that these less costly procedures are reliable predictors of <α> for the first series of molecules, but less reliable for the larger molecules.
Introduction
The electric dipole moment p e of a molecule is a quantity of fundamental importance in structural chemistry.When a molecule is subject to an external electric field E, the molecular charge density may rearrange and hence the dipole moment may change [1].This change can be described by the tensor equation ( 1 Here α is a second rank tensor property called the dipole polarizability, and β is the first of an infinite series of dipole hyperpolarizabilities and p e,0 , the permanent electric dipole moment, is the electric dipole moment in the absence of a field.Because the electric dipole moment may change when an external field is applied, the molecular potential energy U may also change according to the tensor equation (2): Hyperpolarizabilities are known to be small in magnitude, and their effect is minimal for weak electric fields.They are however important quantities when the electric field is large, and molecules that exhibit large β values are of current interest because of their applications in electro optical devices [2,3] and in the general field of non-linear optics (NLO).Many such molecules can be represented by the idealized structure shown in Figure 1.It is thought that better materials can be made by modifying the strength of the activating group, the size and complexity of the conjugated system and the strength of the deactivating group.It is also thought that the presence of an activating group together with a deactivating group in a suitable ring position can enhance some electronic properties.The well-known phrase 'push-pull mechanism' is used to describe such phenomena.
A large number of such push-pull systems have been studied in recent years.Azoarene and stilbene derivatives are known to exhibit large nonlinear optical properties, which imply that they have large hyperpolarizabilities.There have been a number of theoretical studies of these quantities [4][5][6].The aim of this paper is to report a theoretical study of the dipole polarizabilities for the three series of molecules shown in Figure 2. X and Y are a selection of simple activating and deactivating groups, as discussed below.The experimental determination of a molecular polarizability is far from straightforward, especially if the molecule has little or no symmetry.The principal routes are studies of refractive index and relative permittivities, through Rayleigh and Raman scattering and through the quadratic Stark effect.These have been well reviewed elsewhere [7], but a few words of background are in order.
For a molecule with symmetry, the principal axes of the polarizability tensor correspond to the symmetry axes; for molecules such as those discussed here workers often refer to the principal axes as L (long), M (medium) and N (normal to the molecular plane) and so the principal values of the tensor are written α LL , α MM and α NN .The mean value ( ) (which is invariant to rotation of coordinate axes) can be determined from the refractive index n of a gas of non-interacting particles according to the equation where p is the pressure, k B the Boltzmann constant, T the thermodynamic temperature and ∈ 0 the permittivity of free space [1].
In a condensed phase, the problem is more complicated because the separation between molecules is of the order of molecular dimensions and their interactions can no longer be ignored.As a result both the external field and the field due to the surrounding molecules polarize each molecule.The Lorenz-Lorentz equation: applies to non-polar molecules in condensed phases and it can be derived from a detailed consideration of these ideas [1].Here, N is the number of molecules in volume V.In the case of molecules with a permanent dipole moment, it is necessary to take account of the orientation polarization.The Debye equation: permits polarizabilities and dipole moments to be determined from measurements of the relative permittivity ∈ r and the density ρ as a function of temperature.The anisotropy κ, usually defined as: gives a measure of deviations from spherical symmetry since it would be zero for a spherically symmetric charge distribution.An alternative route to polarizability is direct calculation, and there is a large literature on this topic [8].The general idea is to find a level of theory that gives highly accurate values for the polarizability tensor, but naturally this aim has to moderated with the cost of such calculations.
The principles involved in Ab Initio polarizability calculations are well understood and can be illustrated by our results for azoarene (Series III with X = Y = H), shown in Table 1.The atomic unit (au) of dipole polarizability is e 2 a 0 2 E h -1 so that 1 atomic unit = 1.6488 × 10 -41 C 2 m 2 J -1 .The calculations here refer to an isolated gas-phase molecule at 0 K. Naturally, such a molecule has zero-point energy and there is a small but finite contribution to the polarizability from this effect [8].No attempt was made to estimate the vibrational contributions in this paper.All calculations were done using Gaussian 03W [9], with standard basis sets, integration points, cutoffs, etc.With the exception of the final row labelled 'Standard', all calculations were begun by optimizing the molecular geometry at the level of theory specified; polarizabilities were then calculated at the same level of theory using the standard Gaussian 03W keyword 'Polar'.This keyword means that the polarizabilities were obtained analytically rather than by numerical differentiation.
The true experimental value for an isolated molecule of azoarene in the gas phase is unknown, but the Table 1 entries suggest values of about 186 au for <α>, and 0.13 for the anisotropy.The first Table 1 entry shows why HF-LCAO calculations with standard basis sets are no longer thought adequate for accurate polarizability calculations; the mean value of 149.31 is poor and the perpendicular component is particularly badly represented.Addition of diffuse functions (second entry) gives a pleasing increase in the perpendicular component, but the mean value is still poor.
The third entry is the KS-LCAO equivalent to the first entry, and illustrates the importance of electron correlation for these calculations.The fourth entry shows once again the importance of diffuse functions.The fifth and sixth entries suggest that there is no real advantage to be gained by trying to improve the polarization functions beyond the usual (2d,1p) set.
The rows of Table 1 are arranged in order of computer resource (measured as time taken to run the calculation using Gaussian03 W on a standard AMD Athlon XP2600+ PC).On detailed comparison, it turns out that the geometries predicted by the very sophisticated basis sets including ++ and extended polarization functions are almost identical to those given by B3LYP/6-311G(2d,1p), so a compromise was chosen in order to save computer resources.Ab Initio geometries reported from now on are at the B3LYP/6-311G(2d,1p) optimized level of theory, whilst the dipole polarizabilities are at the B3LYP/6-311++G(2d,1p) level of theory but with the same geometry.This is the meaning of the term 'Standard' in Table 1.In a commonly used notation [9], the polarizabilities are therefore calculated at the B3LYP/6-311G(2d,1p)// B3LYP/6-311++G(2d,1p) level of theory.The anisotropy κ does not appear to be a very sensitive descriptor, and will not be mentioned again.
Calculations on monosubstituted benzenes
The first step is to consider the monosubstituted benzenes, which correspond to Series I with X = H.We will report only the mean polarizabilities for this series, but draw attention to any interesting behaviour in the tensor components.Table 2 shows various data for the molecules studied.The second column of Table 2 shows the mean polarizability, and the third column is the polarizability difference on substitution of Y into benzene.These values give us an 'increment' for use when discussing possible additivities in the polarizability mean values for later series of molecules.The table values are arranged in order of increasing increment.The largest increment is seen to be due to NO 2 , and the two smallest increments are due to OH and NH 2 .
According to a recent undergraduate organic chemistry textbook [10], the order of our chosen groups from highest activating to highest deactivating is as follows: NH 2 > OH > OCH 3 > (zero) -C(=O)H > CN > NO 2 , with the point on the scale where groups are neither activating or deactivating denoted 'zero'.There is no particular reason why our increments should correlate exactly with Loudon's scheme; the latter is to do with the chemical reactivity of a given substituted molecule C 6 H 5 X compared to benzene, ours to do with the response of the ground state molecule C 6 H 5 X to an applied electric field.We will see later that the scheme gives a good indication of increments for disubstituted species.Dipole polarizabilities are often used in QSAR studies, where the aim is to give a reliable but quick estimate of <α>, as part of the process of high-throughput screening.Ab Initio polarizability calculations are prohibitively expensive in a QSAR context, even for such simple molecules.One therefore looks to less rigorous but reliable procedures.Consider therefore a typical neutral atom modelled as the sphere of charge shown in Figure 3.The radius is a and the nuclear charge is Q.We switch on the electric field E which displaces the nucleus by a relative distance d from the original atomic centre.At this point there is a force on the nucleus QE due to the applied field and one due to the electron density.According to Gauss' electrostatic theorem [1], the latter force is At equilibrium these two forces must be equal and so the displacement d satisfies The induced electric dipole moment is Qd and the polarizability is Qd / E hence The derivation can be easily extended to a closed volume of arbitrary shape, not necessarily a sphere, and apart from the factor 4πε 0 the polarizability of an atom is determined in this model by its volume.For this reason, workers in the field speak about 'polarizability volumes' and quote their results in volume units.
Molecular volumes are routinely determined in QSAR studies, and typical values are shown in column 4. The numerical conversion factor between Å 3 and atomic units of polarizability is and so the prediction from this simple model would be <α> = 6.748 / × 332.58 au = 2244 au for benzene.Whilst the quantitative agreement with experiment is clearly nonexistent, our molecular volumes do give a good least square fit to the Ab Initio mean polarizability values, with a correlation coefficient of 0.94.
Rewriting equation ( 5) in terms of molar quantities defines the molar refractivity Here M is the molar mass, N A the Avogadro constant and ρ the density.It is an experimental fact that molar refractivities are additive properties at the molecular level, and a view has long prevailed that the molar refractivity of a molecule is a sum of the molar refractivities of the constituent parts (atoms/ groups).Extensive tables of additive atom and group molar refractivities are available [11].These tables have been extended to molecular polarizabilities with the compilations of Denbigh [12] and others.Such values are thought to be unreliable for molecular polarizability calculations.
The definitive reference in this field appears to be that due to K. J. Miller [13].Miller pointed out the need to take account of the atomic environment in molecular calculations, and this is usually done by assigning parameters in which each atom is characterized by its state of atomic hybridization.Miller and Savchik [14] proposed a functional form 2 0 4 4 where τ A is an atomic hybrid component for each atom A in a given state of hybridization.N is the total number of electrons.In fact, Miller and Savchik omitted the factor 4πε 0 and so most computer packages quote the results as polarizability volumes (typically Å 3 ).These are shown in column 5 of Table 2.The Miller method gives a mean polarizability of 70.38 au for benzene, in much better agreement with the Ab Initio value than the crude molecular volume.It is clear that polarization volumes are not to be interpreted as molecular volumes.A linear regression between the Miller and the Ab Initio <α> values as shown in Table 2 gives a regression coefficient of 0.93.Finally we should consider the use of semiempirical HF-LCAO techniques such as CNDO/2, MINDO3, PM3 and AM1 for calculations of dipole polarizabilities.The final column of Table 2 shows AM1 calculations of <α>.All geometries were optimized before calculation of <α>.There is a good least squares correlation between the AM1 and the Ab Initio results, with a correlation coefficient of 0.96, but semiempirical calculations of the tensor α (rather than the mean value) tend to give extremely poor values for the normal component α NN .For benzene we find
Calculations on disubstituted benzenes (Series I)
Table 3 records values of Ab Initio calculations on Series I C 6 H 4 X with a further X substituted in the para position (giving X-C 4 H 4 -X).The second column gives the mean polarizability, the third column the difference between the disubstituted molecule and benzene, whilst the final column shows the sum of increments that would be expected for a purely additive model (with results taken from Table 2).The values in columns 3 and 4 are little different, but there is no obvious trend.We now consider the selection of para-substituted X-C 6 H 4 -Y molecules shown in Table 4, where X and Y are activating/deactivating groups.Again, column 2 shows the Ab Initio mean polarizabilities, column 3 the difference between the molecule and benzene and column 4 the value that this increment would have in a purely additive model using the values given in Table 2.In every case, disubstitution produces a larger enhancement of <α> than we would have expected from pure additivity of the two functional groups.This behaviour is consistent with the push-pull mechanism.The largest push-pull enhancement is for the pair X = NH 2 , Y = NO 2 (7.14 au), which is entirely consistent with the order of activating/ deactivating groups mentioned above.For clarity, the enhancements are shown in Table 5, arranged in order of increasing value.A linear regression between the molecular volumes and the Ab Initio mean polarizabilities gives a correlation coefficient of 0.93, with a corresponding correlation coefficient of 0.95.Although not shown here, AM1 calculations of the mean polarizability give values that correlate well with the Ab Initio values (with a correlation coefficient of 0.99), but the AM1 α NN absolute values are as usual extremely low.
Calculations on disubstituted stilbene and azoarene (Series II and III)
In Table 6, we record the polarizability principal components for the 10 disubstituted stilbenes.These are given primarily for reference, given the compute-intensive nature of the calculations and the sensitivity of the normal component to level of theory.In Table 7, we record a number of quantities for discussion.Tables 8 and 9 are the corresponding Tables for the disubstituted azobenzenes.Once again, we focus on the push-pull contribution to the mean polarizability, defined as the difference between column 2 and column 3, i.e., the differences between the substituted and unsubstituted molecules.For clarity, these are recorded in Table 10, in order of increasing magnitude for the two series.For both series, the smallest enhancement is due to the pair OH/ CN, and the largest enhancement due to the pair NH 2 / NO 2 .The overall combinations vary between the two series, but the enhancements are consistent with the standard activating/ deactivating order mentioned above.
Finally we consider the likely reliability of various easily-computed indices such as the molecular volume, the Miller empirical volume polarizabilities and AM1 polarizabilities discussed above.Linear regressions were done for each of these quantities against the B3LYP mean polarizabilities <α>, and the regression coefficients r are given in Table 11.The correlation coefficients are all well below 0.95, which value if often taken to justify a straight line relationship.It therefore seems that none of the three simpler procedures gives a reliable estimate of <α> for these two latter series of molecules.
• Our Ab Initio calculations give further support to the well-reported 'push-pull' mechanism.
• The simple models considered do not give acceptable least squares correlations with the Ab Initio results for the larger molecules.
• There are good least squares correlations between the Ab Initio results and those given by cheaper procedures such as the calculated molecular volume, the Miller empirical polarizability and semiempirical models such as AM1.
• Semiempirical models grossly underestimate the normal component of the polarizability tensor.
Disubstituted benzenes (Series I)
• Polarizability increments are in accord with the standard organic chemistry 'activatingdeactivating' sequence.
• Disubstitution produces a larger effect than we would expect on the basis of pure additivity, consistent with the push-pull mechanism.
• There are reasonable least squares correlations between the Ab Initio results and those given by the computationally cheaper procedures.
Series II and III
• Polarizability increments are in accord with the standard organic chemistry 'activatingdeactivating' sequence.
• Inclusion of the extra -CH=CH-and -N=N-group enhances the increment, but there is little to choose between these two groups.
• Least squares analysis show that there are essentially no correlations between the Ab Initio results and those given by the computationally cheaper procedures.
Figure 3 .
Figure 3. Atom in an external electric field
Table 2 .
Various quantities for Series I with X = H
Table 3 .
Various quantities for the X-C 6 H 4 -X series
Table 4 .
Various quantities for the X-C 6 H 4 -Y series
Table 6 .
Principal values of α for the X-C 6 H 4 -CH=CH-C 6 H 4 -Y series
Table 7 .
Various quantities for the X-C 6 H 4 -CH=CH-C 6 H 4 -Y series
Table 8 .
Principal values of α for the X-C 6 H 4 -N=N-C 6 H 4 -Y series
Table 9 .
Various quantities for the X-C 6 H 4 -N=N-C 6 H 4 -Y series
Table 10 .
Push-pull enhancements to the mean polarizability.
Table 11 .
Linear regression coefficients r for the Stilbene/ azoarene series II and III. | v3-fos-license |
2019-04-09T13:06:38.117Z | 2018-02-01T00:00:00.000 | 102917083 | {
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} | pes2o/s2orc | Risk and exposure assessment for agricultural workers during treatment of cucumber with the fungicide fenarimol in greenhouses
The exposure pattern and potential risk of fenarimol emulsifiable concentrate to agricultural workers were investigated during the preparation of the pesticide suspension and the application of the prepared suspension to the cucumber in a greenhouse environment. The dermal exposure to fenarimol was 0.17 ± 0.11 mg (0.001 ± 0.001% of prepared active ingredient) for mixing/loading and 0.22 ± 0.15 mg (0.003 ± 0.002% of applied active ingredient) for application, respectively. The most exposed part of body was the hand (100%) during mixing/loading, whereas the primary sites during application were the back and legs. In particular, 54.8% of dermal exposure occurred on the shins. The inhalation exposure to fenarimol was detected as 3.7 ± 1.0 μg for the applicator. In comparison with the exposure patterns to pesticides for agricultural workers in greenhouse reported in previous studies, lower dermal and inhalation exposures to fenarimol were observed during mixing/loading and application, respectively. The results of the risk assessment demonstrated that the possibility of risk to fenarimol exposure was lowest during mixing/loading and application in the greenhouse environment.
Introduction
Pesticides are toxic compounds used to control many pests, diseases and weeds in the agricultural industry [1]. Agricultural workers are occupationally exposed to pesticides during the preparation of the spray suspension and the application to the crops through representative routes such as dermal deposition and inhalation [2][3][4]. As these exposures may be injurious to their health, it is necessary to determine whether the actual working conditions of the agricultural workers were sufficiently safe from pesticide intoxication.
In Korea, the risk possibility of pesticides has increased because farmers now use the pesticides at higher concentrations and for a longer time than previously to maintain or improve the crop production [5]. Moreover, the increase of greenhouse cultivation indicates that greater pesticide exposure may occur owing to the closed environment [6]. Many studies on pesticide exposure have been conducted for several decades. However, the studies were mainly focused on open fields, such as paddy fields and mandarin or apple orchards [2,3,[7][8][9][10][11][12]. Only two cases of greenhouse exposure have been reported in Korea [7,8]. Therefore, many studies and great investment are needed with regard to the pesticide exposure in a closed environment such as a greenhouse.
Fenarimol is a pyrimidine fungicide that acts against pests such as powdery mildews, scabs, rusts and leaf spots in pome fruit, stone fruit, vines and turf. It inhibits ergosterol biosynthesis in fungi, as systemic foliar fungicide and testosterone aromatase activity, which resulted in irreversible infertility in male rats [13]. The acute percutaneous LD 50 for rabbits is [ 2000 mg/kg, and the acceptable daily intake is 0.01 mg/kg b.w. [14]. Fenarimol was selected for this study because this was one of the pesticides mainly used for cultivation of cucumber and was formulated as emulsifiable concentrate (EC). Therefore, the effect of formulation on pesticide exposure could be evaluated by comparison with previous studies.
The present study was performed to assess the pesticide exposure of agricultural workers during the application of fenarimol EC to cucumbers in a greenhouse environment. The major body parts of exposure were identified, and the exposure pattern to fenarimol was compared with those of imidacloprid wettable powder (WP) and thiophanatemethyl WP in cucumber greenhouses as previously reported [7,8].
Reagents and materials
The pesticide used in the field study was the commercial fungicide Fenari EC 12.5% (fenarimol, Dongbu-hannong Chemical, Seoul, Republic of Korea) and obtained through a local vendor. The analytical standards of fenarimol (99.4%) were obtained from the manufacturing company (Dongbu-hannong Chemical) and Rural Development Administration, Korea. HPLC-grade acetone was used (Fisher Scientific Korea Ltd, Seoul, Republic of Korea).
Dermal exposure matrices and measurement
Dermal patches made from cellulose thin-layer chromatography paper (17CHR, 1-mm thickness, Whatman International Ltd, Maidstone, UK) were attached to a worker's forehead, front of neck, back of neck, chest/abdomen, back, upper arms, forearms, thighs and shins of the protective garment to measure the dermal exposure [7,15]. Hand, feet and facial exposures were monitored using cotton gloves, socks and a mask [16]. The exposure parts of the dermal patch and mask were 50 and 200 cm 2 , respectively.
Inhalation exposure matrices and measurement
The XAD-2 resin (ORBO TM 609 Amberlite XAD-2, 400/ 200 mg, Supelco, Bellefonte, PA, USA) was used for the measurement of inhalation exposure, which was connected to an air pump (GilAir-3, Sensidyne, Clearwater, FL, USA) and 37-mm open-faced cassettes equipped with a glass fiber filter (type AE, SKC, Eighty Four, PA, USA). The spray drift of the pesticides was collected by using the XAD-2 resin at flow rate of 2 L/min.
Field study
The cucumber greenhouse for field studies is located in Pyeongtaek, Gyeonggi, Republic of Korea. The cucumber was 200 cm high, in a fruiting stage, and planted at intervals of 20 9 130 cm. The temperature was measured with the thermometer at the start and end of each experiment, whereas the relative humidity was determined using a hygrometer. The temperature and the relative humidity in the field were 24-27°C and 51-57%, respectively. The applicator was a man with a height of 168 cm and a weight of 70 kg. The spray suspension was prepared from Fenari 12.5% EC (100 mL, 1 bottle) and 400 L water for 2 min. The applicator sprayed 280 L of prepared solution with a power sprayer at flow rate of 4.7 L/min for 60 min in a cucumber field of 250 m 2 . The total prepared and sprayed amounts contained 12.5 and 8.75 g a.i. (active ingredient), respectively. Each mixing/loading and application was repeated twice.
Extraction and analysis from exposure matrices
Pesticide residue on the exposure matrices were extracted with acetone by agitation at 200 rpm for 60 min on a shaker (Wooju Scientific, Gimpo, Republic of Korea). After concentration with a nitrogen evaporator (Reacti-Vap, Pierce, Rockford, IL, USA), fenarimol residue was determined with a gas chromatograph equipped with a nitrogen-phosphorous detector (HP 5890 series II plus, Hewlett-Packard Co., Avondale, PA, USA) and a 30 m 9 0.53 mm, i.d., (d f = 1.5 lm) HP-5 capillary column (Agilent Technologies, Inc., Santa Clara, CA, USA). The flow rates of the gases were 15 mL/min in splitless mode for nitrogen as the carrier gas and 105 and 3.6 mL/ min for air and hydrogen, respectively. The injection volume was 1 lL. The injector and detector temperatures were set at 260 and 320°C, respectively. The oven temperature was held at 210°C for 1 min, raised to 280°C at a rate of 10°C/min and maintained for 2 min.
Method validation
The instrumental limit of detection (ILOD) and instrumental limit of quantitation (ILOQ) were determined through the analysis of 0.001-1.0 ppm standard solutions. The method limit of quantitation (MLOQ) was calculated through the consideration of ILOQ, injection volume and extraction solvent volume [3]. The integrated peak area after five repeated injections of 0.1 and 1 ppm of standard solution were compared for instrumental reproducibility. The linearity of the calibration curve in the range from the ILOQ to 10 ppm was checked on the day of preparation and 3 days later. The control exposure matrices were fortified to MLOQ, 5 MLOQ and 10 MLOQ levels and analyzed three times to validate the matrix extraction efficiency (recovery). The trapping efficiency and breakthrough of XAD-2 resin was investigated by spiking on the bottom of a U-shaped glass tube (Daejung Chemical, Daejeon, Republic of Korea) at 70°C and the 1°-part of the XAD-2 resin at 100 and 10 MLOQ, respectively. This test was conducted at 2 L/min for 4 h and repeated three times. The U-shaped glass tube was heated to 70°C.
Calculation of dermal and inhalation exposure
Dermal exposure intensity (lg/cm 2 ), dermal exposure amount per body part (lg) and inhalation exposure amount (ng) of the mixer/loader and applicator were determined based on the approach suggested by Choi et al. [7]. The area of the matrix was 50, 935, 1266 and 200 cm 2 , for the patch, glove, sock and mask, respectively. The body surface area and the respiration rate (1270 L/h) used for the exposure assessment for an adult Korean in this study as suggested by Kim et al. [16].
Risk assessment
The potential dermal or inhalation exposures (PDE or PIE) were calculated through the extrapolation of the corresponding dermal exposure amount (lg) of the whole body or inhalation exposure amount (ng) per activity with five working activities per day [7]. The actual dermal exposure (ADE), the absorbable quantity of exposure (AQE) and the margin of safety (MOS) for the mixer/loader and applicator were determined based on the approach suggested by Choi et al. [7]. For exposure assessment, the actual exposure (AQE) was estimated from the ADE and PIE through dermal contact and inhalation, respectively. MOS values were determined for risk assessment through the comparison of AQE and acceptable operator exposure level (AOEL). The penetration rates of fenarimol through clothes were 1 and 10% for the mixer/loader and applicator, respectively [16], whereas the skin absorption rate was 2.6% [17]. The AOEL, reported as the relevant risk value, was 0.02 mg/kg/day for fenarimol [17], and the body weight for an average Korean adult male was 70 kg [7].
Statistical analysis
The significant differences between the exposure to fenarimol EC in this study and previously reported exposures to imidacloprid WP and thiophanate-methyl WP in cucumber greenhouses [7,8] were determined through an independent t test (two-tailed) using SPSS version 23 software (SPSS, Chicago, IL, USA).
Method validation
The ILOD and ILOQ for fenarimol were 0.01 and 0.05 ng, respectively, which indicated that the analytical sensitivity was sufficient for the analysis. The reproducibility of the instrumental analysis was good (CV \ 3%). The linearity of the calibration curve for fenarimol was consistent in the range from the ILOQ to 10 mg/L over 3 days (R 2 [ 0.9999). The matrix extraction efficiency (recovery) was 84-111% with a small relative standard deviation of 0.2-11.2%, which indicated that the extraction procedures were reasonable (Fig. 1). The trapping efficiency of the XAD-2 resin was reliable, with a mass balance of 94.9 ± 4.9% (Table 1). Fenarimol was not transferred to the 2°-part of the resin in the breakthrough test (Table 1), which demonstrated that the XAD-2 resin has a powerful retention capacity for fenarimol.
Dermal exposure during mixing/loading
Cotton gloves were used to determine the hand exposure of the agricultural workers [16], because they are generally used for measurements of potential dermal exposure, even though overestimation may occur if the cotton gloves absorb more of the pesticide product through direct contact, especially that of liquid formulations, such as EC. The amount of dermal exposure to fenarimol EC was 0.17 ± 0.11 mg for mixing/loading, which corresponded to 0.001 ± 0.001% of the total prepared amount (fenarimol, 12.5 g a.i.). The ratio of dermal exposure to fenarimol EC was similar to that of imidacloprid WP (0.001 ± 0.001%) or thiophanate-methyl WP (0.001%), reported in previous studies [7,8]. Dermal exposure only occurred through the hand owing to the lower scattering possibility of EC in contrast to WP. Hand exposure was reported to reach 92.5-99.9% of the dermal exposure during mixing/loading, especially when using the liquid formulation as a soluble liquid (SL) [7-9, 18, 19]. Therefore, the exposure level during mixing/loading remarkably decreased through the use of appropriate gloves as hand protection.
Dermal exposure during application
Generally, the applicator is exposed to pesticides mainly through dermal absorption. The dermal exposure amount of fenarimol EC was 0.22 ± 0.15 lg (0.003 ± 0.002% of the total applied a.i.) during the application in the greenhouse (Table 2). However, the higher exposure ratios of the dermal to the total applied amount were reported as 0.013 ± 0.003 and 0.016 ± 0.010% in the cucumber greenhouse for imidacloprid WP and thiophanate-methyl WP, respectively [7,8]. The possibility of dermal exposure for WP compared with EC was approximately five times higher (P \ 0.05). In contrast, in open fields such as an apple orchard, the dermal exposure of EC was higher than that of WP, which was higher that of SL [9,12]. These results demonstrated that the formulation type was one of the significant factors that affect the exposure level for applicators, which was in contrast with that of Vidal et al. [20]. However, owing to insufficient exposure cases, further studies are needed to clarify the effects of the formulations on pesticide exposure.
Meanwhile, the patterns of exposure distribution of the pesticide formulations were similar. The dermal exposure on the back and shins, the primary sites of applicator contamination, was 0.12 ± 0.05 (0.06 ± 0.02 lg/cm 2 ) and 0.06 ± 0.09 mg (0.02 ± 0.03 lg/cm 2 ), respectively, which showed the large fluctuation of dermal exposure in the working environment, as described in previous studies (Table 2) [21,22]. As shown in Fig. 2, the contribution ratio of the legs was 63.1% (shin, 54.8%), as a result of indirect contamination through contact with the sprayed plants [21,23]. Other studies with imidacloprid (legs, 51.2%) and thiophanate-methyl (legs, 52.4%) reported similar exposure patterns, despite the different formulations [7,8].
Inhalation exposure
The airborne spay drift resulted in health concerns in the working environment. The inhalation exposure to fenarimol for mixing/loading was not observed because the EC was not dispersed. During application, the inhalation exposure of fenarimol was determined as 3.7 ± 1.0 lg, which corresponded to 0.00004 ± 0.00001% of the total applied amount. Generally, 1% dermal exposure was assumed for respiratory exposure [24]. In this study, inhalation exposure to fenarimol was detected as average 2.3% of the dermal exposure, because of increased inhalation exposure and reduced dermal exposure, which was higher than that reported in previous studies on imidacloprid and thiophanate-methyl [7,8]. However, owing to the very high variation as standard deviation of 2.0% between repetitions, statistically significant differences at the 0.05 level in the levels of inhalation exposure or ratios of the inhalation exposure to the total dermal exposure during application were not observed among the pesticides (fenarimol, imidacloprid and thiophanate-methyl) or between formulation types (EC and WP).
Risk assessment
In the exposure assessment, agricultural workers were only exposed to fenarimol through dermal contact during mixing/loading. However, they were exposed mainly through the inhalation during application ( Table 3). The PDEs were similar between the mixer/loader and the applicator, but the ADE for the applicator was approximately 10 times higher, owing to the different rates of penetration through the clothes of 1 and 10% for mixer/loader and applicators, respectively. The ratio of PIE to AQE during application was 86.4%, which indicated that in a closed environment, such as a greenhouse, the applicator was mainly exposed to pesticides through inhalation. Whereas 6.4-10.4% of AQE were reported to occur through inhalation in the previous studies with imidacloprid and thiophanate-methyl, owing to reduced inhalation exposure and increased dermal exposure in comparison with fenarimol exposure [7,8].
The MOS values for the risk assessment were [ 1, which resulted from the low exposure possibility, the low toxicity (AOEL, 0.02 mg/kg) and the low skin absorption (2.6%) of fenarimol [17]. These results demonstrated that the agricultural workers in the cucumber greenhouse were safe from fenarimol toxicity during mixing/loading and application.
Acknowledgments N.D. not detected a Potential dermal exposure and potential inhalation exposure on the assumption of five working activities per day b Actual dermal exposure = [PDE 9 1% (mix/loader) or 10% (applicator) of penetration rate through clothes] 9 2.6% of skin absorption c Absorbable quantity of exposure, the sum of the ADE and PIE d Margin of safety = AOEL (0.02 mg/kg b.w./day) 9 70 kg b.w./ AQE f Mean ± SD, n = 2 | v3-fos-license |
2020-06-11T09:02:02.302Z | 2020-06-01T00:00:00.000 | 225848541 | {
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} | pes2o/s2orc | Intermedin promotes vessel fusion by inducing VE‐cadherin accumulation at potential fusion sites and to achieve a dynamic balance between VE‐cadherin‐complex dissociation/reconstitution
Abstract To create a closed vascular system, angiogenic sprouts must meet and connect in a process called vessel fusion, which is a prerequisite for establishment of proper blood flow in nascent vessels. However, the molecular machinery underlying this process remains largely unknown. Herein, we report that intermedin (IMD), a calcitonin family member, promotes vessel fusion by inducing endothelial cells (ECs) to enter a “ready‐to‐anchor” state. IMD promotes vascular endothelial cadherin (VEC) accumulation at the potential fusion site to facilitate anchoring of approaching vessels to each other. Simultaneously, IMD fine‐tunes VEC activity to achieve a dynamic balance between VEC complex dissociation and reconstitution in order to widen the anastomotic point. IMD induces persistent VEC phosphorylation. Internalized phospho‐VEC preferentially binds to Rab4 and Rab11, which facilitate VEC vesicle recycling back to the cell‐cell contact for reconstruction of the VEC complex. This novel mechanism may explain how neovessels contact and fuse to adjacent vessels to create a closed vascular system.
INTRODUCTION
Angiogenesis, a process by which the primary vessel network is expanded via sprouting of new vessels from the preexisting vasculature, plays important roles in many physiological and pathological processes, particularly in wound healing and cancer. [1][2][3] To effectively provide blood supply, the angiogenic vessels must be organized into an elaborate hierarchical system of appropriate density. 3,4 Although the majority of cancerous tumors are highly vascularized, the vessels within the tumors are abnormal with regard to almost all aspects of their structure and function. 5,6 The tumor vasculature contains numerous angiogenic sprouts that form dead ends and do not fuse to the established circulatory network, which severely impairs blood perfusion.
To create a closed vascular system, angiogenic sprouts must meet and connect in a process called vessel anastomosis or vessel fusion, which is a prerequisite for blood flow through the vessels. 3,4 Unfortunately, few studies have focused on the process of vessel fusion. According to Fantin et al, macrophages act as cellular chaperones for vascular anastomosis. 7 However, the molecular machinery that stimulates and regulates vessel fusion remains largely unknown. 3,4 Our previous work [8][9][10] revealed that intermedin (IMD; also named adrenomedullin 2 [ADM2]), a member of the calcitonin family, 11 normalizes the tumor vasculature and effectively improves tumor blood supply. IMD dramatically remodels the vasculature morphology into a hierarchical architecture that is well organized with relatively fewer sprouts, 9 larger lumens, 8 and more anastomosed vessels. 10 Because IMD significantly reduces vessel density while increasing the number of anastomotic vessels, 9,10 we hypothesize that IMD may be an important molecule that stimulates vessel fusion, thus contributing to vessel normalization and blood perfusion improvement.
IMD induces hierarchical and functional vasculature development, indicating that it may contribute to vessel fusion
As shown in Figures 1A and 1B, in normal tissues and physiological angiogenic (ie, wound healing) zones, vessels sprouted from the existing vasculature and connected to adjacent vessels to form closed vascular systems allowing blood flow. However, in tissues with pathological angiogenesis, particularly in tumors ( Figures 1A and 1B; right panels), the sprouting vessels were often blind-ended and did not fuse to the circulatory networks. The decreased vessel connectivity led to stagnant or completely dead ends, even in highly vascularized areas ( Figure 1B). Compared with wild-type (WT) mice, IMD-knockout (KO) (IMD −/− ) mice exhibited significantly more blind-ended vessels in both the physiological and tumor vasculatures ( Figure 1C). To confirm the specificity of the vascular phenotype observed in IMD −/− mice, a rescue experiment was performed. Injection of the mature IMD peptide into IMD −/− mice increased vessel connectivity under both physiological and pathological conditions, enhancing the hierarchical nature of the vasculature ( Figure 1D). Impaired vessel hierarchical structure was also observed in the developing retinal vasculature in IMD −/− mice (Figure 1E). We counted the vessel rings between the arteriole and venule to indirectly assess successful vessel anastomosis ( Figure 1F). The number of vessel rings was approximately 48% lower in IMD −/− mice than in WT mice (Figure 1H). Because increases in vessel ring numbers may be due to increased vessel branching, we counted the tips of vessels that sprouted from the leading front of the retinal vasculature. The number of vessel sprouts in IMD −/− mice was significantly greater than that in WT mice ( Figure 1G and 1I). This finding is consistent with our previous observation that IMD restricts rather than promotes vessel branching. 9 Our previous work has revealed that IMD increases tumor blood perfusion, whereas IMD blockade decreases tumor blood perfusion. 10 Herein, in the retinal model, we found that effective perfusion of the capillaries between the arteriole and venule was severely impaired in the IMD −/− mice ( Figures 1J and 1K), consistent with our previous observation.
IMD promotes vessel fusion in in vitro and in vivo models
Histological images can provide only snapshots of vessel growth at certain time points. To observe the vessel fusion process more dynamically, we established a threedimensional in vitro angiogenic model (fibrin bead assay) 12 to monitor the continuous growth of vascular sprouts and the approach, contact, and fusion of the sprouts with other vessels ( Figure 2A). Vascular endothelial growth factor (VEGF) is believed to be the most important growth factor during angiogenesis. However, VEGF alone was unable to induce the sprouting vessels to form the fine connections necessary for establishment of a hierarchical vessel system. In the vehicleand VEGF-treated groups, the adjacent vessels approached reciprocally, but most of them crossed over without connecting or fusing (Figure 2A). On the other hand, IMD dramatically promoted the contact of approaching vessels and the formation of fine connections in the presence or absence of VEGF. In addition, treatment with a monoclonal antibody targeting IMD (anti-IMD) inhibited this effect (Figure 2A). Compared with the control, IMD significantly increased the rate of successful vessel fusion by approximately 2.5-fold, and anti-IMD decreased the rate by approximately threefold F I G U R E 1 IMD promotes vessel fusion in vitro and in vivo. A, H&E histological analysis of the normal dermis, healing wound, and tumor tissue (subcutaneously inoculated Lewis lung cancer) from C57/BL6 mice. B-D, Immunofluorescent analysis (stained for CD31) of the vascular system in normal dermis, healing wounds, and tumor tissues from WT, IMD −/− , and IMD −/− mice rescued by IMD peptide injection. The white arrows in panel (B) indicate the representative blind-ended vessels. E-I, Retinal vasculature of WT and IMD −/− mice (stained with IB4). F, Asterisks indicate the vessel circles between the retinal arteriole and venule. G, Asterisks indicate the vessel sprouts at the leading front of retinal vasculature. H and I, Quantification of vessel circles and sprout tips using 20 randomly chosen fields from five samples. J, WT or IMD −/− mice were injected with FITC-dextran to mark the perfused vessels. Retinas were stained with IB4. Dash line circles the area between arteriole and venule. K, The effective perfusion (FITC-positive area) between arteriole and venule was quantified by the software Image-Pro Plus v5.0.2.9 (similarly hereinafter) using 10 randomly chosen fields. The data were presented as scatter plots with mean ± SEM. Significance was assessed by unpaired, two-tailed parametric t test with Welch's correction (H, I, and K). IMD −/− : intermedin knockout; FITC: fluoresceine isothiocyanate; WT: wildtype ( Figure 2B). IMD is expressed in blood vessels and endothelial cells (ECs). 10 Thus, we examined fusion in ECs isolated from IMD −/− mice and found that the successful fusion rate was significantly lower in IMD −/− mouse ECs than in normal WT mouse ECs; in addition, the reduction was reversed by exogenous IMD administration ( Figure 2C).
The experimental accessibility and optical clarity of zebrafish embryos make these animals good models for observation of the dynamic vessel fusion process in vivo. 13 In normal embryos, the intersomitic vessels (ISVs) sprout from the dorsal aorta and then fuse with the dorsal longitudinal anastomosing vessels (DLAVs). 14 We used morpholinos (IMD-MO and Ctrl-MO as a control; sequences provided in Section 4) to knock down IMD expression in Tg (flk1:eGFP) transgenic zebrafish. The gene knockdown (KD) efficiency was verified by real-time RT-PCR ( Figure 2D). For a gene rescue experiment, the mature zebrafish IMD (zIMD) peptide was synthesized and added to the fish water to reach a final concentration of 2 µM. The vessel fusion between ISVs and DLAVs was severely impaired in the IMD-MO-injected zebrafish; 56 of 66 embryos showed abnormal anastomosis. The abnormal vessel fusion in IMD-MO-injected zebrafish was significantly alleviated by zIMD administration, after which only five of 59 embryos showed abnormal anastomosis. No abnormalities were observed in the Ctrl-MO-treated zebrafish ( Figures 2E and 2F). Time-lapse imaging showed the progression of vessel fusion in the Ctrl-MO-and IMD-MO-treated zebrafish ( Figure 2G).
Given the ability of IMD to promote vessel anastomosis, we hypothesized that IMD may help transplant grafts regain F I G U R E 2 The dynamic process of IMD-induced vessel fusion. A, Fibrin beads assay: VEGF (50 ng/mL), IMD (2 µM), or anti-IMD (25 µg/mL; same doses hereinafter) were added at day 1. Images in the same field were captured at day 11 and day 14. B, The successful fusion rates were calculated using 10 randomly chosen vessels from two independent experiments. C, Fibrin beads assay was performed using endothelial cells function, as vascular anastomosis between the graft and host is critical for restoration of blood supply. To test this hypothesis, we performed skin transplant surgery using WT and IMD −/− mice. Compared with WT mice, IMD −/− mice exhibited significant delays in skin graft function restoration (as indicated by hair regrowth). IMD injection reversed this effect ( Figures 2H and 2I). To directly observe anastomosis between the graft vessels and the host vessels, we acquired skin grafts from GFP-transgenic mice (labeled with green fluorescence) and transplanted them onto the backs of WT and IMD −/− mice. After the grafts began to regrow hair, the grafts and surrounding tissues were removed entirely and stained with CD31 (labeled with red fluorescence) to display all blood vessels. In theory, with this method, only the blood vessels from the skin graft will show both green and red fluorescence, whereas the blood vessels from the host will show only red fluorescence. Therefore, connections between red and green blood vessels indicate that the host and graft vessels are successfully fused. Upon counting the successfully anastomosed blood vessels, we found that IMD KO significantly impaired the process of vessel fusion, whereas IMD peptide supplementation alleviated this defect (Figures 2J and 2K). According to the above results, IMD may be a key molecule in vessel fusion and blood supply restoration.
IMD enhances the anchoring ability of adjacent vessels to facilitate successful vessel fusion
We then investigated how IMD facilitates the process of vessel fusion. Two major patterns of vessel fusion, tip-to-tip fusion and tip-to-stalk fusion, were observed ( Figures 3A and 3B). During successful vessel fusion, vessel tips approached and anchored to adjacent vessel stalks or vessel tips and formed fine anastomoses with connected hollow lumens (Figures 3A and 3B); however, during unsuccessful fusion, although adjacent vessels approached and contacted reciprocally, they were unable to anchor to each other and instead crossed over each other ( Figures 3C and 3D). Statistical analysis showed that IMD markedly increased the successful fusion rates of both the tip-to-stalk and tip-to-tip patterns and indicated that these effects could be inhibited by anti-IMD ( Figures 3E and 3F).
IMD has been reported to regulate vascular endothelial cadherin (VEC), an endothelial-specific transmembrane component of adherens junctions, 10,15,16 which are the major junctional structures at EC cell-cell contact zones. 17,18 VEC is recruited to the fusion point when an ISV fuses to a DLAV in zebrafish, 13 and stable vessel anastomosis requires full expression of VEC. A partial reduction in VEC expression compromises the capacity for establishment of successful reciprocal contacts. 19 Thus, VEC-medicated EC-EC adhesion may be crucial for successful vessel fusion. We hypothesized that IMD may promote the formation of the VEC complex between reciprocally approaching ECs, thereby enhancing the anchoring ability of the adjacent vessels and facilitating successful vessel fusion. We performed an RNA interference and rescue experiment to test this hypothesis. VEC transcription levels were assessed by real-time RT-PCR ( Figure 3G). KD of VEC via targeting of the 3 ′ -UTR of VEC mRNA significantly impeded the ability of IMD to induce vessel fusion ( Figure 3H).
We next investigated whether VEC KD would affect already established anastomotic points. Cells in fibrin beads were transfected with siRNA-VEC at day 11. The VEC KD efficiency was determined by western blot (WB) assay (Figure 3I, lower panels). We found that the already established vessel fusion sites dissociated after VEC KD for 3 days (Figure 3I, upper panels). To determine whether VEC expression would repair this disruption, we performed a VEC rescue experiment by transfecting the cells in this system with Lv.VEC after VEC KD at day 11. The VEC expression levels at day 15 and day 19 (after VEC KD or VEC KD rescue for 4 days and 8 days, respectively) were evaluated by WB assay ( Figure 3J, lower panels). We found that VEC rescue reestablished the impaired fusion sites of adjacent vessels ( Figure 3J, upper panels) and facilitated successful vessel fusion. (1 cm × 1 cm), and the red circle marked the hair regrowth area. I, The hair regrowth area in the transplant site was quantified (n = 6). J, Skin grafts from the GFP-transgenic mice (green) were transplanted onto the backs of WT or IMD −/− mice. After the grafts begun to regrow hair, the grafts and surrounding tissues were entirely removed and stained with CD31 (red) to display all blood vessels. The blood vessels from GFP + -grafts show green/red overlapped fluorescence, whereas the host vessels show only red fluorescence. K, The connection between red and green/red double-positive vessels was quantified, which indicates that the host and graft vessels are successfully fused (n = 12). The data were presented as columns with mean ± SD. Significance was assessed by one-way ANOVA (Kruskal-Wallis test) followed by nonparametric Dunn's post hoc analysis. anti-IMD: antibody to intermedin; Ctrl: control; FITC: fluoresceine isothiocyanate; IMD −/− : intermedin knockout; MO: morpholino; VEGF: vascular endothelial growth factor; WT: wildtype F I G U R E 3 The IMD-induced vessel fusion is VEC dependent. A and B, Fibrin beads assay (described in Section 4) was performed to observe the process of vessel fusion in vitro. Representative patterns of successful and unsuccessful tip-to-stalk and tip-to-tip vessel fusion. The dash-line box outlined the partial enlarged view of the area of vessel fusion. The dash-line circles outlined the contact and fusion sites. The white arrows indicated the widening of the fusion site. C and D, Representative patterns of successful and unsuccessful tip-to-stalk and tip-to-tip vessel fusion. The 2.4 IMD induces ECs to enter a "ready-to-anchor" state by fine-tuning the behavior of VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution IMD has been reported to stabilize endothelial junctions 10,15,20,21 ; however, it is unclear whether IMD facilitates the formation of new VEC complexes between ECs that are adjacent but not yet fully contacting each other, which is a prerequisite for successful vessel anchoring. Herein, we investigated whether IMD affects VEC distribution at cell-cell contact areas. We found that IMD treatment significantly increased the accumulation of VEC in filopodial bridges that connected adjacent ECs, whereas anti-IMD treatment markedly decreased VEC accumulation ( Figures 4A and 4B). These results indicate that IMD may facilitate vesicular VEC transport to potential contact points. Thus, IMD may help VEC translocate to potential contact points, therefore inducing ECs to enter a ready-to-anchor state. This process may help adjacent vessels make good contacts and form VEC complexes, resulting in an increased chance of successful vessel fusion.
We then realized that good contact between adjacent ECs is necessary but not sufficient to achieve successful vessel fusion. As shown in Figure 3A-D, successful vessel fusion requires three steps: contact, anchoring, and widening of the anastomotic point. After the approaching sprouts anchor to each other, the anastomotic point must be expanded to enable interconnection of adjacent lumenized vessels ( Figure 4C). If IMD only strengthens the VEC complex at the cell-cell contact point and prevents the dissociation of this complex, the adjacent ECs will be firmly fixed to the anastomotic point, which will restrict the widening of the anastomotic site and lead to unsuccessful vessel fusion. Thus, the dynamic dissociation and reconstitution of the VEC complex are indispensable processes for successful vessel fusion.
Autophosphorylation of VEC leads to VEC complex dissociation and VEC endocytosis. 18 Because IMD has been reported to stabilize the endothelial barrier, 10,15,20,21 IMD might affect VEC phosphorylation. WB and immunoprecipitation (IP) assays revealed that IMD increased VEC phosphorylation at Y685 in HUVECs, similar to the effect of VEGF ( Figures 4D and S1A). However, IMD did not phosphorylate VEC at Y658 or Y731 as VEGF did ( Figure S1B). The cytoplasmic domain of VEC contains nine tyrosine residues that represent potential phosphorylation sites. According to Wallez, VEC is a direct substrate for Src kinase, and Y685 is the unique site phosphorylated by active Src. 22 Our recent work has revealed that IMD induces Src phosphorylation in vitro and in vivo and that the specific Src inhibitor SU6656 blocks constitutive and IMD-induced Src phosphorylation. 8 Herein, the IP/WB assay revealed that SU6656 inhibited IMD-induced VEC phosphorylation ( Figure 4E). As positive and negative controls, VEGF and anti-IMD induced and inhibited the phosphorylation of VEC, respectively (Figure 4E). According to the results, IMD may activate VEC via the Src signaling cascade. An IP time course assay revealed that 5 min of IMD treatment induced VEC phosphorylation; this effect became gradually stronger with longer treatment durations ( Figure 4F). Tyrosine phosphorylation usually peaks approximately 5-10 min after stimulation and then declines rapidly. Thus, IMD may exert a persistent effect on VEC activity.
VEC internalization is considered a result of VEC phosphorylation. Herein, we used two VEC antibodies, one recognizing the extracellular domain of VEC (named VEC-ext) and another recognizing the intracellular domain of VEC (named VEC-int). HUVECs were labeled with VEC-ext or nonspecific IgG for 30 min at 4 • C, stimulated with vehicle and IMD (with or without pretreatment with SU6656) for 20 min at 37 • C, and then stained using an Alexa Fluor 488-conjugated secondary antibody (green). After successful staining was confirmed under a microscope, the cells were incubated with the VEC-int antibody and subsequently stained with Alexa Fluor 568 (red). We calculated the numbers of internalized VEC vesicles and found that IMD significantly increased them; furthermore, SU6656 nearly completely inhibited this effect ( Figures 5A and 5B). We then performed a surface biotinylation assay to confirm this effect. The results showed that IMD stimulation significantly reduced the levels of biotin-labeled VEC on the cell surface ( Figure 5C), indicating that the increased numbers of VEC vesicles in the cytoplasm were indeed coming from the cell surface.
VEC internalization is considered a result of VEC complex dissociation. Thus, although IMD promoted the formation of dash-line box outlined the partial enlarged view, and the dash-line circles indicated that the approaching vessels contact but do not anchor with each other, resulting in unsuccessful vessel fusion. E and F, The successful tip-to-stalk and tip-to-tip fusion rates (treated with vehicle, IMD, or anti-IMD) were calculated using 10 randomly chosen vessels from two independent experiments. G, The VEC-knockdown and VEC-knockdown and rescue efficiency was tested using real-time RT-PCR (n = 10). H, The successful fusion rates (VEC-knockdown or VEC-knockdown and rescue) were calculated using 10 randomly chosen vessels from two independent experiments. I, Representative images showing the impaired vessel fusion site after VEC-knockdown for 3 days (upper panels). The VEC expression level was assessed by western blot (WB) assay (lower panels). Arrows indicated the detachment of the already established anastomotic point. J, Representative images showed that the VEC rescue (VEC-knockdown followed by Lv.VEC transfection) repaired the anastomotic point and leads to successful vessel fusion (upper panels). VEC expression level was assessed by WB (lower panels). KD: knockdown; VEC: vascular endothelial cadherin F I G U R E 4 IMD relocates VEC to the potential contact point and induces VEC phosphorylation. A, Double staining for filopodia (Phalloidin, red) and VEC (green) showed the newly formed VEC complexes between close but not tightly touched ECs. B, The staining density of VEC relative to phalloidin was quantified, as described in Section 4. C, Schematic: the widening of vessel fusion point. First, adjacent vessels contact and anchor to each other; second, the anastomotic point expand continuously for the interconnection of adjacent lumenized vessels. The whole process is accompanied by VEC vesicle transportation and redistribution. D, HUVECs treated with IMD or VEGF were immunoprecipitated (IP) by anti-total-VEC and immunoblotted (IB) by anti-pY685-VEC. E, The HUVECs were pretreated with SU6656 (5 µM for 30 min), followed by IMD treatment for 10 min were subjected to the IP-IB assay; the cells treated by VEGF and anti-IMD were tested as controls. F, Samples from HUVECs treated by IMD for 5, 10, 20, and 60 min were subjected to the IP-IB assay using anti-total-VEC and anti-pY685-VEC. D and F, The levels of p-VEC (referred to t-VEC) were presented relative to control; n = 3. Data were presented as scatter plots with mean ± SD. Significance was assessed by one-way ANOVA (Kruskal-Wallis test) followed by nonparametric Dunn's post hoc analysis (B) or unpaired, two-tailed parametric t test with Welch's correction (D-F). IP: immunoprecipitation; p-VEC: phosphorylated VEC; t-VEC: total-VEC; VEC: vascular endothelial cadherin; WB: western blot F I G U R E 5 IMD fine-tunes the behavior of VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution. A, HUVECs were labeled with anti-VEC-ext (recognizing the extracellular domain of VEC), and stimulated with vehicle and IMD (with or without pre-treatment of SU6656), followed by detection using secondary antibody (green). After successful staining was confirmed, the cells were incubated with the anti-VEC-int (recognizing the intracellular domain of VEC; red). The white box outlined the partial enlarged view of the area near the cell-cell contact. The hollow arrows pointed the constitutive VEC vesicles labeled by anti-VEC-int, and the solid arrows pointed the internalized VEC vesicles labeled by anti-VEC-ext. B, The number of internalized VEC was calculated. C, Surface-biotinylation assay was performed, and the VEC complexes, it concurrently induced VEC complex destabilization and VEC internalization. According to these results, IMD may fine-tune the behavior of VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution, which is a prerequisite for anastomotic point expansion, as demonstrated in Figure 4C.
We next examined the EC monolayer because the ECs that constitute blood vessel walls are in contact with each other, similar to confluent cultured ECs. Interestingly, other than the linear and zipper-like VEC complexes, we observed unique web-like structures between the ECs in close contact with one another ( Figure 5D). The presence of these unique structures suggested the occurrence of a dynamic process of simultaneous VEC complex dissociation and reconstruction. Our recent study revealed that IMD enlarges the vessel lumen by inducing the proliferation of quiescent ECs. 8 Because the ECs that constitute blood vessels wall are in close contact with each other, the proliferation of new ECs from preexisting ECs and the consequent enlargement of the vessel lumen inevitably lead to the dissociation of "old" VEC complexes followed by the reconstitution of "new" VEC complexes, as demonstrated in Figure 5F. This is unlikely to be a unique phenomenon caused by a certain growth factor; rather, it is likely a naturally occurring phenomenon. Stimulation with IMD significantly increased the frequency and the areas covered by these weblike structures, whereas treatment with anti-IMD markedly decreased these parameters. VEGF, although considered to be the most important growth factor in angiogenesis, showed no obvious effect on the formation of the structures (Figures 5D and 5E).
IMD promotes VEC transportation via both the Rab4 and Rab11 recycling routes
As shown in Figure 5B-G, IMD potently induced VEC phosphorylation and subsequent VEC internalization. These findings raised the following question: after VEC is internalized into the cytoplasm, how is it transported back to the cell membrane? The small GTPases Rab4 and Rab11 facilitate rapid and slow canonical recycling routes for membrane proteins, respectively. 23 Using multiple immunofluorescence staining, we found that VEC endosomes colocalized with either Rab4 or Rab11 in the cytoplasm of resting ECs (Fig-ure 6A). This result indicates the existence of a constitutive recycling process for VEC. Treatment with IMD did not affect the expression of either Rab4 or Rab11 in HUVECs ( Figure S2). However, IMD significantly increased the number of Rab4 + and Rab11 + vesicles that accumulated precisely on the linear, zipper-like, and web-like VEC complexes ( Figure 6B-E). According to these results, IMD may induce Rab4and Rab11-carrying VEC endosomes to return to cell-cell contact points to rebuild VEC complexes.
To test whether IMD can directly promote binding of internalized VEC to Rab4/Rab11, thus entering the membraneprotein recycling process, we designed a three-step experiment. In Step 1, cells were labeled with VEC-ext (an antibody recognizing the extracellular domain of VEC). In Step 2, the labeled cells were incubated with vehicle or IMD for 20 min to stimulate VEC internalization and stained with an Alexa Fluor® 488-conjugated (green) secondary antibody for 30 min for detection of internalized VEC. In Step 3, after successful staining was confirmed, the cells were double-stained with anti-Rab4 or anti-Rab11 antibodies and an Alexa Fluor® 568-conjugated (red) secondary antibody. The results showed that IMD significantly promoted binding of internalized VEC to Rab4/Rab11 ( Figure 6F-H). These findings raised another question: why does internalized VEC tend to bind to Rab4/Rab11? Interestingly, we noticed that IMD treatment did not induce merely transient VEC phosphorylation; rather, it induced persistent VEC phosphorylation, which became gradually stronger with longer treatment durations (Figure 5D). This is not a common phenomenon. It is well known that tyrosine phosphorylation usually peaks for 5-10 min after stimulation and then declines rapidly. Thus, we sought to investigate the role of persistent phosphorylation, hypothesizing that such phosphorylation helps VEC bind to Rab4/Rab11.
We designed an experiment to test this hypothesis. After cells were labeled with VEC-ext and stimulated with IMD to induce VEC phosphorylation and internalization, the cell lysates were collected and subjected to IP with VEC-ext and VEC-int separately. As demonstrated above, IMD induced persistent VEC phosphorylation; thus, at 20 min after IMD stimulation, internalized VEC should still be phosphorylated and able to be recognized by an anti-VEC-Y685 antibody. In addition, if phosphorylated VEC preferentially binds to Rab4/Rab11, the anti-VEC-extimmunoprecipitated protein complex should be able to be recognized by anti-Rab4 and anti-Rab11 antibodies, but the level of biotin-labeled VEC on the cell surface was detected (n = 3). D, The representative images showing the web-like VEC complex in HUVEC monolayer treated by vehicle, IMD, VEGF, or anti-IMD. E, The area covered by web-like structure was quantified (n = 10). F, The expansion of anastomotic point was needed to enlarge the vessel lumen via EC proliferation. The newly proliferated ECs from the pre-existing ECs will inevitably lead to the "old" VEC complex dissociation and the "new" VEC complex reconstitution, resulting in coexistence of three types of VEC complex: line-shape, zipper-like, and web-like structure. All quantifications use 10 randomly chosen fields from two independent experiments. Data were presented as scatter plots with mean ± SEM. Significance was assessed by one-way ANOVA (Kruskal-Wallis test) followed by nonparametric Dunn's post hoc analysis. IP: immunoprecipitation; VEC-ext: antibody recognizing the extracellular domain of VEC; VEC-int: antibody recognizing the intracellular domain of VEC; WB: western blot anti-VEC-int-immunoprecipitated protein complex should not. A diagram of the experimental design is shown in Figure 6I. The results confirmed our hypothesis that upon IMD stimulation, internalized phospho-VEC preferentially bound to Rab4/Rab11, but nonphosphorylated VEC did not ( Figure 6J-M). Taken together, the experimental findings described above show that IMD directly promotes VEC internalization from the cell surface by inducing persistent VEC-Y685 phosphorylation. The internalized phospho-VEC preferentially binds to Rab4 and Rab11, which can facilitate VEC vesicle transport in the cytoplasm and recycling back to cell-cell contact areas.
The active form of Rab4 or Rab11 is required for VEC transportation but may not be necessary for VEC cargo binding
As mentioned above, immunofluorescence analysis of cell slides can provide only a snapshot of the cell status at a certain time point. To acquire more direct evidence of whether VEC is recycled via Rab4 or Rab11 in living cells, HUVECs were transfected with lentiviruses expressing VEC (red) and either Rab4 or Rab11 (green) and observed under time-lapse microphotography at 15-s intervals. The VEC vesicles were bound to either Rab4 ( Figure 6F) or Rab11 ( Figure 6G), indicating that VEC endosomes were concurrently recycled through either a Rab4-or Rab11-dependent route. The continuous microphotography time lapse revealed that over certain time periods, one or several endosomes in a field could travel long distances across the cytoplasm, whereas others usually vibrated in a Brownian manner (Videos 1 and 2 in the Supporting Information). One Rab4-bound VEC vesicle took approximately 8 min to travel from the cytoplasm to the cell border, and a Rab11-bound VEC vesicle took approximately 18 min to travel across a similar distance (Figures 6F and 6G; Videos 1 and 2 in the Supporting Information). IMD significantly increased the numbers, ranges, and speeds of long-range-moving Rab4-and Rab11-bound VEC vesicles (Figures 6H and 6I; Videos 3 and 4 in the Supporting Information). KD of Rab4 or Rab11 partially impeded the ability of IMD to promote VEC transportation ( Figures 6J-L and S3). To exclude possible off-target effects, cells were transfected with small hairpin RNA (shRNA) targeting the 3 ′ -UTR of either Rab4 or Rab11; the KD was subsequently reversed by transfection of Rab4 WT or Rab11 WT constructs that did not contain the 3 ′ -UTR, respectively. The constitutive and IMD-induced VEC movement were both restored ( Figure 6J-L). When Rab4 and Rab11 were silenced at the same time, the movement of VEC endosomes was nearly blocked, and IMD lost its ability to promote VEC movement ( Figure 6J-L). According to these results, IMD may promote VEC transportation via both the Rab4 and Rab11 recycling routes.
Small GTPases, including Rab4 and Rab11, have two states: active and inactive, during which they bind GTP or GDP, respectively. To determine whether the activity status of Rab4 and/or Rab11 affects VEC transportation, GTPbound constitutively active mutant forms of Rab4 and Rab11 (Rab4[Q67L] and Rab11[Q70L], abbreviated as Rab4 Act and Rab11 Act , respectively) and GDP-bound dominant-negative mutant forms (Rab4[S27N] and Rab11[S25N], abbreviated as Rab4 Neg and Rab11 Neg , respectively) were constructed (labeled with green). HUVECs were co-transfected with VEC-expressing lentiviruses (red) and the mutant constructs. The constitutively active forms (Rab4 Act /Rab11 Act ) bound to nearly all VEC endosomes, similar to the respective WT constructs (Figures 7A and 7B). Surprisingly, Rab4 Neg and Rab11 Neg also bound to VEC (Figures 7A and 7B); this was unexpected because the dominant-negative mutants of Rab4 and Rab11 were assumed to be unable to bind their cargoes. Sequential microphotography revealed that the movement of VEC/Rab4 Act and VEC/Rab11 Act vesicles was slightly greater than that of VEC/Rab4 WT and VEC/Rab11 WT vesicles ( Figure 7C-H and Videos 5 and 6 in the Supporting Information). However, the movement of VEC/Rab4 Neg and VEC/Rab11 Neg vesicles was significantly decreased F I G U R E 6 IMD promotes VEC vesicle transportation via Rab4 and Rab11. A, HUVECs were double stained with VEC/Rab4 or VEC/Rab11. Arrows indicate the co-localization of VEC/Rab. (B) The accumulation of Rab4 + or Rab11 + vesicles on three types of VEC complexes: line-shape, zipper-like, and web-like structure. Arrows indicate Rab4 + or Rab11 + vesicles co-localization with VEC complex. C-E, Quantification of Rab4 + or Rab11 + vesicles accumulated on the line-shape (C), zipper-like (D), and web-like VEC complex (E). F, The HUVECs were labeled with VEC-ext (an antibody recognizing extracellular domain of VEC, green) or nonspecific IgG, and incubated with vehicle or IMD-stimulated VEC internalization. After successful staining was confirmed, the cells were double stained for Rab4 or Rab11 (red). G and H, The number of VEC + /Rab4 + and VEC + /Rab11 + vesicles were counted (n = 10). I, The diagram shows how to detect the interaction of phospho-VEC and Rab4/Rab11: HUVECs were labeled with VEC-ext and stimulated with IMD to induce VEC phosphorylation. The cells lysates were collected and immunoprecipitated with VEC-ext and VEC-int, respectively. The precipitated proteins were then immunoblotted using anti-Rab4 or anti-Rab11 antibodies. If the phosphorylated VEC preferentially binds to Rab4/Rab11, the anti-VEC-ext-immunoprecipitated protein complex will be recognized by anti-Rab4 and anti-Rab11 antibodies, but the anti-VEC-int-immunoprecipitated protein complex will not. J, The IP-IB assay detected the interaction between phospho-VEC and Rab4/Rab11. K-M, The density of the band (referred to total-VEC) was presented relative to that of the control. The mean level in the control group was set to 1.0; n = 3. Significance was assessed by unpaired, two-tailed parametric t test with Welch's correction. IB: immunoblotting; IP: immunoprecipitation; VEC: vascular endothelial cadherin; Veh: vehicle; VEC-ext: antibody recognizing the extracellular domain of VEC; VEC-int: antibody recognizing the intracellular domain of VEC ( Figure 7C-H and Videos 7 and 8 in the Supporting Information). Thus, the active form of Rab4 or Rab11 is required for VEC transportation but may not be necessary for VEC cargo binding.
DISCUSSION
During vascular development and angiogenesis, vessel sprouts must meet, connect, and fuse to adjacent vessels to create a closed vascular system. The vessel fusion process is crucial in establishing a hierarchical and functional vasculature. However, the molecular mechanism underlying this process remains largely unknown. In this study, we found that an endogenous peptide, IMD, functions as a key mediator to stimulate vessel fusion by precisely regulating the behavior of VEC. According to previous studies, VEC is recruited to the EC interface during the fusion of ISVs and DLAVs, 13,24 and successful vessel fusion requires stable cell-cell junctions that are defective in the absence of VEC. 19 Although the importance of VEC in vascular fusion has been demonstrated, the molecular mechanism by which VEC participates in the regulation of vascular fusion remains unclear.
In this study, we found that IMD regulates the behavior of VEC in two ways. First, IMD increases VEC accumulation at the potential fusion point, thereby inducing ECs to enter a ready-to-anchor state that facilitates the anchoring of approaching vessels to each other via VEC complex formation. Second, IMD not only induces VEC complex dissociation and VEC endocytosis via Src-mediated VEC-Y685 autophosphorylation but also facilitates VEC complex rebuilding by promoting VEC endosome transport back to the cell-cell contact point via Rab4-and Rab11-mediated vesicle trafficking routes.
Our results indicated that IMD directly promoted VEC internalization from the cell surface by inducing persistent VEC-Y685 phosphorylation. The internalized phospho-VEC preferentially bound to Rab4 and Rab11, but nonphosphorylated VEC did not. The role of IMD in promoting VEC vesicle movement may be due to the characteristics of Rab4 and Rab11. In this process, perhaps the most important effect of IMD is promotion of VEC and Rab4/Rab11 binding; the rest of the process is mediated by Rab4 and Rab11. As the cargo vehicle molecules of fast and slow canonical recycling routes, respectively, Rab4 and Rab11 can facilitate VEC vesicle transport in the cytoplasm and recycling back to cell-cell contact areas.
Previous studies have noted the importance of VECmediated formation of stable reciprocal contact between adjacent vessels during vascular fusion. 19 Although good contact between adjacent ECs is indeed necessary, it is not sufficient for successful vessel fusion. If the VEC complex at a cellcell contact point does not dissociate, the adjacent ECs will become firmly fixed at the anastomotic point, anastomotic site widening will be prohibited, and functional connection of the lumenized vessels will be impossible. Our results suggest that vascular fusion is a highly dynamic process in which IMD fine-tunes the behavior of VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution. The unique effect of IMD on VEC also ensures that when fusion sites are widened, the ECs at both ends remain connected with newly formed VEC complexes. This activity ensures that vascular leakage does not occur, which may result when the old VEC complexes dissociate.
Although vessel fusion is a crucial step in angiogenesis and vascular development, only a few studies have focused on this process. Fantin and colleagues have reported that macrophages may act as cellular chaperones for vascular anastomosis. 7 However, vascular fusion can still occur without the presence of macrophages near vascular junctions. According to a study by Kubota and colleagues, although macrophage defects lead to decreases in the density of vascular networks at the beginning of vascular development, a relatively normal vascular system is eventually formed. 25 In this study, we observed two patterns of vessel fusion (tip-to-tip and tip-to-stalk) in the three-dimensional in vitro angiogenesis model (fibrin bead assay). This system contained only ECs and skin fibroblasts; it did not contain macrophages. These results suggest that macrophages may facilitate the process of vascular fusion but may be dispensable.
In the first week after birth, the retinal blood vessels in mice are still in the developmental stage and do not cover the entire retinas. After postnatal day 7 (P7), the retinal vasculature begins to mature, and some small branches regress. As F I G U R E 7 The active form of Rab4 or Rab11 is required for VEC transportation. A-D, HUVECs were co-transfected with lentivirus expressing VEC/Rab4 WT or VEC/Rab11 WT , treated without IMD (A and B) or with IMD (C and D), and observed under continuous microphotography with a 15-s interval. Arrows indicate the long-range moving VEC + /Rab4 WT+ or VEC + /Rab11 WT+ vesicles. E-G, HUVECs were transfected with shRNA to knockdown Rab4 (shR-47596 targets ORF; shR-47597 targets 3'-UTR) or Rab11 (shR-377861 targets ORF; shR-411101 targets 3'-UTR), and rescued by transfection of Lentiviral Rab4 WT or Rab11 WT , and incubated with or without IMD. The number of long-range moving VEC + vesicles and their moving distance and speed were quantified. H and I, HUVECs were co-transfected with Lentiviral VEC (red) and Rab4 WT /Rab4 Act /Rab4 Neg (green) or Rab11 WT /Rab11 Act /Rab11 Neg (green), respectively. J-O, The number of long-range moving VEC + /Rab + vesicles and their moving distance and speed were quantified. All quantifications use 10 randomly chosen fields from two independent experiments. Data were presented as scatter plots with mean ± SEM. Significance was assessed by one-way ANOVA (Kruskal-Wallis test) followed by nonparametric Dunn's post hoc analysis. Act: active; IMD −/− : intermedin knockout; KD: knockdown; Neg: negative; shRNA: small hairpin RNA; VEC: vascular endothelial cadherin; WT: wildtype reported by Fantin and colleagues, 7 retinal vascular regression is detectable from P9 to P21. In this study, the eyes of neonatal WT and IMD-KO C57BL/6 mice were collected at P6. At this time point, the occurrence of vascular regression is very rare. Therefore, vessel regression was unlikely to be the main cause of the reduced blood perfusion. According to the results, impaired lumen enlargement and decreased vessel fusion may have been the main causes of the reduced blood perfusion in IMD-KO mice.
Shear stress caused by blood flow is also important for vascular remodeling. 26,27 Here, we observed successful vessel fusions with connected open lumens occurring in the absence of blood flow (Figures 2A and 3, fibrin bead assay). This result suggests that at least in the early stages of angiogenesis and vascular development when neovessels form a primitive vasculature, development of a closed vascular network may be induced by a flow-independent mechanism. At this stage, molecular manipulation may be the primary way to regulate the vessel fusion process. In the later stage, when the primitive vessel network is remodeled into a hierarchical and functional vasculature, blood flow is established, and mechanical stress may begin to participate in the vascular reshaping process.
Taken together, our data suggest a crucial role of the IMD-Rab4/Rab11-VEC signaling axis in vessel fusion. To the best of our knowledge, IMD is the first endogenous molecule observed to be capable of promoting vascular fusion without the aid of other angiogenic factors or cells. IMD acts as a driver, VEC acts as an effector, and Rab4/Rab11 acts as a "cargo vehicle" to transport VEC endosomes to the areas in which EC contact is most likely to occur. Importantly, we have revealed that vessel fusion is a dynamic process in which IMD precisely controls VEC to achieve a dynamic balance between VEC complex dissociation and reconstitution. This machinery is consistent with the natural process of vascular fusion and is supported by our experimental data. Thus, a flow-independent molecular mechanism underlying vessel fusion has been revealed. The ability of IMD to promote vessel fusion may be useful for blood supply restoration after transplantation or during wound healing and for broadening of antiangiogenic drug screening via targeting of the vascular anastomotic process.
Cells and culture conditions
Lewis lung cancer cell line LL/2 was obtained from ATCC and routinely cultured in complete DMEM. HUVECs were isolated from umbilical cords and cultured in EGM-2 (Lonza, Cat. CC-3162). Human skin fibroblasts (HSFs) for coculturing with HUVECs were isolated from surgical specimens and routinely grown in complete DMEM. All cells were tested for mycoplasma contamination, and routinely culture at 37 • C and 5% CO 2 .
IMD(ADM2) −/− mice
The IMD(ADM2) −/− mice were obtained using CRISPR/Cas9 system as described previously. 8 In brief, two sgRNAs were designed targeting the specific locations in the third exon of murine-IMD gene. The IMD/Cas9 plasmids were injected into the zygotes of C57BL/6J mice. The mutation of IMD was identified by PCR in 21 of 28 mice. The PCR products were then cloned and sent to sequence. The sequencing result showed that there was a 317-bp deletion (highlighted) in 27# mice, and coding sequence of IMD was entirely removed. No fetal death or neonatal death was found in the IMD-KO line.
Zebrafish experiment
Tg (flk1:eGFP) transgenic zebrafish were bred and maintained in normal condition (28 • C; pH 7.2-7.4; 14 h on and 10 h off light cycles). A total of 5-10 nL suspension containing 10 ng ctrl-Morpholino (MO) or IMD-MO were injected into zebrafish embryo 48 h post fertilization through the perivitelline space in a single injection by using an electronically regulated air-pressure microinjector (Harvard Apparatus, NY, PL1-90). For each implantation, about 50-60 fish were transferred to six-well plate containing 2 mL of fresh fish water. In the IMD rescue group, zIMD peptide (synthesized mature zebrafish-IMD, VGCVLGTCQVQNL-SHRLYQLVGQSGREDSPINPRSPHSY) was added into the fish water to reach a final concentration of 2 µM. The fish water was changed daily.
The living zebrafish embryos were anesthetized using 0.003% tricaine and embedded in 3% methylcellulose. The digital micrographs were acquired using Zeiss Imager Z1 fluorescence microscope (Carl Zeiss Microimaging Inc, Germany) equipped with an AxioCam MRc5 digital CCD camera or a Zeiss LSM 510 Meta Confocal Microscope. Images were taken in the same focal plane in brightfield and transmitted light passing through GFP filters (488 nm); 0.5-2 µm step z-stacks (512 × 512 focal planes, 50-200 µm in depth) were acquired by using 10× or 20× objective lens. Image capture and processing were performed by using ZEISS Axiovision rel.4.8 software.
Morpholino information
Ctrl-MO: 5 ′ -CCTCTTACCTCAGTTACAATTTATA-3 ′ is a standard negative control sequence, targeting a human betaglobin intron mutation that causes beta-thalassemia. This MO causes little change in phenotype in any known test system except human beta-thalassemic hematopoietic cells, and been broadly used as a negative control. IMD-MO: 5 ′ -AAACCGGGAAAAGCGCTCTCATTGT-3 ′ (targeting IMD or ADM2) is designed and provided by Gene Tools LLC.
Fibrin beads assay
Dextran-coated Cytodex-3 beads (GE) were coated with HUVECs at a concentration of 400 cells/bead. The following day, the HUVEC-coated beads were washed and resuspended at a concentration of 200 beads/mL in 2.5 mg/mL of fibrinogen (Sigma) with 0.15 units/mL of aprotinin (Sigma). A total of 0.625 units of thrombin (Sigma) was added into the solution containing 500 µL fibrinogen/bead in one well of a 24-well culture plate. Fibrinogen/bead solution was allowed to clot, and 2 × 10 4 HSFs were plated on top of the gel clot. Medium was replaced every other day until desired growth is achieved. Reagents were added as indicated in the figure legends.
IP analysis
Cells were washed by ice-cold PBS and lysed with nondenaturing lysis buffer that contains cocktail proteinase inhibitor. After centrifugation, the supernatant was extracted, and incubated with 2 µg anti-VEC (Cat. #2500, 1:50) under continuous rotation at 4 • C overnight. The mixture was then incubated with the agarose beads (Cat. D00118065, Calbiochem) under continuous rotation at 4 • C for 4 h. After centrifugation, the supernatant was removed. The beads were washed by nondenaturing lysis buffer, mixed with loading buffer, and then boiled for 5 min. After centrifugation, the supernatant was collected and subjected to WB analysis.
Antibody feeding assay
Two VEC antibodies, one recognizing the extracellular domain of VEC (named as VEC-ext; from BD PharMingen, Cat. 555661) and another recognizing the intracellular domain of VEC (named as VEC-int; from CST, Cat. #2500), were chosen for this experiment. The HUVECs were plated on coverslips coated with 0.1% gelatine, starved overnight with 0.5% FBS, and labeled with VEC-ext or nonspecific IgG for 30 min at 4 • C, and stimulated with vehicle or IMD (2 µM) with or without pretreatment of SU6656 (5µM) for 20 min at 37 • C. The cells were then fixed with ice-cold acetone, permeabilized with 0.1% Triton X-100 for 10 min, and incubated with an Alexa Fluor 488-conjugated secondary antibody for 30 min to detect VEC-ext antibodies. After successful staining was confirmed under the microscope, cells were incubated with the VEC-int antibody followed by Alexa Fluor 568-conjugated secondary antibody staining.
Cell surface biotinylation assay
The protocol was described previously. 28 In brief, the cells were stimulated with IMD (2 µM) at 37 • C for 10 min to induce VEC internalization. The surface receptors were then labeled with 0.5mg/mL sulfo-NHS-SS-biotin (Life Technology) according to the manufacturer's instructions. After quenching the excess biotin with 100 mM glycine in PBS, the cells were dissolved in lysis buffer (25 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA-NaOH at pH 8.5, 0.5% Triton X100, 0.5% NP-40, 100 mM NaF, 10 mM Na4 P2 O7, and 1 mM Na3 VO4) and protease inhibitor cocktail (Sigma, P2714, 1:100). The lysates were precipitated with avidin agarose beads (Life Technology) and probed for the proteins of interest.
Antibody feeding assay to test the interaction of internalized VEC and Rab4/Rab11
Step 1: The HUVECs were plated on coverslips coated with 0.1% gelatine, starved overnight with 0.5% FBS, and labeled with VEC-ext (an antibody recognizing extracellular domain of VEC) or nonspecific IgG for 30 min at 4 • C Step 2: The labeled cells were incubated with vehicle or IMD for 20 min at 37 • C to stimulate VEC internalization, and stained with an Alexa Fluor 488-conjugated (green) secondary antibody for 30 min to detect the internalized VEC Step 3: After successful staining was confirmed under microscope, the cells were double stained with anti-Rab4 or anti-Rab11 antibodies followed by Alexa Fluor 568conjugated (red) secondary antibody.
IP-IB assay to test the interaction of phospho-VEC and Rab4/Rab11
The HUVECs were plated on coverslips coated with 0.1% gelatine, starved overnight with 0.5% FBS, and labeled with VEC-ext or nonspecific IgG for 30 min at 4 • C, and stimulated with IMD for 20 min at 37 • C to induce VEC phosphorylation and internalization. The cells lysates were collected and immunoprecipitated with VEC-ext and VEC-int, respectively. The precipitated proteins were then immunoblotted (IB) using anti-Rab4 or anti-Rab11antibodies. If the phosphorylated VEC preferentially binds to Rab4/Rab11, the anti-VEC-ext-immunoprecipitated protein complex will be recognized by anti-Rab4 and anti-Rab11 antibodies, but the anti-VEC-int-immunoprecipitated protein complex will not. The diagram of the experimental design is shown in Figure 6I
Continuous microphotography of the living cells
HUVECs were routinely culture at 37 • C and 5% CO 2 , until reached 70-90% confluent. The lentivirus express-ing mCherry-VEC (red) was co-transfected with the lentivirus expressing eGFP-Rab4 WT , eGFP-Rab4 Act , and eGFP-Rab4 Neg or eGFP-Rab11 WT , eGFP-Rab11 Act , and eGFP-Rab11 Neg (green), as indicated in the main text. The living cells were then placed under the inverted confocal microscope (Nikon TI-DH). The double-fluorescent images (Red/Green) were continuously acquired in a 15-s interval, and lasted for 30-60 min. The videos (GIF animation) were generated using the frames with a 15-s interval. The number of long-range moving VEC + , VEC + /Rab4 + , or VEC + /Rab11 + vesicles in each randomly chosen field was counted. The moving distance ( m) and speed ( m/s) of the long-range moving vesicles were also quantified. All quantifications use 10 randomly chosen fields from two independent experiments (n = 10).
Animal studies
All animal experiments were approved by the Animal Ethics Committee of Sichuan University and performed according to institutional and international guidelines.
Skin transplant
WT or IMD −/− C57BL/6 mice (6-8 weeks of age) were used in the skin transplantation. Mice were anesthetized using isoflurane. Hair was removed, and the surgical area was prepared with povidone-iodine following by 70% alcohol. Fullthickness skin was harvested from the dorsum of the donor mouse and sectioned into 1 × 1 cm grafts for subsequent transplantation. The square graft (1 × 1 cm) was placed on a graft bed prepared on the back of the recipient mouse. The graft was covered with protective bandages for 3 days. The hair regrowth in the healed graft area was considered as the skin graft regains its function.
Tumor study
Lewis lung cancer was established in WT or IMD −/− C57BL/6 mice (6-8 weeks of age) by subcutaneous injection with 2.5 × 10 6 cells into the shaved right flank. The tumor volume was measure every 3 days after the inoculation of tumor cells. The tumor volume was determined by the following formula: Volume (mm 3 ) = ½ × length (mm) × width (mm) × width (mm). Mice were anesthetized and euthanized at the end point of the tumor experiment (when the largest tumor reached about 1500 mm 3 according to the ethical standards for animal welfare), and perfused transcardially with 2% PFA in PBS for 10 min to fix the tissues. Tumors along with adjacent normal tissues were removed, and cryosectioned at 120 µm thickness for confocal microscopy and three-dimensional reconstruction.
Retina study
Eyes of the neonatal WT or IMD −/− C57BL/6 mice (6-8 weeks of age) were collected at postnatal day 6 (P6), and fixed with 2% PFA in PBS for 2 h. For the blood perfusion assay, the mice were anesthetized and injected with FITC-dextran through left ventricle and allowed to circulate for 15 min for staining the perfused vessels. Retinas were carefully dissected, washed with PBS, and blocked with 10% goat serum in PBST for 3 h, and then incubated overnight with AlexaFluor568-conjugated IB4 to label the whole retinal vasculature.
Acquisition of microscopic images
The microscopic images from the Fibrin beads assay were acquired using Nikon TE2000 inverted microscope. Images of tissue slides, including the normal dermis, wound healing, and tumor tissues, were acquired using Nikon A1RMP+ upright confocal microscope. Images of a whole retinal vasculature were acquired using Zeiss Z2 upright fluorescence microscope and automatically combined with 25 continuous shooting images by the software Axiovision. Images of cell slides, including the immunostaining of VEC, Rab4, Rab11, and Phalloidin, were acquired using Zeiss Z2 upright fluorescence microscope. The continuous microphotography (to observe the movement of VEC + /Rab4 or VEC + /Rab11 + vesicles in living cells) was performed using Nikon TI-DH inverted confocal microscope with a 15-s interval.
The staining density of VEC relative to phalloidin (Figure 3F) was quantified using Image-Pro Plus v5.0.2.9. The quantification method was as follows: Step 1, the area of cell-cell contacts was selected, and the intensity of F-actin (phalloidin-positive) positive signal was quantified; Step 2, the intensity of VEC signal in the same area was quantified; Step 3, the ratio of the intensities of VEC/phalloidin staining in one field was calculated and expressed as a dot in the statistical graph. The relative intensity of the VEC signal to phalloidin signal at cell-cell contacts was quantified from 10 randomly chosen fields in two experiments.
Statistics
When comparing two groups for which a Gaussian distribution was not assumed, the unpaired, two-tailed nonparametric Mann-Whitney U test was used; when a Gaussian distribution was assumed, the unpaired, two-tailed parametric t test with Welch's correction was used. Data from multiple groups were compared using one-way ANOVA (Kruskal-Wallis test) followed by nonparametric Dunn's post hoc analysis. A P value < .05 was considered statistically significant. *P < .05, **P < .01, and ***P < .001. The n-number of each experiment was indicated in figures. In animal studies, no randomization was applied because all mice used were genetically defined, inbred mice. | v3-fos-license |
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